CN101495641A - Apparatus for making bio-organic compounds - Google Patents

Abstract

Systems and methods for producing bioorganic compounds comprising a container, a first phase comprising an aqueous medium containing host cells capable of producing a bioorganic compound, wherein the bioorganic compound constitutes a second phase.

Description

Equipment for the manufacture of biological organic compounds

Cross References to Related Applications

This application claims U.S. Provisional Application 60/808,989, filed May 26, 2006, entitled "MICROOGRANISMS FOR PRODUCTION OF ISOPRENOIDS"; Fuel and its production method (BIOFUELS AND METHODS FOR PRODUCTION)" U.S. Patent US60/808,666; December 12, 2006, the title of the U.S. Patent U.S.60/ 870,592; and the priority of U.S. Patent U.S. 60/922,782, entitled "APPARATUS FORMAKING BIO-ORGANIC COMPOUNDS," filed April 10, 2007, the entire contents of which are incorporated herein by reference.

Background of the invention

Traditionally, bioorganic compounds of interest are produced by extraction from natural sources such as plants, microorganisms and animals. However, extraction yields are usually very low because most bioorganic compounds accumulate in small amounts in nature. While these quantities are far less than required for many commercial applications, there remains a need for systems and methods for producing bioorganic compounds on an industrial scale.

The present invention fulfills this need. Provided by the present invention are various industrial-scale systems for the production of bioorganic compounds using host cells. These bioorganic compounds have at least five carbon atoms and can be carbohydrates such as mono- or polyols, esters, ethers, aldehydes, ketones, or hydrocarbons such as alkanes, alkenes, or alkynes. The bioorganic compound may be linear or cyclic, and may be saturated or unsaturated.

Summary of the invention

The present invention provides production systems for various bioorganic compounds. In one aspect, a production system for bioorganic compounds is provided, comprising:

a. At least one container with a capacity of at least 100 liters;

b. The aqueous culture medium in the container forms the first phase;

c. a plurality of host cells in an aqueous medium capable of manufacturing, producing or synthesizing at least one bioorganic compound; and,

d. A liquid organic second phase in contact with the first phase comprising said at least one bioorganic compound.

In another aspect, the present invention provides methods of producing at least one bioorganic compound. The method includes:

a. cultivating a plurality of host cells that manufacture, produce or synthesize at least one bioorganic compound in an aqueous medium, wherein the aqueous medium constitutes the first phase;

b. forming an organic second phase in contact with the first phase, the organic second phase comprising said bioorganic compound;

c. separating at least a portion of the organic second phase from the first phase; and,

d. separating said at least one bioorganic compound from the organic second phase.

Description of drawings

Figure 1 is a container with a capacity of at least 100 liters for use in the present invention.

Figure 2 is another embodiment of the container.

Figure 3 is a schematic illustration of the mevalonate ("MEV") pathway to produce isopentenyl diphosphate ("IPP").

Figure 4 is a schematic illustration of the DXP pathway for the production of IPP and dimethylpropylene pyrophosphate ("DMAPP"). Dxs is 1-deoxy-D-xylulose-5-phosphate synthase; Dxr is 1-deoxy-D-xylulose-5-phosphate reductoisomerase (also known as IspC); IspD is 4-diphosphate Cytidine-2C-methyl-D-erythritol synthase; IspE is 4-diphosphocytidine-2C-methyl-D-erythritol synthase; IspF is 2C-methyl-D-erythritol synthase thritol 2,4-cyclic diphosphate synthase; IspG is 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG); and ispH is isopentene/ Dimethylallyl diphosphate synthase.

Figure 5 is a graphical representation of the conversion of IPP and DMAPP to geranyl pyrophosphate ("GPP"), farnesyl pyrophosphate ("FPP"), and geranylgeranyl pyrophosphate ("GGPP").

Figure 6 shows a map of the expression plasmid pMBIS-gpps.

Figure 7 shows a map of the expression plasmid Pam00408.

Figure 8 shows a map of the expression plasmid pAM424.

Figure 9 shows expression plasmids pTrc99A-ADS, pTrc99A-FSA, pTrc99A-LLS, pTrc99A-LMS, pTrc99A-GTS, pTrc99A-APS, pTrc99A-BPS, pTrc99A-PHS, pTrc99A-TS, pTrc99A-CS, pTrc99A-SS, and maps of pAM373.

Figure 10 is a plasmid construction diagram of plasmids pAM489-pAM498.

Detailed description of the invention

definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Many terms cited herein are defined to have the following meanings:

"Bioorganic compound" refers to an organic compound having at least five carbon atoms that can be produced by a host cell by taking up a carbohydrate carbon source and converting the carbohydrate carbon source into a desired product.

"Deoxyxylulose 5-phosphate pathway" or "DXP pathway" as used herein refers to the pathway that converts glyceraldehyde-3-phosphate and pyruvate into IPP and DMAPP. The DXP pathway is illustrated schematically in FIG. 4 .

"Endogenous"refers to a substance or process that may exist naturally, eg, in a non-recombinant host cell.

As used herein, "exogenous nucleic acid" refers to a nucleic acid that meets at least one of the following conditions: (a) the nucleic acid is foreign to a given host cell ("exogenous") (i.e., obtained from the cell not naturally found in the given host cell); (b) the nucleic acid comprises a nucleotide sequence that is naturally present in a given host cell (i.e., is "endogenous" to the cell), but which is present in the (c) the nucleic acid comprises a nucleotide sequence that differs in sequence from the endogenous nucleotide sequence, but the nucleoside the nucleic acid sequence encodes the same protein (having the same or substantially the same amino acid sequence) and is produced in abnormal amounts (e.g., more than expected or found in nature) in said cell; or (d) said nucleic acid Contains two or more nucleotide sequences that are not found in the same relationship to each other in nature (eg, the nucleic acid is recombinant).

"Host cell" and "microorganism" are used interchangeably herein to refer to any archaeal, bacterial, or eukaryotic living cell into which exogenous nucleic acid can or has been inserted. The term also refers to the progeny of the original cell, which may not necessarily be completely morphologically identical to the original parent, or completely complementary in genomic or total DNA, due to natural, accidental, or deliberate mutation.

"Isoprenoid" and "isoprenoid compound" are used interchangeably herein to refer to compounds derived from isopentenyl diphosphate.

"Isolating" when referring to a bioorganic compound means enriching a composition in a certain amount of the bioorganic compound. Thus, after the bioorganic compound has been isolated or subjected to an isolation step, the amount of bioorganic compound in the composition will be greater than the amount present in the composition prior to this step.

"Mevalonate pathway" or "MEV pathway" as used herein refers to the biosynthetic pathway that converts acetyl-CoA to IPP. The MEV pathway is illustrated schematically in FIG. 3 .

When "naturally occurring" is applied to nucleic acids, enzymes, cells, or organisms, it refers to nucleic acids, enzymes, cells, or organisms found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism that can be isolated from a natural source and has not been intentionally modified by man in a laboratory is naturally occurring.

"Optional" or "optionally" means that the subsequently described feature or structure may or may not exist, or that the subsequently described event or circumstance may or may not occur, and that the description includes the specified feature or structure A situation where there is and a feature or structure is not, or a situation where an event or situation occurs and a situation where the event or situation does not occur.

"Pyrophosphoric acid" and "diphosphoric acid" are used interchangeably herein.

As used herein, a composition that is a "substantially pure" compound is substantially free of one or more other compounds, that is, the composition comprises more than 80% by volume, more than 90% by volume of the total volume of the composition. % by volume, more than 95% by volume, more than 96% by volume, more than 97% by volume, more than 98% by volume, more than 99% by volume, more than 99.5% by volume, more than 99.6% by volume, more than 99.7% by volume , more than 99.8% by volume, more than 99.9% by volume of the compound; or less than 20% by volume, less than 10% by volume, less than 5% by volume, less than 4% by volume, less than 3% by volume, less than 2% by volume, less than 1% by volume, less than 0.5% by volume, less than 0.1% by volume, or less than 0.01% by volume of one or more other compounds.

In the following description, all numbers disclosed herein are approximations, regardless of whether the word "about" or "approximately" is used in conjunction therewith. They can vary by 1%, 2%, 5%, or sometimes 10% to 20%. Whenever a numerical range having a lower limit RL and an upper limit RU is disclosed, then any number falling within that range is specifically disclosed. Specifically, the following numbers in this range are specifically disclosed: R=RL+k*(RU-RL), where k is a variable ranging from 1% to 100% in 1% increments, ie k is 1% , 2%, 3%, 4%, 5%, ..., 50%, 51%, 52%, ..., 95%, 96%, 97%, 98%, 99%, or 100%. In addition, any numerical range bounded by two R numbers, as defined above, is also specifically disclosed.

In addition to the above definitions, certain compounds described herein have one or more double bonds which may exist as Z or E isomers. In some embodiments, the invention includes these compounds in substantially pure form as individual isomers, as well as mixtures of various isomers, eg, racemic mixtures of stereoisomers.

Equipment for the manufacture of biological organic compounds

The present invention provides various production systems for producing bioorganic compounds. In some embodiments, the bioorganic compound can be produced using a batch, continuous, fed-batch, or semi-continuous fermentation process.

Batch fermentation can be a closed system where the medium components are fixed at the start of the fermentation and are not artificially altered during the fermentation. Thus, at the start of the fermentation, the medium is inoculated with the desired organism and nothing is added to the system for the fermentation to occur. However, in some embodiments, "batch" fermentations are batchwise to the addition of the carbon source, and often attempts are made to control control factors such as pH and oxygen concentration. In a batch system, the metabolite and biomass composition of the system can change continuously until the fermentation is stopped. Within a batch culture, cells can vary from a static lag phase, through a logarithmic phase of high growth, to a stationary phase, where the final growth rate decreases or ceases. Cells in stationary phase will eventually die if left untreated. Cells in log phase are usually responsible for the production of most end products or intermediates.

A variation on the standard batch system is the fed-batch system. Fed-batch fermentation processes are also suitable for the present invention and comprise typical batch systems, but with the addition of additional carbon sources or substrates in increments during the fermentation. Fed-batch systems are useful when degradant repression tends to inhibit cell metabolism and when it is desired to have a certain amount of substrate in the medium. In fed-batch systems, the actual substrate concentration is difficult to measure and therefore is estimated based on changes in measurable factors such as pH, dissolved oxygen, and partial pressure of exhaust gases such as _CO2 .

Continuous fermentation is an open system in which a defined fermentation medium is fed continuously to one or more bioreactors, which can be connected in series, and equal amounts of conditioned medium can be withdrawn from the system at the same time for feeding Other processing. Continuous fermentation generally maintains the culture at a constant high density so that the cells grow primarily in log phase. Continuous fermentation allows adjustment of a single factor or any number of factors that affect cell growth or final product concentration. For example, one approach would keep a certain amount of nutrients, such as carbon sources or nitrogen levels, at a fixed ratio and let all other parameters attenuate. In other systems, many factors affecting growth can be changed continuously while cell concentrations remain constant. Continuous systems strive to maintain steady-state growth conditions, so cell losses due to media withdrawal must be balanced with the cell growth rate of the fermentation.

Accordingly, in some embodiments of the invention there is provided a bioorganic production system comprising:

a. At least one container with a capacity of at least 100 liters;

b. an aqueous medium within said at least one container, which constitutes the first phase;

c. A plurality of host cells in an aqueous medium capable of manufacturing, producing or synthesizing at least one bioorganic compound; and

d. A liquid organic second phase in contact with the first phase comprising said at least one bioorganic compound.

Suitable containers for use in the present invention can be any container that can hold host cells and an aqueous medium for fermentation. For example, the vessel may be a tank used as a reactor or fermenter, or it may be part of a centrifuge that separates heavier feedstock from lighter feedstock in subsequent processing steps. Alternatively, one or more vessels can be used in a continuous or semi-continuous process.

A general exemplary embodiment of a suitable container 100 is shown in FIG. 1 . Vessel 100 includes an inlet port 120 for adding host cells, fermentation media, and other compounds, nutrients, or components to the vessel to assist, regulate, or improve host cell fermentation, production of bioorganic compounds, and otherwise The production steps are carried out; the outlet interface 130 is used to take out raw materials during or after the fermentation process; and the gas outlet 140 is used to discharge waste gas, such as carbon dioxide produced during or after the fermentation process. Vessel 100 may be completely filled with host cells, fermentation medium, and other materials such that there is no room for gas at the top of the vessel. Alternatively, container 100 may be partially filled, leaving room for gas to be contained. The quantity, pressure, and composition of gases in the space can be controlled to optimize or maximize host cell growth and production of bioorganic compounds. For example, during fermentation in an aerobic host cell, the gas typically may comprise air or other oxygen-containing gas at various pressures above, at or below atmospheric pressure; for example, for microaerophilic and anaerobic host cells, the gaseous Oxygen concentrations can be controlled to a range below atmospheric concentrations but still above zero, and during anaerobic host cell fermentation, the gas typically contains little or no oxygen and can completely contain most or all nitrogen or other suitable gases.

In a closed system, the inlet port 120, outlet port 130, and gas outlet port 140 of the vessel 100 as shown in Figure 1, may be closed or under positive pressure during the fermentation process. Alternatively, especially when using aerobic host cells, the container 100 can be used as an open system, opening one or more ports and outlets to the atmosphere to provide a gas/liquid material transfer (intake of air or oxygen and out of carbon dioxide) system. If desired, the gas outlet 140 can serve as both a gas outlet and a gas inlet, from which oxygen or air or other gases can be introduced into the system. In some embodiments, vessel 100 includes a single gas inlet and a single gas outlet. In such an open system, other hardware may be included on the vessel to prevent contamination or infiltration of other organisms or other materials into the vessel during fermentation.

Another container embodiment is shown in FIG. 2 . In addition to an inlet port 220, an outlet port 230, a gas inlet 235, and a gas outlet 240 similar to the container in FIG. 1, the container 200 in FIG. 2 includes a stirrer bar 250 for mixing. In some embodiments, the stirrer 250 includes a motor drive shaft 252 that includes a shaft seal 251 and is coupled to one or more impellers 254 . Stir bar 250 may typically be attached to the top or bottom of container 200 . Optionally, each impeller 254 may end with one or more blades 256 . The impeller 254 may be of any suitable shape and may be specifically selected to control the amount of mixing, growth rate of host cells, production rate of bioorganic compounds, harvesting rate, and transfer rate of oxygen or other gases. Additionally, one or more baffles 258 may be added to vessel 200 to further enhance mixing. In another embodiment, agitation may be by way of a recirculation line with a pump that draws the feedstock from one part of the vessel, such as the bottom, and reintroduces the feedstock to another part of the vessel, such as the top. Agitation within the vessel of the host cells and fermentation medium helps ensure that the host cells are exposed to sufficient nutrients to allow them to grow and produce bioorganic compounds.

If the fermentation process is an aerobic process, oxygen or air may be bubbled in through sparger 260 to improve gas/liquid material transfer. The sparger 260 may include one or more gas outlets (not shown) submerged in the fermentation medium, preferably at or near the bottom of the vessel. In some embodiments, the showerhead 260 may be a spray ring having multiple gas outlets, typically arranged in a ring or circular configuration. Optionally, to harvest sensitive organisms or reduce foaming, the container can be passively aerated, such as using various ventilation screens, membranes, fibers, or other passive aeration devices, or a portion of the culture medium can be removed from the container and supplied with oxygen , and return it to the container.

If temperature control is desired, heaters or heat exchangers can be used to heat or cool the fermentation reaction. In one embodiment, the temperature can be controlled using a heating/cooling jacket 270 surrounding and/or attached to at least a portion of the vessel 200, which can be connected to a heat exchanger (not shown) to allow temperature controlled Heat exchange liquid is circulated through the sleeve 270 . Alternatively, heaters or heat exchangers may be submerged in the fermentation medium. Exemplary embodiments of this type of heater or heat exchanger include: an electric immersion heater, an immersion heat exchanger with helical or linear tubes of a heat exchange liquid such as hot water or oil, and one or more A nozzle for injecting a hot stream, such as air and/or water, into the fermentation medium. Alternatively or additionally, a heater or heat exchanger may be attached to the outer surface of the container. Such heaters and heat exchangers include electrical heating strips on the outside walls of the vessel, and heated or jacketed recirculation piping attached to the vessel.

Container 200 may contain additional inlet and outlet interfaces. In some embodiments, additional inlet and outlet ports may be located on the top, sides, or bottom of vessel 200 . In some embodiments, additional inlet ports include feed lines for adding oxygen or other gases, nutrients, foam, and pH control agents during the fermentation reaction. Any inlet and outlet connections may contain sterilization means for a variety of purposes, including in-process use and multiple connections or reconnections during fermentation.

Additionally, one or more probe interfaces 280 and/or sample valves 290 can be placed at various locations on the vessel 200 to help monitor critical parameters such as concentrations of various products and metabolites, pH, liquid levels during fermentation , pressure, foam, dissolved oxygen concentration, temperature, agitation rate, power, voltage, valve position, and cell density.

It should be understood that the vessels in Figures 1 and 2 are for exemplary purposes and that many different vessel configurations may be employed for the fermentation process, e.g., depending on the type of host cell, bioorganic compound produced, production volume, fermentation type of process, type of downstream processing, separation process, and other considerations.

A vessel, such as that shown in Figure 2, is suitable for use in a batch fermentation process. If it is desired to conduct a continuous or semi-continuous fermentation process (as opposed to a batch fermentation process) in which feedstock is continuously added to and withdrawn from the vessel, the vessel will typically include additional inlet and outlet connections, which may be located at the top, bottom of the vessel , or side. These additional inlet and outlet ports facilitate the flow of material into and out of the container. In some embodiments, one or more vessels continuously receive host cells, fermentation medium, and optional additives while continuously withdrawing host cells, by-products, and/or bioorganic compounds from the vessels. In these embodiments, the discharge from one vessel can be used as a feed to another vessel, which optionally also receives fresh host cells, fermentation medium, nutrients, and/or other additives . A single vessel, or a series of vessels together can be configured to provide a desired average residence time of the host cells. A portion of the effluent from one of the downstream vessels may be returned to one or more upstream vessels, thereby recirculating the effluent to an earlier stage of processing, or other feedstock from further downstream processing steps may be reintroduced into the vessel middle.

Containers used in some embodiments of the present invention contain additional hardware attachable to the container to facilitate processing. Such hardware may include additional hardware for facilitating CIP and SIP processing. In some embodiments, one, some, or each of the ports, outlets, inlets, valves, and all hardware within the container may be sterilizable in situ. In some embodiments, sterilization can be performed using steam sterilization. For example, any of the interface, outlet, or sample valve may include, or have additional hardware attached to, additional hardware that provides steam supply and condensate return to the interface outlet or valve so that it can be used or reused. Steam sterilized before use.

The container may have a capacity of at least 100 liters. In some embodiments, the container has a capacity of 100 to 3,000,000 liters, such as at least 1000 liters, at least 5,000 liters, at least 10,000 liters, at least 25,000 liters, at least 50,000 liters, at least 75,000 liters, at least 100,000 liters, at least 250,000 liters, at least 500,000 liters, or at least 1,000,000 liters.

The vessel may contain or have sensors and probes attached thereto for measuring various parameters such as pressure, pH, dissolved oxygen concentration, temperature, gas flow rate, liquid flow rate, liquid level, valve position, foam, agitation , power, voltage and any other parameters used to control or optimize the growth of host cells and the production of bioorganic compounds. Sensors and probes can provide information to one or more automated systems, used to control and record various measured parameters, and control the temperature of the vessel, stirring speed, pump, vessel or any inlet by controlling the air flow rate, power, heating or cooling , outlets, injection ports, sample valves, or other interfaces for in-situ sterilization or cleaning, outlet flow control, or any other related mechanism for controlling fermentation parameters, to adjust any of the various parameters. Such regulation may occur using any known control mechanism, eg, controlling or actuating various valves, pumps or motors, and may use proportional, proportional-integral or proportional-integral-derivative control systems.

The automated system may additionally be controlled and monitored by a central control system, which may be a local or plant-wide control system, and may control a process producing only one bioorganic compound or a process producing multiple bioorganic compounds. The automation system and central control system may comprise any suitable software, firmware and/or hardware, which may be proprietary or off-the-shelf, or a combination of both, and may communicate using any suitable communication system. Non-limiting examples of such communication systems include hardwired systems, which may be digital or similar, and may involve direct connections or be in the form of a network such as a LAN or WAN or Ethernet. Additionally, in some embodiments, the communication system may be wireless, and may be proprietary, Bluetooth, Ultra Wideband, 802.11a, b, g, or n, or ZigBee, including TDMA, FDMA, OFDM, and CDMA, and may be in Any suitable band operation, such as 2.4GHz or 5.8GHz.

Any vessel used in the production of bioorganic compounds may contain additional hardware such as additional stir bars, additional inlet ports, outlet ports, sample ports, additional heating/cooling devices such as additional heating coils, additional Aeration equipment such as additional spray heads, additional sensors and probes, additional cleaning or sterilization equipment to facilitate processing or any other fermentation parameters.

In some embodiments of the invention, there are provided isoprenoid production systems comprising:

a. At least one container with a capacity of at least 100 liters;

b. an aqueous medium within the at least one container, which constitutes the first phase;

c. A plurality of host cells in an aqueous medium capable of making, producing, or synthesizing one or more isoprenoid compounds; and

d. A liquid organic second phase in contact with the first phase comprising said one or more isoprenoid compounds.

In some embodiments, the isoprenoid compound is a _C5 isoprenoid. These compounds are derived from one isoprene unit and are also known as hemiterpenes. An exemplary example of a semiterpene is isoprene. In other embodiments, the isoprenoid compound is a _C10 isoprenoid. These compounds are derived from two isoprene units and are also known as monoterpenes. An exemplary example of a monoterpene is myrcene. In other embodiments, the isoprenoid compound is a _C15 isoprenoid. These compounds are derived from three isoprene units and are also known as sesquiterpenes. An exemplary example of a sesquiterpene is phytol (which is also known as patchouli alcohol). In other embodiments, the isoprenoid compound is a _C20 isoprenoid. These compounds are derived from four isoprene units and are also known as diterpenes. An exemplary example of a diterpene is taxene. In other embodiments, the isoprenoid compound is a C20 ₊ isoprenoid. These compounds are derived from more than four isoprene units and include: triterpenes (C ₃₀ isoprenoids, derived from 6 isoprene units) such as squalene; tetraterpenes (C ₄₀ isoprenoids) Pentadiene compounds, derived from 8 isoprenoids) such as β-carotene; and polyterpenes (C40 ₊ isoprenoid compounds, derived from more than 8 isoprenoid units) such as polyisoprene Diene. In some embodiments, the isoprenoid compound can be any combination of two or more isoprenoid compounds.

In another aspect of the present invention, there is provided a method of producing at least one bioorganic compound comprising:

a. cultivating a large number of host cells in an aqueous medium to produce, manufacture or synthesize at least one bioorganic compound, wherein the aqueous medium constitutes the first phase;

b. forming a liquid organic second phase in contact with the first phase, comprising said at least one bioorganic compound;

c. separating at least a portion of the second phase from the first phase; and,

d. separating the at least one bioorganic compound from the second phase.

The isoprenoid production system may comprise one or more additional processing components including: 1) one or more separation systems for separating the at least one biological species from the aqueous medium and the organic second phase organic compound; 2) one or more reactors for biologically or chemically modifying said at least one biological organic compound, for example by addition, substitution, hydrogenation, alkylation, hydroxylation, condensation, halogenation, or any Other suitable reactions; 2) one or more mixing vessels or systems for mixing said at least one bioorganic compound with one or more additional components; 3) and one or more additional purification or separation A system for further purifying said bioorganic composition or said at least one bioorganic compound.

The second phase may comprise the at least one bioorganic compound. Bioorganic compounds can make up a portion, a substantial portion, or substantially all of the second phase. In some embodiments, bioorganic compounds make up 1% to 99%, such as 5% to 95%, 10% to 90%, 20% to 80%, 25% to 75%, 35% to 65%, or 40% % to 50% of the second phase. In some embodiments, the second phase consists essentially of bioorganic compounds.

In some embodiments, the plurality of host cells comprises more than one type of host cell, e.g., more than one species or strain of host cells, e.g., 2-5 species or strains of host cells, e.g., 2, 3, 4 Or 5 species or strains of host cells. In some embodiments, the plurality of host cells can produce more than one bioorganic compound, eg 2-5 bioorganic compounds, eg 2, 3, 4 or 5 bioorganic compounds.

The bioorganic compounds may be separated from the first phase and/or the second phase using any suitable separation method. In some embodiments, the bioorganic compound is isolated from the second phase and is thus substantially pure.

In some embodiments, the organic second phase forms spontaneously due to chemical and molecular interactions, such as differences in solubility or hydrophobicity, density, concentration, or any other spontaneous phase separation mechanism. In other embodiments, the separation of the first and second phases is induced in a separation vessel, or system, which may be the same or a different vessel or process system as the fermentation vessel. In some embodiments, phase separation is induced by centrifugation, eg, continuous or batch centrifugation. In other embodiments, phase separation is induced by introducing demulsifiers or nucleating agents into the fermentation reaction. Demulsifiers prevent or limit the amount of bio-organic compounds that emulsify in the aqueous phase. Exemplary examples of demulsifiers include flocculants and coagulants. Nucleating agents facilitate the aggregation of tiny droplets of bioorganic compounds to coalesce and eventually form separate phases. If a sufficient amount of nucleating agent is used, the nucleating agent itself can form an organic second phase into which bioorganic compounds can migrate. Exemplary examples of nucleating agents include liquid droplets of the bioorganic compound itself and organic solvents such as dodecane, isopropyl myristate, and methyl oleate. Some embodiments may include a combination of one or more of the phase separation materials and methods described above.

Once phase separation has occurred, the separated phases can be withdrawn individually from the separation vessel. Any amount of the second phase can be separated from the first phase, e.g. all, part, 1% to 100%, e.g. 5% to 95%, 10% to 90%, 20% to 80%, 25% to 75% %, 35% to 65%, or 40% to 50% of the second phase can be separated from the first phase. If the organic second phase is not as dense as the aqueous first phase, one or more spigots can be provided or placed on the separation vessel close to the interface between the two phases (preferably in the organic second phase) so that the higher density Drain the organic second phase before the aqueous phase. Alternatively, the aqueous first phase can be withdrawn from the separation vessel through an outlet near the bottom of the separation vessel until the organic second phase emerges. At this point in time, the organic second phase can be transferred to a separate site for further processing or storage. Both the aqueous first and organic second phases can flow from the separation vessel under the influence of gravity, under gas pressure, or by a pump or a combination of pumps.

If the organic second phase is denser than the aqueous first phase, one or more spigots can be provided or placed on the separation vessel close to the interface between the two phases (preferably in the organic second phase) Drain the aqueous first phase before the organic second phase. Alternatively, the organic second phase can be removed from the separation vessel using an outlet near the bottom of the separation vessel.

For continuous processes where the aqueous first phase is denser than the organic second phase, a separate vessel with one or more taps can contain defined volumes of fermentation medium and host cells, while the continuously produced organic second phase can Discharge via tap for storage or further processing. If the organic second phase is denser than the aqueous first phase, the organic second phase can be continuously withdrawn from the bottom of the separation vessel at a rate that prevents complete emptying of the organic second phase from the separation vessel, so as not to draw out the aqueous first phase. Mutually.

In some embodiments, adsorption (the process by which molecules move from a bulk liquid to the surface of an adsorbent) can be used to separate bioorganic compounds from the organic second phase. Illustrative examples of adsorbents include activated carbon; alumina; aluminosilicates such as zeolites; clays such as Fuller's earth; molecular sieves; organic polymers such as polystyrene and resins; Depending on the sorbent used, the sorbent can be used to capture desired bioorganic products or unwanted by-products. Separation by adsorption can be achieved using batch, continuous or semi-continuous processes.

In other embodiments, it is possible to separate bioorganic compounds from the organic second phase using distillation, a method of separating substances based on differences in their volatilities. In batch distillation, the entire batch of liquid is first filled into a vessel, where it is heated or depressurized. Vapor is thus continuously produced and condensed to form a collected liquid distillate. In continuous equilibrium distillation, a continuously flowing liquid feed is heated or depressurized in order to cause partial evaporation of the mixture and separate recovery of the liquid and vapor components. During passage through the distillation column, the liquid and vapor separate and the product appears as a vapor stream and a liquid stream. When the vapor and liquid are close to phase equilibrium, it is called a flashing process. The vapor product can be concentrated to form a liquid distillate, if desired.

In other embodiments, the bioorganic compounds are separated from the organic second phase using gas-liquid extraction. Also known as stripping, this process is the transfer of components dissolved in a liquid stream into a vapor stream in a more concentrated form. Temperature and pressure can be optimized for the desired transfer of bioorganic compounds. Exemplary examples of steam streams include air and steam. Typically, a liquid stream flows down the column, and at the same time a vapor stream bubbles up (opposite the direction of the liquid stream).

In other embodiments, liquid-liquid extraction is used to separate the bioorganic compounds from the organic second phase. Liquid-liquid extraction, also known as solvent extraction, is the transfer of substances from one liquid phase to another immiscible liquid phase.

In a batch liquid-liquid extraction system, the feed liquid (organic second phase) is mixed with an immiscible second liquid phase in a suitable vessel. The mixture is then allowed to stand to separate into an extract and a residue, and the lighter layer can be drained from the container. Depending on the product and the solvent used, the desired bioorganic compound can be present in the extract or in the raffinate.

In continuous liquid-liquid extraction systems, differences in density, vapor pressure at a given temperature, or boiling point can be used to separate desired bioorganic products from feed liquid (organic phase). Such systems may use mixing/settling tanks, columns or columns, centrifuges, and combinations thereof to achieve separation.

In other embodiments, ultrafiltration, a pressure-driven membrane process that separates solution components based on molecular size and shape, is used to separate bioorganic compounds from the organic second phase and/or the aqueous first phase. With differential pressure applied across the UF membrane, solvent and minor solute species permeate the membrane and are collected as permeate, while larger solute species are retained by the membrane and recovered as concentrated retentate. Ultrafiltration involves solute molecules that are ten times or more the size of the solvent, and are typically less than 1/2 micron in size. The solute or material to be separated usually has a molecular weight greater than 500 amu, such as polymers, colloidal dispersions and emulsions. A non-limiting example of an ultrafiltration system is a tangential flow ultrafiltration system.

In some embodiments, the host cell is capable of producing about 10 grams to about 50 grams, greater than about 15 grams, greater than about 20 grams, greater than about 25 grams, or greater than about 30 grams of bioorganic compound per liter of fermentation medium.

In some embodiments, the host cell is capable of producing about 50 to about 1500 milligrams per gram of dry cell weight, such as greater than about 100 milligrams, greater than about 150 milligrams, greater than about 200 milligrams, greater than about 250 milligrams, greater than about 500 milligrams, greater than about 750 milligrams milligrams, or greater than about 1000 milligrams of biological organic compounds.

fuel composition production system

In some embodiments, the present invention comprises a fuel composition production system comprising:

a. At least one container with a capacity of at least 100 liters;

b. The aqueous culture medium in the container forms the first phase;

c. A plurality of host cells in an aqueous medium capable of manufacturing, producing or synthesizing at least one bioorganic compound; and

d. A liquid organic second phase in contact with the first phase comprising said at least one bioorganic compound.

The fuel composition production system may comprise one or more additional processing components including: 1) one or more separation systems for separating the at least one bioorganic compound from the aqueous medium and the organic second phase; 2) One or more reactors for biologically or chemically modifying the at least one bioorganic compound, for example by addition, substitution, hydrogenation, alkylation, hydroxylation, condensation, halogenation, or any other suitable reaction 2) one or more mixing vessels or systems for mixing the at least one bioorganic compound and one or more additional fuel components, such as petroleum-based fuels, fuel additives, and combinations thereof; and 3) a or additional purification or separation systems for further purification of the fuel composition or the at least one bioorganic compound.

In some embodiments, the fuel additive is selected from the group consisting of: oxidizing agents, antioxidants, environmental protection agents, thermal stability improvers, cetane number improvers, stabilizers, low temperature fluidity improvers, combustion enhancers, antifoaming agents, anti Smoke additives, corrosion inhibitors, lubricity improvers, icing inhibitors, injector cleaning additives, smoke suppressants, drag reducing additives, metal deactivators, dispersants, detergents, demulsifiers, dyes, markers, static electricity Dispersants, biocides, and combinations thereof.

In some embodiments, the fuel composition production system comprises:

a) one or more batch, fed-batch, or continuous flow fermentation systems comprising:

i) at least one container with a capacity of at least 100 liters;

ii) an aqueous medium in the at least one container, constituting the first phase;

iii) a plurality of host cells in an aqueous medium capable of manufacturing, producing or synthesizing at least one bioorganic compound; and,

iv) a liquid organic second phase in contact with the first phase, comprising said at least one bioorganic compound;

b) one or more first phase separation systems, thereby separating the first phase and the second organic phase or one or more components of the second organic phase;

c) optionally one or more second phase separation systems, thereby separating the at least one bio-organic compound from the second organic phase;

d) optionally one or more reactors or vessels, wherein at least one bioorganic compound is chemically or biologically modified;

e) optionally one or more purification systems, wherein said bioorganic compound or modified bioorganic compound is purified or further purified;

f) optionally one or more mixing vessels or systems for mixing said at least one bioorganic compound and one or more additional fuel components; and

g) Optionally one or more further purification systems, wherein the mixture of said at least one bioorganic compound and said one or more additional fuel components is purified or further purified.

In some embodiments, the one or more first phase separation systems comprise one or more systems, vessels, or other phase separation components detailed herein that are specifically configured to separate from the second organic phase first phase. In some embodiments, the one or more second phase separation systems comprise one or more systems, vessels, or phase separation modules specifically configured as detailed herein for separating bioorganic compounds from a second organic phase.

In some embodiments, the one or more reactors in which at least one bioorganic compound is chemically or biologically modified comprise the same or different vessels as used in the fermentation or separation system. Alternatively, the one or more reactors may comprise one or more distinct vessels, which may contain additional hardware, sensors, interfaces, probes, and/or control systems adapted to conduct specific reactions of bioorganic compounds or other grooming. The reactor can be a batch, fed-batch or continuous reactor.

In some embodiments, the bioorganic compound, or modified bioorganic compound, or fuel composition may be purified or further purified using one or more purification systems. The purification system may comprise any suitable purification system, including any system that can remove undesired compounds from bioorganic compounds, or that can separate undesired compounds from bioorganic compounds. In some embodiments, the purification system may comprise one or more systems, vessels, or phase separation modules specifically configured as detailed herein for obtaining a desired purity of bioorganic compounds. In some embodiments, the purification can be accomplished using one or more separation systems in series to achieve the desired purity. In some embodiments, the separation systems may be configured differently from each other in order to achieve purity in a stepwise fashion.

In some embodiments, purification may be performed to meet the specifications or requirements of federal, state, or local laws, regulations, or regulations for bioorganic compounds or for fuel compositions. In some embodiments, the purification may improve the function of the bioorganic compound or fuel composition beyond the requirements of federal or state laws, regulations, or regulations. In some embodiments, federal, state, or local laws, rules, or regulations may relate to environmental emissions, fuel performance, tax incentives, and other economic incentives. In some embodiments, the purification can reduce the environmental impact, carbon footprint, fuel efficiency derived therefrom, reliability derived therefrom, energy derived therefrom, or long-term economic cost of the bioorganic compound or fuel composition.

In some embodiments, the fuel composition system comprises one or more mixing vessels or systems for mixing the at least one bioorganic compound with one or more additional fuel components. The mixing vessel or mixing system can be any suitable vessel or system. The mixing vessel may contain any or all of the inlets, outlets, fittings, sensors, probes, stirrers, and other related hardware identified for use in the bioorganic compound production vessel. The mixing vessel can mix one or more fuel components with the bioorganic compound. For example, 2-5 fuel components, such as 3 or 4 fuel components. Mixing systems can be batch, continuous or fed-batch.

In some embodiments, the present invention encompasses a method of making a fuel composition comprising:

a. cultivating a large number of host cells in an aqueous medium to produce, manufacture or synthesize at least one bioorganic compound, wherein the aqueous medium constitutes the first phase;

b. forming a liquid organic second phase in contact with the first phase, comprising said at least one bioorganic compound;

c. separating at least a portion of the second phase from the first phase;

d. separating said at least one bioorganic compound from the second phase;

e. optionally chemically or biologically modify said at least one bioorganic compound;

f. Optionally purifying bioorganic compounds or modified bioorganic compounds;

g. optionally mixing the at least one bio-organic compound and one or more additional fuel components; and

g) optionally purifying the mixture of the one or more bioorganic compounds and the one or more additional fuel components.

In some embodiments, the fuel composition comprises a biofuel composition. In some embodiments, the biofuel further comprises at least one bioorganic compound, and a petroleum-based fuel, a fuel additive, or a combination thereof. In further embodiments, the petroleum-based fuel is gasoline, jet fuel, kerosene, diesel, or combinations thereof.

In some embodiments, the bioorganic compound production system or fuel composition production system can be constructed or created by retrofitting an ethanol production facility.

In some embodiments, the fuel composition production system may comprise one or more automated control systems. The automatic control system may be the same as or different from the control system of the bio-organic production system, and may include various sensors, probes and other equipment for measuring and controlling the relationship between each system in the fuel composition system and the fuel composition production method Various process parameters associated with each step. The automated system may additionally be controlled and monitored by a central control system, which may be a local or plant-wide control system, and may control a production process that produces only one bioorganic compound or that of multiple bioorganic compounds production process. The automation system and central control system may comprise any suitable software, firmware and/or hardware, which may be proprietary or off-the-shelf, or a combination thereof, and may communicate using any suitable communication system. Non-limiting examples of such communication systems include digital or similar hardwired systems, and may include direct connections or in the form of networks such as LAN or WAN or Ethernet. Additionally, in some embodiments, the communication system may be wireless, and may be proprietary, Bluetooth, Ultra Wideband, 802.11a, b, g, or n, or ZigBee, including TDMA, FDMA, OFDM, and CDMA, and Can operate on any suitable band, such as 2.4GHz or 5.8GHz.

host cell

Any suitable host cell can be used to practice the present invention. In some embodiments, the host cell is a genetically modified host microorganism in which nucleic acid molecules have been inserted, deleted, or modified (i.e., mutated; for example, by insertions, deletions, substitutions, and/or inversions of nucleotides) to produce the desired bioorganic compounds, or effectively increase the yield of desired bioorganic compounds.

Exemplary examples of suitable host cells include all archaeal, bacterial, or eukaryotic cells. Examples of archaeal cells include, but are not limited to, those belonging to the following genera: Aeropyrum, Archaeoglobus, Halobacterium, Methanococcus, Methanobacterium (Methanobacterium), Pyrococcus, Sulfolobus, and Thermoplasma. Exemplary examples of Archaeal species include, but are not limited to: Aeropyrum pernix, Archaeoglobus fulgidus, Methanococcus jannaschii, Methanobacterium thermoautotrophicum , Pyrococcus abyssi, Pyrococcus horikoshii, Thermoplasma acidophilum, Thernoplasma volcanium.

Examples of bacterial cells include, but are not limited to, those belonging to the following genera: Agrobacterium, Alicyclobacillus, Anabaena, Anacystis , Arthrobacter, Azobacter, Bacillus, Brevibacterium, Chromatium, Clostridium, Corynebacterium, Enterobacter, Erwinia, Escherichia, Lactobacillus, Lactococcus, Mesorhizobium, methyl Metbylobacterium, Microbacterium, Phormidium, Pseudomonas, Rhodobacter, Rhodopseudomonas, Rhodospirillum Rhodospirillum, Rhodococcus, Salmonella, Scenedesmun, Serratia, Shigella, Staphylococcus ), Streptomyces, Synnecoccus, and Zymomonas.

Exemplary examples of bacterial species include, but are not limited to: Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium ammoniagenes, Brevibacterium immariophilum, Clostridium beijerinckii (Clostridium beigerinckii), Enterobacter sakazakii, Escherichia coli, Lactococcus lactis, Mesorhizobium loti, Pseudomonas aeruginosa ), Pseudomonas mevalonii, Pseudomonas putida, Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodospirillum rubrum, Salmonella enterica, Salmonella typhi, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneri, Shigella sonnei bacteria (Shigella sonnei), Staphylococcus aureus, etc.

In general, if bacterial host cells are used, non-pathogenic strains are preferred. Exemplary examples of non-pathogenic strain types include, but are not limited to: Bacillus subtilis, (Escherichia coli), Lactibacillus acidophilus, Lactobacillus helveticus, Pseudomonas aeruginosa, Pseudomonas mevalonii, Pseudomonas putida, Rhodobacter sphaeroides, Rodobacter capsulatus, Crimson Rhodospirillum rubrum etc.

Examples of eukaryotic cells include, but are not limited to, fungal cells. Examples of fungal cells include, but are not limited to, those belonging to the following genera: Aspergillus, Candida, Chrysosporium, Cryptococcus, Fusarium, Kluyveromyces, Neotyphodium, Neurospora, Penicillium, Pichia, Saccharomyces, Trichoderma ( Trichoderma), and Xanthophyllomyces (formerly known as Phaffia).

Exemplary examples of eukaryotic species include, but are not limited to: Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Candida albicans, Chrysosporium lucknowense), Fusarium graminearum, Fusarium venenatum, Kluyveromyces lactis, Neurospora crassa, Pichia angusta , Pichia finlandica, Pichiakodamae, Pichia membranaefaciens, Pichiamethanolica, Pichia opuntiae, Pasteur Pichia pastoris, Pichia pijperi, Pichia quercuum, Pichia salictaria, Pichia thermotolerans, seaweed Pichia trehalophila, Pichia stipitis, Streptomyces ambofaciens, Streptomycesaureofaciens, Streptomyces aureus, Saccaromycesbayanus , Saccaromyces boulardi, Saccharomyces cerevisiae, Streptomyces fungicidicus, Streptomyces griseochromogenes, Streptomyces griseus, Streptomyces lividus lividans), Streptomyces olivogriseus, Streptomyces rameus, Streptomyces tanashiensis, Streptomyces burgundy Streptomyces vinaceus, Trichoderma reesei, and Xanthophyllomyces dendrorhous (formerly known as Phaffia rhodozyma).

In general, if eukaryotic cells are used, non-pathogenic strains are preferred. Exemplary examples of non-pathogenic strain species include, but are not limited to: Fusarium graminearum, Fusarium venenatum, Pichia pastoris, Saccaromyces boulardi), and Saccharomyces cerevisiae.

In some embodiments, the host cells of the invention have been designated GRAS or generally recognized as safe by the Food and Drug Administration. Illustrative examples of such strains include: Bacillus subtilis, Lactibacillus acidophilus, Lactobacillus helveticus, and Saccharomyces cerevisiae.

Designing pathways to manufacture bioorganic compounds

Illustrative examples of a class of bioorganic compounds are isoprenoids. They contain diverse families of more than 40,000 individual products, many of which are essential to living organisms. Isoprenoids serve to maintain cell fluidity, electron transport, and other metabolic functions. In addition to their use in the manufacture of fuel compositions, a vast number of natural and synthetic isoprenoids are used as pharmaceuticals, cosmetics, fragrances, pigments and colorants, fungicides, preservatives, nutraceuticals, and Fine chemical intermediates to use.

Isoprenoids are produced in nature by two different metabolic pathways that converge on IPP and its isomer DMAPP. In general, unlike plants, eukaryotes uniquely use the MEV isoprenoid pathway to convert acetyl-CoA to IPP, which subsequently isomerizes to DMAPP. Prokaryotes, with some exceptions, use mevalonate-independent or DXP pathways to produce IPP and DMAPP via a branch point, respectively. Typically, plants use both the MEV and DXP pathways to synthesize IPP. The methods described herein for designing MEV and DXP pathways to make desired isoprenoid compounds can be readily adapted to similarly design other pathways for making other bioorganic compounds.

MEV pathway

A schematic illustration of the MEV pathway is shown in Figure 3. Typically, the pathway generally consists of six steps.

In the first step, two molecules of acetyl-CoA are combined by the enzyme to form acetoacetyl-CoA. An enzyme known to catalyze this step is, for example, acetyl-CoA thiolase (also known as acetyl-CoA acetyltransferase). Exemplary examples of nucleotide sequences include, but are not limited to, the following GenBank accession numbers and organisms from which the sequences are derived: (NC_000913 region: 2324131..2325315; Escherichia coli), (D49362; Paracoccus denitrificans), and ( L20428; Saccharomyces cerevisiae).

In the second step of the MEV pathway, acetoacetyl-CoA is enzymatically condensed with another molecule of acetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). An enzyme known to catalyze this step is, for example, HMG-CoA synthetase. Exemplary examples of nucleotide sequences, including but not limited to: (NC_001145, complementary 19061..20536; Saccharomyces cerevisiae), (X96617; Saccharomyces cerevisiae), (X83882; Arabidopsis thaliana), (AB037907; bacteria), (BT007302; Homo sapiens), and (NC_002758, locus marker SAV2546, GeneID 1122571; Staphylococcus aureus).

In the third step, HMG-CoA is enzymatically converted to mevalonate. An enzyme known to catalyze this step is, for example, HMG-CoA reductase. Exemplary examples of nucleotide sequences, including but not limited to: (NM_206548; Drosophila melanogaster), (NC_002758, site marker SAV2545, GeneID1122570; Staphylococcus aureus), (NM_204485; Jungle chicken), (AB015627; Streptomyces sp. KO 3988), (AF542543; Nicotiana acuminate), (AB037907; Lispora sp. cinereus), (AX128213, provided sequence encoding truncated HMGR; Saccharomyces cerevisiae), and (NC-001145: Complementary 115734. .118898; Saccharomyces cerevisiae).

In the fourth step, mevalonate is phosphorylated by the enzyme to form mevalonate 5-phosphate. An enzyme known to catalyze this step is, for example, mevalonate kinase. Exemplary examples of nucleotide sequences include, but are not limited to: (L77688; Arabidopsis thaliana) and (X55875; Saccharomyces cerevisiae).

In the fifth step, a second phosphate group is enzymatically added to mevalonate 5-phosphate to form mevalonate 5-pyrophosphate. An enzyme known to catalyze this step is, for example, phosphomevalonate kinase. Exemplary examples of nucleotide sequences include, but are not limited to: (AF429385; Hevea brasiliensis), (NM_006556; Homo sapiens), and (NC_001145, complement 712315..713670; Saccharomyces cerevisiae).

In the sixth step, mevalonate 5-pyrophosphate is enzymatically converted to IPP. An enzyme known to catalyze this step is, for example, mevalonate pyrophosphate decarboxylase. Exemplary examples of nucleotide sequences include, but are not limited to: (X97557; Saccharomyces cerevisiae), (AF290095; Enterococcus faecium), and (U49260; Homo sapiens).

If IPP is to be converted to DMAPP, then a seventh step is required. An enzyme known to catalyze this step is, for example, IPP isomerase. Exemplary examples of nucleotide sequences include, but are not limited to: (NC_000913, 3031087..3031635; Escherichia coli) and (AF082326; Haematococcus pluvialis). If conversion to DMAPP is required, increased expression of IPP isomerase will ensure that the conversion of IPP to DMAPP is not the rate-limiting step representing the overall pathway.

DXP pathway

A schematic illustration of the DXP pathway is shown in Figure 4. The DXP pathway generally consists of seven steps. In the first step, pyruvate is condensed with D-glyceraldehyde 3-phosphate to produce 1-deoxy-D-xylulose-5-phosphate. An enzyme known to catalyze this step is, for example, 1-deoxy-D-xylulose-5-phosphate synthase. Exemplary examples of nucleotide sequences, including but not limited to: (AF035440; Escherichia coli), (NC_002947, site marker PP0527; Pseudomonas putida KT2440), (CP000026, site marker SPA2301; paratyphi enterica Salmonella, see ATCC 9150), (NC_007493, site marker RSP_0254; Rhodobacter sphaericus 2.4.1), (NC_005296, site marker RPA0952; Rhodopseudomonas palustris CGA009), (NC_004556, site marker PD1293; Xylella fastidiosa Temeculal), and (NC_003076, locus marker AT5G11380; Arabidopsis thaliana).

In the second step, 1-deoxy-D-xylulose-5-phosphate is converted to 2C-methyl-D-erythritol-4-phosphate. An enzyme known to catalyze this step is, for example, 1-deoxy-D-xylulose-5-phosphate reductoisomerase. Exemplary examples of nucleotide sequences, including but not limited to: (AB013300; Escherichia coli), (AF148852; Arabidopsis), (NC_002947, site marker PP1597; Pseudomonas putida KT2440), (AL939124, site Dot markers SCO5694; S. coelicolor A3(2)), (NC_007493, site marker RSP_2709; Rhodobacter sphaeroides 2.4.1), and (NC_007492, site marker Pf1_1107; Pseudomonas fluorescens PfO-1).

In the third step, 2C-methyl-D-erythritol-4-phosphate is converted to 4-diphosphocytidine-2C-methyl-D-erythritol. An enzyme known to catalyze this step is, for example, 4-diphosphocytidine-2C-methyl-D-erythritol synthase. Exemplary examples of nucleotide sequences, including but not limited to: (AF230736; Escherichia coli), (NC_007493, site marker RSP_2835; Rhodobacter sphaeroides 2.4.1), (NC_003071, site marker AT2G02500; Arabidopsis thaliana) , and (NC_002947, locus marker PP1614; Pseudomonas putida KT2440).

In the fourth step, 4-diphosphocytidine-2C-methyl-D-erythritol is converted to 4-diphosphocytidine-2C-methyl-D-erythritol-2-phosphate. An enzyme known to catalyze this step is, for example, 4-diphosphocytidine-2C-methyl-D-erythritol kinase. Exemplary examples of nucleotide sequences include, but are not limited to: (AF216300; Escherichia coli) and (NC_007493, site marker RSP_1779; Rhodobacter sphaeroides 2.4.1).

In the fifth step, 4-diphosphocytidine-2C-methyl-D-erythritol-2-phosphate is converted to 2C-methyl-D-erythritol 2,4-cyclodiphosphate. An enzyme known to catalyze this step is, for example, 2C-methyl-D-erythritol 2,4-cyclic diphosphate synthase. Exemplary examples of nucleotide sequences, including but not limited to: (AF230738; E. coli), (NC_007493, site marker RSP_6071; Rhodobacter sphaeroides 2.4.1), and (NC_002947, site marker PP1618; putrid pseudomonas Bacteria KT2440).

In the sixth step, 2C-methyl-D-erythritol 2,4-cyclic diphosphate is converted to 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate . An enzyme known to catalyze this step is, for example, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase. Exemplary examples of nucleotide sequences, including but not limited to: (AY033515; Escherichia coli), (NC_002947, site marker PP0853; Pseudomonas putida KT2440), and (NC_007493, site marker RSP_2982; Rhodobacter sphaeroides 2.4.1).

In the seventh step, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate is converted to IPP or its isomer DMAPP. An enzyme known to catalyze this step is, for example, isopentyl/dimethylallyl diphosphate synthase. Exemplary examples of nucleotide sequences include, but are not limited to: (AY062212; Escherichia coli) and (NC_002947, site marker PP0606; Pseudomonas putida KT2440).

In some embodiments, "crosstalk" (or interference) between the host cell's own metabolic processes and those involved in IPP production, as provided herein, is minimized or completely eliminated. For example, when the host microorganism relies exclusively on the DXP pathway to synthesize IPP, and introduces the MEV pathway to provide additional IPP, crosstalk is minimized or eliminated entirely. Such a host organism would not be able to alter the expression of MEV pathway enzymes or process intermediates associated with the MEV pathway. Organisms that rely exclusively or predominantly on the DXP pathway include, for example, Escherichia coli.

In some embodiments, the host cell produces IPP via the MEV pathway, either exclusively or in combination with the DXP pathway. In other embodiments, the host's DXP pathway is functionally inactive, such that the host cell produces IPP exclusively through the exogenously introduced MEV pathway. The DXP pathway can be functionally inactive by blocking gene expression or inactivating the function of one or more naturally occurring DXP pathway enzymes.

In other embodiments, the host cell produces IPP via the DXP pathway, either exclusively or in combination with the MEV pathway. In other embodiments, the host's MEV pathway is functionally inactive such that the host cell produces IPP exclusively through the exogenously introduced DXP pathway. The MEV pathway can be functionally inactive by blocking gene expression or inactivating the function of one or more naturally occurring enzymes of the MEV pathway.

_C5 compound

Exemplary _C5 bioorganic compounds are hemiterpenes, which are usually derived from IPP or DMAPP. An exemplary example of a semiterpene is isoprene.

Isoprene

Isoprene, whose structure is

Found in many plants. Isoprene is produced from IPP using the enzyme isoprene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (AB198190; Populus alba) and (AJ294819; Populus alba x Populus alba).

C ₁₀ compound

Exemplary _C10 bioorganic compounds are monoterpenes typically derived from geranyl pyrophosphate (GPP), which is produced by the condensation of IPP and DMAPP. An enzyme known to catalyze this step is, for example, geranyl pyrophosphate synthase.

Figure 5 schematically shows how IPP and DMAPP produce GPP, which can be further processed into monoterpenes.

Exemplary examples of nucleotide sequences of geranyl pyrophosphate synthases include, but are not limited to: (AF513111; Abies abies), (AF513112; Abies abies), (AF513113; Abies abies), (AY534686; Antirrhinum) , (AY534687; Snapdragon), (Y17376; Arabidopsis), (AE016877, site AP11092; Bacillus cereus; ATCC 14579), (AJ243739; sweet orange), (AY534745; Bohui Xiuyi), (AY953508 Pine-toothed beetle), (DQ286930; tomato), (AF182828; peppermint), (AF182827; peppermint), (MPI249453; peppermint), (PZE431697, locus CAD24425; Paracoccus zeaxanthin), (AY866498; Coptis chinensis), (AY351862; Grape), and (AF203881, locus AAF12843; Zymomonas mobilis).

GPP is subsequently converted to various _C10 compounds. Exemplary examples of _C10 compounds include, but are not limited to:

Carene

Carene, whose structure is

Found in the resin of many trees, especially pine. Carenes are produced from GPP using carene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (AF461460, region 43..1926; Picea spruce) and (AF527416, region: 78..1871; Sage angustifolia).

Geraniol

Geraniol (also known as geraniol), the structure of which is

It is the main component of rose oil and palmarosa oil. It is also found in geranium, lemon, and citronella. Geraniol is produced from GPP using geraniol synthase. Exemplary examples of suitable nucleotide sequences include, but are not limited to: (AJ457070; Camphor camphora), (AY362553; Basil), (DQ234300; Perilla strain 1864), (DQ234299; Lemon perilla strain 1861), ( DQ234298; Lemon Perilla line 4935), and (DQ088667; Lemon Perilla).

Linalool

Linalool, whose structure is

Found in many flowers and spice plants such as coriander seed. Linalool is manufactured from GPP using linalool synthase. Exemplary examples of suitable nucleotide sequences include, but are not limited to: (AF497485; Arabidopsis), (AC002294, site AAB71482; Arabidopsis), (AY059757; Arabidopsis), (NM_104793; Arabidopsis Mustard), (AF154124; Artemisia annua), (AF067603; Bohui Embroidery), (AF067602; short spring flowers), (AF067601; Bohui Embroidery), (U58314; Bohui Embroidery), (AY840091; tomato) , (DQ263741; lavender), (AY083653; lemon mint), (AY693647; basil), (XM_463918; rice), (AP004078, locus BAD07605; rice), (XM_463918, locus XP_463918; rice), (AY917193; lemon perilla), (AF271259; perilla), (AY473623; European spruce), (DQ195274; North American spruce), and (AF444798; Perilla frutescens var. crispa variety number 79).

Limonene

Limonene, whose structure is

Found in the rinds of citrus fruits and mints. Limonene is produced from GPP using limonene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (+)-limonene synthase (AF514287, Region: 47..1867; Lemon) and (AY055214, Region: 48..1889; Ageratum) , and (-)-limonene synthase (DQ 195275, region: 1..1905; North American spruce), (AF006193, region: 73..1986; North American fir), and (MHC4SLSP, region: 29..1828; spearmint).

myrcene

Myrcene, whose structure is

Found in the essential oils of many plants including laurel, verbena and bay leaf, from which it gets its name. Myrcene is produced from GPP using myrcene synthase. Exemplary examples of suitable nucleotide sequences include, but are not limited to: (U87908; Abies), (AY195609; Antirrhinum), (AY195608; Antirrhinum), (NM_127982; Arabidopsis TPS10), (NM_113485; Arabidopsis thaliana ATTPS-CIN), (NM_113483; Arabidopsis ATTPS-CIN), (AF271259; Perilla), (AY473626; Picea eucalyptus), (AF369919; Picea punctatus), and (AJ304839; Quercus holly).

ocimene

α- and β-ocimene, their structures are

Found in a variety of plants and fruits including basil (Ocimum basilicum), and is manufactured from GPP using ocimene synthase. Exemplary examples of suitable nucleotide sequences include, but are not limited to: (AY195607; Antirrhinum), (AY195609; Antirrhinum), (AY195608; Antirrhinum), (AK221024; Arabidopsis), (NM_113485; Arabidopsis Arabidopsis ATTPS-CIN), (NM_113483; Arabidopsis ATTPS-CIN), (NM_117775; Arabidopsis ATTPS03), (NM_001036574; Arabidopsis ATTPS03), (NM127982; Arabidopsis TPS10), (AB110642; Citrus CitMTSL4 ), and (AY575970; Lotus japonicus).

α-pinene

α-pinene, whose structure is

Found in pine and eucalyptus. α-pinene is produced from GPP using α-pinene synthase. Exemplary examples of suitable nucleotide sequences include, but are not limited to: (+) α-pinene synthase (AF543530, Region: 1..1887; Loblolly pine), (-) α-pinene synthase ( AF543527, region: 32..1921; loblolly pine), and (+)/(-) α-pinene synthase (AGU87909, region: 6111892; North American fir).

β-pinene

β-pinene, whose structure is

Found in pine, rosemary, parsley, dill, basil, and roses. β-pinene is produced from GPP using β-pinene synthase. Exemplary examples of suitable nucleotide sequences include, but are not limited to: (-) β-pinene synthase (AF276072, Region: 1..1749; Artemisia annua) and (AF514288, Region: 26..1834; lemon).

sabinene

sabinene, whose structure is

Found in black pepper, carrot seeds, sage and tea tree. Sabinene is produced from GPP using sabinene synthase. Exemplary examples of suitable nucleotide sequences include, but are not limited to, AF051901, Region: 26..1798, from Salvia officinalis.

γ-terpinene

Gamma-terpinene, whose structure is

It is a constituent of essential oils derived from citrus fruits. Biochemically, γ-terpinene is produced from GPP using γ-terpinene synthase. Illustrative examples of suitable nucleotide sequences include: (AF514286, region: 30..1832 from lemon) and (AB110640, region 1..1803 from mandarin).

Terpinolene

Terpinolene, whose structure is

Found in blackcurrant, cypress, guava, lychee, papaya, pine, and tea trees. Terpinolene is produced from GPP using terpinolene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (AY693650 from Ocimum basil) and (AY906866, region: 10..1887 from Douglas fir).

C ₁₅ compound

An exemplary _C15 bioorganic compound is a sesquiterpene generally derived from farnesyl pyrophosphate (FPP), which is produced by the condensation of two molecules of IPP and one molecule of DMAPP. An enzyme known to catalyze this step is, for example, farnesyl pyrophosphate synthase.

Figure 5 also schematically shows how IPP and DMAPP are combined to produce FPP, which can be further processed into sesquiterpenes.

Exemplary examples of farnisyl pyrophosphate synthase nucleotide sequences include, but are not limited to: (ATU80605; Arabidopsis), (ATHFPS2R; Arabidopsis), (AAU36376; Artemisia annua), (AF461050; ), (D00694; Escherichia coli K-12), (AE009951, site AAL95523; Fusobacterium nucleatum subsp. nucleatum ATCC25586), (GFFPPSGEN; Fujikura Gibberella), (CP000009, site Points AAW60034; Gluconobacter oxidans 621H), (AF019892; sunflower), (HUMFAPS; Homo sapiens), (KLPFPSQCR; Kluyveromyces lactis), (LAU15777; white lupin), (LAU20771; white lupine), ( AF309508; Mus musculus), (NCFPPSGEN; Neurospora crassa), (PAFPS1; guayule), (PAFPS2; guayule), (RATFAPS; Rattus norvegicus, (YSCFPP; Saccharomyces cerevisiae), (D89104; Millet S. cerevisiae), (CP000003, site AAT87386; S. pyogenes), (CP000017, site AAZ51849; S. pyogenes), (NC_008022, site YP_598856; S. pyogenes MGAS10270), (NC_008023, site Site YP 600845; S. pyogenes MGAS2096), (NC_008024, site YP_602832; S. pyogenes MGAS10750), and (MZEFPS; maize).

Alternatively, FPP can also be made by adding IPP to GPP. Exemplary examples of nucleotide sequences encoding enzymes capable of catalyzing this reaction include, but are not limited to: (AE000657, site AAC06913; Haemogenes aeruginosa VF5), (NM_202836; Arabidopsis thaliana), (D84432, site BAA12575; Bacillus subtilis), (U12678, site AAC28894; Bradyrhizobium soybean USDA 110), (BACFDPS; Geobacillus stearothermophilus), (NC_002940, site NP_873754; Haemophilus ducrei 35000HP), (L42023 , site AAC23087; Haemophilus influenzae Rd KW20), (J05262; Homo sapiens), (YP_395294; Lactobacillus sake subsp. interrogans serovar Copenhageni str. Fiocruz) L1-130), (AB003187; Micrococcus luteus), (NC_002946, site YP_208768; Neisseria gonorrhoeae FA 1090), (U00090, site AAB91752; Rhizobium sp. NGR234), (J05091; S. cerevisiae), (CP000031, site AAV93568; Silicobacter bordetii DSS-3), (AE008481, site AAK99890; S. pneumoniae R6), and (NC_004556, site NP779706; Temecula 1 fastidious Xylella fastidiosa Temeculal).

FPP is subsequently converted to various _C15 compounds. Exemplary examples of _C15 compounds include, but are not limited to:

Amorphadiene

Amorphadiene, whose structure is

is a precursor of artemisinin produced by Artemisia annua. Amorphadiene is produced from FPP using amorphadiene synthase. An exemplary example of a suitable nucleotide sequence is the sequence of SEQ ID NO. 37 in US Patent Application No. 2004/0005678.

Figure 5 schematically shows how IPP and DMAPP are combined to produce FPP, which can then be further processed to produce amorphadiene.

α-farnesene

α-farnesene, whose structure is

Found in a variety of biological resources, including but not limited to Dufour's glands of ants, and the peel coating of apples and pears. α-farnesene is produced from FPP using α-farnesene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to, DQ309034 from Pear variety d'Anjou (Pear; gene name AFS1) and AY182241 from Apple (Apple; gene AFS1). Pechous et al., Planta 219(1):84-94 (2004).

β-farnesene

β-farnesene, whose structure is

Found in a variety of biological sources, including but not limited to aphids, and essential oils such as from peppermint. In certain plants such as wild potatoes, β-farnesene is synthesized as a natural insect repellant. β-farnesene is produced from FPP using β-farnesene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to, GenBank accession numbers AF024615 from Peppermint (Peppermint; gene Tspall), and AY835398 from Artemisia annua. Picaud et al., Phytochemistry 66(9):961-967 (2005).

Farnesol

Farnesol, whose structure is

Found in a variety of biological sources, including insects, and in essential oils such as from citronella, orange blossom, cyclamen, lemongrass, tuberose, and rose. Farnesol is manufactured from FPP using hydroxylases such as farnesol synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to, GenBank Accession No. AF529266 from maize, and YDR481C from Saccharomyces cerevisiae (gene Pho8). Song, L., Applied Biochemistry and Biotechnology 128:149-158 (2006).

Nerolidol

Nerolidol, whose structure is

Also known as nerolidol, it is found in a variety of biological sources including, for example, essential oils from neroli, ginger, jasmine, lavender, tea tree, and lemongrass. Nerolidol is manufactured from FPP using hydroxylases such as nerolidol synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to, AF529266 from maize (maize; gene tps1).

Green phytol

Green phytol, whose structure is

Also known as patchouli alcohol, it is a constituent of patchouli essential oil. Chlorophytol is produced from FPP using phytol synthase. Exemplary examples of suitable nucleotide sequences include, but are not limited to, AY508730 from Patchouli, region: 1..1659.

Valencia tangerene

Valencia tangerene, whose structure is

is one of the main chemical constituents of orange smell and aroma, found in orange peel. Valencia tangerene is produced from FPP using cedarone synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to, AF441124 from sweet orange, region: 1..1647, and AY917195 from perilla, region: 1..1653.

_C20 compound

Exemplary _C20 bioorganic compounds are diterpenes typically derived from geranylgeranylpyrophosphate (GGPP), which is produced by the condensation of three molecules of IPP and one molecule of DMAPP. An enzyme known to catalyze this step is, for example, geranylgeranyl pyrophosphate synthase.

Figure 5 also schematically shows how IPP and DMAPP combine to produce GGPP, which can be further processed to diterpenes, or can be further processed to produce carotenoids.

Exemplary examples of geranylgeranyl pyrophosphate synthase nucleotide sequences include, but are not limited to: (ATHGERPYRS; Arabidopsis), (BT005328; Arabidopsis), (NM_119845; Arabidopsis), ( NZ_AAJM01000380, site ZP_00743052; Bacillus thuringiensis serotype Israel, ATCC 35646 sq1563), (CRGGPPS; periwinkle), (NZ_AABF02000074, site ZP_00144509; Fusobacterium nucleatum subsp. Gibberella), (AY371321; Ginkgo biloba), (AB055496; Hevea brasiliensis), (AB017971; Homo sapiens), (MCI276129; Mucor volvulus), (AB016044; Mus musculus), (AABX01000298, locus NCU01427; rough Neurospora), (NCU20940; Neurospora crassa), (NZ_AAKL01000008, site ZP_00943566; Ralstonia solanacearum UW551), (AB118238; Rattus norvegicus), (SCU31632; Saccharomyces cerevisiae), (AB016095; Synechococcus elongatus bacteria), (SAGGPS; white mustard), (SSOGDS; Sulfolobus acidocaldarius), (NC_007759, site YP_461832; acidotrophic syntroph SB), and (NC_006840, site YP_204095; Vibrio fischeri ES114) .

Alternatively, GGPP can also be made by adding IPP to FPP. Exemplary examples of nucleotide sequences encoding enzymes capable of catalyzing this reaction include, but are not limited to: (NM_112315; Arabidopsis thaliana), (ERWCRTE; Pantoea agglomerans), (D90087, site BAA14124; Pantoea pineapple ), (X52291, site CAA36538; Rhodobacter capsulata), (AF195122, site AAF24294; Rhodobacter sphaeroides), and (NC_004350, site NP_721015; Streptococcus mutans UA159).

GGPP is subsequently converted into various _C20 isoprenoids. Exemplary examples of _C20 compounds include, but are not limited to:

Geranylgeraniol

Geranylgeraniol, whose structure is

Constituent of wood oil and linseed oil from Cedrela toona. Geranylgeraniol can be produced, for example, by adding a phosphatase group behind the GGPP synthase gene in the expression construct.

Rosin Diene

Rosin diene contains the following isomers:

Found in trees such as North American fir. Abietin diene is produced by abietidene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (U50768; Abies) and (AY473621; Spruce).

C ₂₀₊ compounds

C20 ₊ bioorganic compounds are also included within the scope of the present invention. Illustrative examples of such compounds include sesquiterpenes ( _C25 compounds made from five isoprene units), triterpenes ( _C30 compounds made from six isoprene units), and tetraterpenes ( _C40 compound made from eight isoprene units). These compounds are made using methods analogous to those described herein and substituting or adding the nucleotide sequence of the appropriate synthetase.

design approach

Although for purposes of illustration, the invention has been described with reference to the design of MEV and/or DXP pathways, which can be adapted to similarly suitable pathway designs for the production of non-isoprenoid bioorganic compounds. These pathways are typically engineered using recombinant DNA techniques by expressing appropriate exogenous sequences encoding one or more enzymes.

Nucleotides of interest can be expressed by single or multiple vectors. The nucleic acids can be arranged in a single operon, or in separate operons on one or more vectors. When desired, two expression vectors may be used, each containing one or more foreign sequences operably linked to a single operator. While the choice of single or multiple vectors and single or multiple operons may depend on the size of the foreign sequence and the capacity of the vector, it depends to a greater extent on the generosity that the vector is able to provide when expressed in the chosen host cell. Determine the total production of biological organic compounds. In some cases, two-operon expression systems provide higher yields of bioorganic compounds. Destination vectors can remain episomally replicable, or as an integral part of the host cell genome. Typically, the latter is preferred for sustained proliferation of the host cell.

In certain host cells, a nucleic acid of interest may be controlled by one or more operators. In some cases, two or three operon systems provide higher yields of bioorganic compounds than single operon systems.

When desired, the nucleic acid sequence of interest can be modified to reflect the codon bias of the selected host cell to achieve higher expression of the sequence in the host cell. For example, the nucleotide sequence of interest will in some embodiments be modified to accommodate the codon bias of yeast. See, eg, Bennetzen and Hall (1982) J: Biol. Chem. 257(6):3026-3031. As another non-limiting example, the nucleotide sequence will in other embodiments be modified to accommodate the codon bias of E. coli. See, eg, Gouy and Gautier (1982) Nucleic Acids Res. 10(22):7055-7074; Eyre-Walker (1996) MoI. Biol. Evol. 13(6):864-872. See also Nakamura et al. (2000) Nucleic Acids Res. 28(1):292. Codon usage tables for many organisms are known, which can be used as a reference in the sequence design of the present invention. The usage of common codons for a given host microorganism generally increases the likelihood of transcription and thus the level of expression of the desired sequence.

The preparation of nucleic acids of interest can be accomplished by various conventional recombinant techniques and synthetic methods. Briefly, target nucleic acids can be prepared genomic DNA fragments, cDNA and RNA, all of which can be directly extracted from cells, or recombinantly produced by various amplification methods, including but not limited to PCR and rt-PCR.

Direct chemical synthesis of nucleic acids typically involves the sequential addition of 3'-blocked and 5'-blocked nucleomonomers to the terminal 5'-hydroxyl of an extending polymeric chain of nucleotides, with each addition passing through the extending This is accomplished by nucleophilic attack on the 3'-position of the added monomer, typically a phosphorus derivative, such as a phosphotriester, phosphoramidite, or the like, by the 5'-hydroxyl at the end of the chain. . Such methods are known to those of ordinary skill in the art and are described in related articles and literature (e.g., Matteuci et al. (1980) Tet. Lett. 521:719; Caruthers et al., U.S. Patent No. 4,500,707; and Southern et al. described in US Patent Nos. 5,436,327 and 5,700,637).

The level of transcription of a nucleic acid in a host microorganism can be increased in a number of ways. For example, it can be achieved by increasing the copy number of the nucleotide sequence encoding the enzyme (for example, by using a higher copy number expression vector comprising the nucleotide sequence encoding the enzyme, or by introducing the enzyme encoding enzyme into the genome of the host microorganism Additional copies of the nucleotide sequence of the nucleotide sequence, for example, by recA-mediated recombination, using "suicide" vectors, recombination using bacteriophage lambda recombinase, and/or by insertion of transposons or transposable elements). In addition, by changing the order of the coding region of the operon on the polycistronic mRNA, either dissolving the operon into separate genes each with its own control elements, or adding a promoter operably linked to the enzyme coding region (transcription initiation or transcriptional control sequences) (for example, in E. coli host microorganisms, use the consensus arabinose or lactose-inducible promoter instead of the modified lactose-inducible promoter, such as that found in the pBluescript and pBBR1MCS plasmids species), or by using an inducible promoter and inducing it by adding chemicals to the growth medium. The level of transcription of a nucleotide sequence in a host microorganism can be increased in a number of ways, including but not limited to increasing the stability of the mRNA, modifying the sequence of the ribosome binding site, modifying the initiation codon of the ribosome binding site and the enzyme coding sequence distance or sequence, modify the entire intercistronic region located "upstream" or adjacent to the 5' side of the start codon of the enzyme coding region, using hairpins and specially designed sequences to stabilize the 3' end of the mRNA transcript , modify the codons used by the enzyme, alter the expression of tRNAs with rare codons used in the biosynthesis of the enzyme, and/or increase the stability of the enzyme, such as, for example, by mutation of its coding sequence. tRNAs for preferred codons and rare codons can be determined based on analysis of the gene sequence derived from the host microorganism.

Destination vectors can be constructed to produce the desired level of copy number of the encoded enzyme. In some embodiments, the destination vector produces at least 10, between 10 and 20, between 20-50, between 50-100, or even greater than 100 copies of the desired enzyme. Low copy number plasmids generally provide less than about 20 plasmid copies per cell; medium copy number plasmids generally provide from about 20 plasmid copies per cell to about 50 plasmid copies per cell, or from about 20 plasmid copies per cell to about 80 plasmid copies per cell; whereas high copy number plasmids typically provide from about 80 plasmid copies per cell to about 200 plasmid copies per cell, or more.

Suitable low copy expression vectors for E. coli include, but are not limited to, pACYC184, pBeloBac11, pBR332, pBAD33, pBBR1MCS and derivatives thereof, pSC101, SuperCos (cosmid), and pWE15 (cosmid). Suitable mid-copy expression vectors for E. coli include, but are not limited to, pTrc99A, pBAD24, and vectors containing the CoIE1 origin of replication and derivatives thereof. Suitable high copy number expression vectors for E. coli include, but are not limited to, pUC, pBluescript, pGEM and pTZ vectors. Suitable low-copy (centromere) expression vectors for yeast include, but are not limited to, pRS415 and pRS416 (Sikorski & Hieter (1989) Genetics 122: 19-27). Suitable high copy 2 micron expression vectors in yeast include, but are not limited to, pRS425 and pRS426 (Christainson et al. (1992) Gene 110: 119-122). Alternative 2-micron expression vectors include non-selectable marker variants of the 2-micron vector (Bruschi & Ludwig (1988) Curr. Genet. 15:83-90), or complete 2-micron plasmids with expression cassettes (as described in US Patent Application 20050084972 exemplified in ), or a 2-micron plasmid with a defective selectable marker, such as LEU2d (Erhanrt et al. Sciences 782(1):202-207).

Regulatory elements, including, for example, promoters and operators, can also be designed to increase the metabolism of engineered pathways by increasing the expression of one or more genes that play an important role in determining the overall yield of bioorganic compound production flow. A promoter is a nucleotide sequence that initiates and controls the transcription of a nucleic acid sequence by RNA polymerase. An operator is a nucleotide sequence adjacent to a promoter that functions to control the transcription of a desired nucleic acid sequence. Operators contain protein-binding regions that can bind specific repressor proteins. In the absence of a suitable repressor protein, transcription is initiated through the promoter. When a suitable repressor is present, the repressor binds to the operator and thus represses transcription from the promoter.

In some embodiments of the invention, the promoter used in the expression vector is inducible. In other embodiments, the promoter used in the expression vector is constitutive. In some embodiments, one or more nucleic acid sequences are operably linked to an inducible promoter and one or more other nucleic acid sequences are operably linked to a constitutive promoter.

Non-limiting examples of suitable promoters for use in prokaryotic host cells include: bacteriophage T7 RNA polymerase promoter; trp promoter; lac operator promoter; hybrid promoters, such as the lac/tac hybrid promoter , tac/trc hybrid promoter, trp/lac promoter, T7/lac promoter; trc promoter; tac promoter, etc.; araBAD promoter; in vivo regulated promoters, such as ssaG promoter or related promoters (see, For example, U.S. Patent Application No. 20040131637), the pagC promoter (Pulkkinen and Miller, J.Bacteriol. (1991) 173(1):86-93; Alpuche-Aranda et al. (1992) Proc.Natl.Acad.Sci.U.S. A.89(21): 10079-83), nirB promoter (Harborne et al. (1992) MoI. Micro. 6: 2805-2813) and the like (see, e.g., Dunstan et al. (1999) Infect. Immun. 67: 5133 -5141; McKelvie et al. (2004) Vaccine 22:3243-3255; and Chatfield et al. (1992) Biotechnol. and AX798183); stationary phase promoters, such as dps promoter, spv promoter, etc.; promoters derived from pathogenic islet SPI-2 (see, for example, WO96/17951); actA promoter (see, for example, Shetron- Rama et al. (2002) Infect. Immun. 70: 1087-1096); rpsM promoter (see, for example, Valdivia and Falkow (1996) MoI. Microbiol. 22: 367-378); tet promoter (see, for example, Hillen et al. (1989), in Saenger W. and Heinemann U. (eds.) Topics in Molecular and Structural Biology, Protein-Nucleic Acid Interaction Vol. 10 pp. 143-162, Macmillan, London, UK); SP6 promoter (see, e.g. Melton et al. (1984) Nucl. Acids Res. 12:7035-7056); et al.

In some embodiments, the overall activity of an exogenous enzyme that plays a more important role in the overall production of a bioorganic compound than other enzymes in the respective pathway is increased by expressing the enzyme from a strong promoter. Suitable strong promoters for E. coli include, but are not limited to, Trc, Tac, T5, T7, and _Pλ . In another embodiment of the invention, the overall activity of one or more enzymes of the designed pathway in the host is increased by expressing the enzymes with a strong promoter in a high copy number plasmid. Suitable examples in E. coli include, but are not limited to, the use of Trc, Tac, T5, T7, and P _lambda promoters, and pBAD24, pBAD18, pGEM, pBluescript, pUC, and pTZ vectors.

Non-limiting examples of suitable promoters for use in eukaryotic host cells include, but are not limited to, the CMV immediate early promoter, the HSV thymidine kinase promoter, the early or late SV40 promoter, LTRs from retroviruses, and small Mouse metallothionein-I promoter.

Non-limiting examples of suitable constitutive promoters for use in prokaryotic host cells include the sigma70 promoter (eg, the consensus sigma70 promoter). Non-limiting examples of suitable inducible promoters for use in bacterial host cells include pL of bacteriophage lambda; Plac; Ptrp; Ptac (Ptrp-lac hybrid promoter); Lactoside (IPTG)-inducible promoters, such as the lacZ promoter; tetracycline-inducible promoters; arabinose-inducible promoters, such as PBAD (see, e.g., Guzman et al. (1995) J. Bacteriol. 177:4121-4130); Xylose-inducible promoters, such as Pxy1 (see, e.g., Kim et al. (1996) Gene 181:71-76); GAL1 promoter; tryptophan promoter; lac promoter; alcohol-inducible promoters, such as methanol-inducible Promoters and ethanol-inducible promoters; raffinose-inducible promoters; heat-inducible promoters, such as the heat-inducible λPL promoter; promoters controlled by heat-sensitive repressors (such as CI857-repressed λ-based expression vectors; See, eg, Hoffmann et al. (1999) FEMS Microbiol Lett. 177(2):327-34); et al.

Non-limiting examples of suitable constitutive promoters for use in yeast host cells include the ADH1, ADH2, PGK, or LEU2 promoters. Non-limiting examples of suitable inducible promoters for use in yeast host cells include, but are not limited to, divergent galactose-inducible promoters, such as the GAL1 or GAL10 promoters (West et al. (1984) MoI. Cell. Biol. 4 (11):2467-2478), or the CUP1 promoter. When desired, the destination vector contains a stronger promoter than the native E. coli Lac promoter.

Non-limiting examples of operators used in bacterial host cells include the lactose promoter operon (the LacI repressor changes conformation upon contact with lactose preventing the LacI repressor from binding to the operator), the tryptophan promoter Operator (the TrpR repressor protein has a configuration that binds to the operator when complexed with tryptophan; when lacking tryptophan, the TrpR repressor has a configuration that does not bind to the operator), and the tac promoter operates (See, eg, deBoer et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80:21-25).

The gene in the expression vector also typically encodes a ribosome binding site to direct the transcription (ie synthesis) of any mRNA gene encoded product. For suitable ribosome binding sites for use in E. coli, see Shine et al. (1975) Nature 254:34, and Steitz, Biological Regulation and Development: Gene Expression (ed. R.F. Goldberger) Vol. 1 p. 349, 1979, Plenum Publishing, NY ). Insertion of a ribosome binding site encoding a 5'-AAAACA-3' nucleotide sequence upstream of the coding sequence facilitates efficient transcription in yeast host microorganisms (Looman et al. (1993) Nuc.Ac.Res.21:4268 -4271; Yun et al. (1996) Mol. Microbiol. 19:1225-1239).

Other regulatory elements that may be used in expression vectors include transcriptional enhancer elements and transcriptional terminators. See, eg, Bitter et al. (1987) Methods in Enzymology, 153:516-544.

Expression vectors may be adapted for use in certain types of host microorganisms but not others. However, one of ordinary skill in the art can readily determine by routine experimentation whether a particular expression vector is suitable for a given host microorganism. For example, expression vectors can be introduced into a host organism and the activity and expression of any genes contained in the vectors subsequently monitored.

Expression vectors may also contain one or more selectable marker genes that, when expressed, exhibit one or more phenotypic characteristics for screening or identifying host cells carrying the expression vector. Non-limiting examples of suitable selectable markers for eukaryotic cells include dihydrofolate reductase and neomycin resistance. Non-limiting examples of suitable selection markers for prokaryotic cells include tetracycline, ampicillin, chloramphenicol, carbenicillin, and kanamycin resistance.

For the production of bioorganic products on an industrial scale, the use of selection markers requiring the addition of antibiotics to the fermentation medium may be impractical or prohibitively expensive. Accordingly, some embodiments of the invention use host cells that do not require the use of selection markers that confer antibiotic resistance to ensure maintenance of plasmids (expression vectors). In these embodiments of the invention, the expression vector comprises a plasmid maintenance system such as the 60-kb IncP(RK2) plasmid, optionally accompanied by an RK2 plasmid replication and/or isolation system, thereby enabling plasmid expression in the absence of antibiotic selection. Retention (see, eg, Sia et al. (1995) J. Bacteriol. 177:2789-97; Pansegrau et al. (1994) J. Mol. Biol. 239:623-63). A suitable plasmid maintenance system for this purpose is encoded by the parDE operon of RK2, which encodes a stable toxin and an unstable antitoxin. Antitoxins can inhibit the lethal effects of toxins through direct protein-protein interactions. Cells lacking an expression vector containing the parDE operon are quickly deprived of this unstable antitoxin, resulting in a stable toxin that then causes cell death. The RK2 plasmid replication system is encoded by the trfA gene, which encodes a DNA replication protein. The RK2 plasmid isolation system is encoded by the parCBA operon, and the function of the encoded protein is to decompose plasmid polymers that may be produced by DNA replication.

Destination vectors can be stably or transiently introduced into host cells using a variety of established techniques. For example, one method involves calcium chloride treatment, wherein the expression vector is introduced by calcium precipitation. Other salts, such as calcium phosphate, can also be used in a similar manner. In addition, electroporation (ie, the application of an electric current to increase the permeability of cells to nucleic acids) can be used. Other transformation methods include microinjection, DEAE-dextran-mediated transformation, and heat shock in the presence of lithium acetate. Lipoplexes, liposomes and dendrimers can also be used to transfect host microorganisms.

At the time of transformation, various methods can be used to identify host cells into which the vector of interest has been introduced. One exemplary screening method involves subculturing independent cells to form independent clones, followed by testing for expression of the desired gene product. Another approach entails screening transformed host cells based on the phenotypic characteristics conferred by the expression of a selectable marker gene contained within the expression vector. Using these or those methods known in the art, one of ordinary skill in the art can identify genetically modified host cells.

Introduction of various pathway sequences of the present invention into host cells can be confirmed by methods such as PCR, Southern or Northern blot hybridization. For example, nucleic acid can be prepared from the resulting host cells, and a particular sequence of interest can be amplified by PCR using primers specific for the sequence of interest. Amplified products are subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, or capillary electrophoresis, followed by staining with ethidium bromide, SYBR Green solution, or the like, or detection of DNA with UV detection. Alternatively, nucleic acid probes specific for the sequence of interest can be used in the hybridization reaction. Expression of a particular gene sequence is determined by reverse transcription PCR, Northern blot hybridization, or by immunoassay using antibodies reactive with the encoded gene product to detect the corresponding mRNA. Examples of immunoassays include, but are not limited to, ELISA, radioimmunoassay, and sandwich immunoassay.

The yield of bioorganic compounds produced via one or more metabolic pathways disclosed herein can be increased by inhibiting reactions that divert intermediates from production steps toward bioorganic product formation. Inhibition of non-productive reactions can be achieved by reducing the expression and/or activity of enzymes involved in one or more non-productive reactions. Such reactions include side reactions of the TCA cycle that lead to fatty acid biosynthesis, alanine biosynthesis, aspartate superpathway, gluconeogenesis, heme biosynthesis at levels affecting the total production of bioorganic compounds biosynthesis, and/or biosynthesis of glutamate.

A variety of methods can be used to knock-out or knock-down a gene of interest. For example, reduction of gene expression can be achieved by deletion, mutation and/or gene rearrangement. It can also be performed using antisense RNA, siRNA, miRNA, ribozymes, triple-stranded DNA, and transcription and/or translation inhibitors. In addition, transposons can be used to interfere with gene expression, for example, to inactivate one or both genes by insertion between a promoter and a coding region, or between two adjacent genes.

The number of microorganisms per liter of ferment, or microbial density, can be measured by measuring the weight of microorganisms isolated from a volume of fermentation medium. A commonly used standard is dry cell weight per liter of fermentation medium. Another method that can be used to monitor a fermentation as it proceeds is by measuring the optical density of the medium. The commonly used method is to measure the optical density at a wavelength of 600nm, which is called OD ₆₀₀ value or OD value. The OD value can be correlated with the density of a particular type of organism in a particular medium, but a specific relationship between OD and number of microorganisms per volume is generally not applicable to all types of organisms in all types of medium. By measuring OD values and dry cell weight over a range of cell densities, a calibration curve can be created. In some cases, these correlations can be used in different fermentations of the same or similar microorganisms in the same or similar media.

Example

Unless otherwise indicated, the practice of the present invention can use conventional techniques of the biosynthesis industry and the like within the technical scope of the art. To the extent these techniques are not fully described in this paper, sufficient relevant references can be found in the scientific literature.

In the following examples, efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.) One of ordinary skill in the art can derive the correct amount based on the remainder of the disclosure herein. Unless otherwise indicated, temperatures are reported in degrees Celsius and pressures are at or near sea level. All reagents, unless otherwise noted, were obtained commercially. The following examples are for illustrative purposes only and do not limit the scope of the invention in any way.

Example 1

This example describes a method for preparing expression plasmids encoding enzymes of the MEV pathway from S. cerevisiae arranged in operons.

The expression plasmid pMevT was prepared by inserting the MevT operator (SEQ ID NO: 1) into the pBAD33 vector. The MevT operon encodes a collection of enzymes of the MEV pathway that together convert the ubiquitous precursor acetyl-CoA to (R)-mevalonate, acetoacetyl-CoA thiolase, HMG-CoA synthetase , and HMG-CoA reductase. The ERG13 gene was PCR amplified from Saccharomyces cerevisiae genomic DNA by PCR amplifying the coding sequence of the atoB gene (GenBank accession number NC_000913 region: 2324131..2325315) (encoding acetoacetyl-CoA thiolase) from the genomic DNA of Escherichia coli Coding sequence (GenBank accession number X96617, region: 220..1695) (encoding HMG-CoA synthetase), and a fragment of the coding region of the HMG1 gene (GenBank accession number M22002, region: 1660. .3165) (encoding a truncated HMG-CoA reductase (tHMGR)), to make the MevT operon. The upstream PCR primers used to amplify the HMG1 gene fragment contained an artificial start codon. The amplified fragments were spliced together using overlap extension (splicing), in which a ribosome binding site was introduced after the atoB and ERG13 coding sequences. After addition of the 3'A overhang, the MevT operon was ligated into the TA cloning vector pCR4 (Invitrogen, Carlsbad, CA) and sequenced to ensure accuracy. The MevT operon was then ligated into the XmaI PstI restriction enzyme site of vector pBAD33 (Guzman et al. (1995) J. Bacteriol. 177(14):4121-4130). To place the operon under the control of the P _Lac promoter, the araC-P _BAD NsiI-XmaI fragment of pBAD33 was replaced with the NsiI-XmaI fragment of pBBR1MCS, resulting in the expression plasmid pMevT (see US Patent No. 7,192,751).

The expression plasmid pAM36-MevT66 was prepared by inserting the MevT66 operon into the pAM36 vector. By inserting an oligonucleotide cassette containing AscI-SfiI-AsiSI-XhoI-PacI-FsIl-PmeI restriction endonuclease sites into the pACYC184 vector (GenBank accession number XO6403) and removing the tet resistance gene in pACYC184 Preparation of vector pAM36. The MevT66 operon is synthesized by using the nucleotide sequence SEQ ID NO: 1 as a template, and the sequence includes the atoB gene from Escherichia coli (GenBank accession number NC_000913 region: 2324131..2325315), the ERG13 gene from Saccharomyces cerevisiae ( GenBank Accession No. X96617, Region: 220..1695), and an abridged version of the HMG1 gene from Saccharomyces cerevisiae (GenBank Accession No. M22002, Region: 1777..3285), all three sequences codon optimized for in the expression. Synthetically generated MevT66 operon is flanked by 5' EcoRI and 3' HindIII restriction enzyme sites and can therefore be cloned into compatible restriction enzyme sites in cloning vectors , such as standard pUC or pACYC-derived vectors. From this construct, the MevT66 operon flanked by SfiI and AsiSI restriction endonuclease sites was PCR amplified, the amplified DNA fragment was completely digested with SfiI and AsiSI restriction endonucleases, the reaction mixture was separated by gel electrophoresis, and Qiagen gel purification kit (Valencia, CA), gel extraction of about 4.2kb DNA fragments, the separated DNA fragments were connected to the SfiI AsiSI restriction endonuclease site of the pAM36 vector to generate expression plasmid pAM36-MevT66.

The expression plasmid pAM25 was prepared by inserting the MevT66 operon into the pAM29 vector. Vector pAM29 was prepared by assembling the pl5A origin of replication, the kan resistance gene from pZS24-MCS1 (Lutz and Bujard (1997) Nucl Acids Res. 25:1203-1210), and the oligonucleotide-generated lacUV5 promoter. DNA synthesis constructs containing the MevT66 operon (see above) were digested to completion using EcoRI and HindIII restriction enzymes, the reaction mixture was separated by gel electrophoresis, the 4.2 kb DNA fragment was gel extracted, and the separated DNA fragments were ligated To the EcoRIHindIII restriction endonuclease site of pAM29, the expression plasmid pAM25 was generated.

The expression plasmid pMevB-Cm was generated by inserting the MevB operon into the pBBR1MCS-1 vector. The MevB operon encodes a collection of enzymes that collectively convert (R)-mevalonate to IPP, namely mevalonate kinase, phosphomevalonate kinase, and mevalonate pyrophosphate carboxylase . ERG12 gene (GenBank accession number X55875, region: 580..1911) (encoding mevalonate kinase), ERG8 gene (GenBank accession number Z49939, region: 3363..4718) ( Encoding phosphomevalonate kinase), and MVD1 gene (GenBank accession number X97557, region: 544..1734) (encoding mevalonate pyrophosphate carboxylase) coding sequence, and using overlap extension (splicing method) to The PCR fragments were spliced together to make the MevB operon. By choosing appropriate primer sequences, the terminators of ERG12 and ERG8 were changed from TAA to TAG during amplification to introduce ribosome binding sites. After addition of the 3'A overhang, the MevB operon was ligated into the TA cloning vector pCR4 (Invitrogen, Carlsbad, CA). The MevB operon was cut by using PstI restriction endonuclease to completely digest the cloning vector, the reaction mixture was separated by gel electrophoresis, the 4.2 kb DNA fragment was gel extracted, and the separated DNA fragment was ligated into the vector pBBR1MCS-1 ( The expression plasmid pMevB-Cm was generated at the PstI restriction endonuclease site of Kovach et al., Gene 166(1):175-176 (1995).

The expression plasmid pMBI was prepared by inserting the MBI operon into the pBBR1MCS-3 vector. The MBI operon encodes the same enzyme as the MevB operon, as well as isopentenyl pyrophosphate isomerase that catalyzes the conversion of IPP to DMAPP. The MBI operon was generated by PCR amplifying the coding sequence of the idi gene (GenBank accession number AF119715) from E. coli genomic DNA using primers containing an XmaI restriction endonuclease site at the 5' end; using the XmaI restriction endonuclease Completely digest the amplified DNA fragment, separate the reaction mixture by gel electrophoresis, gel extract the 0.5kb fragment, and connect the separated DNA fragment to the XmaI restriction endonuclease site of the expression plasmid pMevB-Cm, thereby idi is placed at the 3' end of the MevB operon. The MBI operon was subcloned into the SalI and SacI restriction endonuclease sites of the vector pBBR1MCS-3 (Kovach et al., Gene 166(1): 175-176 (1995)), resulting in the expression plasmid pMBI (see U.S. Pat. No. 7,192,751 ).

The expression plasmid pMBIS was prepared by inserting the ispA gene into pMBI. The ispA gene encodes farnesyl pyrophosphate synthase, which catalyzes the conversion of IPP and DMAPP to FPP. Using the forward primer with the SacII restriction endonuclease site and the reverse primer with the SacI restriction endonuclease site, from Escherichia coli genomic DNA, the coding sequence of the ispA gene was amplified by PCR (GenBank accession number D00694, Area: 484..1383). The amplified PCR product was completely digested with SacII and SacI restriction enzymes, the reaction mixture was separated by gel electrophoresis, and a 0.9 kb DNA fragment was gel extracted. The isolated DNA fragment was ligated into the SacII SacI restriction endonuclease site of pMBI, thereby placing the ispA gene 3' to the idi and MevB operons and generating the expression plasmid pMBIS (see U.S. Patent No. 7,192,751).

The expression plasmid pMBIS-gpps was derived from the expression plasmid pMBIS prepared by replacing the ispA coding sequence with the nucleotide sequence encoding geranyl diphosphate synthase ("gpps"). A DNA fragment containing the nucleotide sequence encoding geranyl diphosphate synthase was prepared synthetically using the gpps gene coding sequence of Arabidopsis thaliana codon-optimized for expression in Escherichia coli (GenBank accession number Y17376, region :52..1320) as a template. The nucleotide sequence is flanked by an initial SacII restriction endonuclease site and an end SacI restriction enzyme site, and is cloned into a compatible restriction enzyme site in a cloning vector, such as the standard Vectors derived from pUC or pACYC. The synthetically prepared geranyl diphosphate synthase sequence was isolated by using SacII and SacI restriction endonucleases to completely digest the DNA synthesis construct, the reaction mixture was separated by gel electrophoresis, and a DNA fragment of approximately 1.3 kb was extracted from the gel. The DNA fragment of the expression plasmid pMBIS was connected to the SacII SacI restriction endonuclease site to generate the expression plasmid pMBIS-gpps (see Figure 6 for the plasmid map).

The expression plasmid pAM45 was prepared by inserting the MBIS operon into pAM36-MevT66 and adding the lacUV5 promoter in front of both operons. The MBIS operon was PCR amplified from pMBIS using primers containing a 5'XhoI restriction endonuclease site and a 3'PacI restriction endonuclease site. The amplified PCR product was completely digested with XhoI and PacI restriction enzymes, the reaction mixture was separated by gel electrophoresis, and a 5.4kb DNA fragment was extracted from the gel, and the separated DNA fragment was ligated into the XhoI PacI restriction of pAM36-MevT66 Dicer site, resulting in plasmid pAM43. A DNA fragment containing the nucleotide sequence encoding the lacUV5 promoter was synthesized with oligonucleotides and subcloned into the AscI SfiI and AsiSI XhoI restriction endonuclease sites of pAM43 to generate the expression plasmid pAM45.

Example 2

This example describes a method for making expression vectors encoding enzymes from the MEV pathway from S. aureus arranged in operons.

The expression plasmid pAM41 was derived from the expression plasmid pAM25 by replacing the HMG1 gene coding sequence encoding Saccharomyces cerevisiae HMG-CoA reductase with the mvaA gene coding sequence encoding Staphylococcus aureus HMG-CoA reductase (GenBank accession number BA000017, region: 2688925..2687648) prepared. Using primers 4-49mvaA SpeI (SEQ ID NO: 2) and 4-49mvaAR XbaI (SEQ ID NO: 3), from Staphylococcus aureus subsp. aureus (ATCC 70069) genomic DNA, PCR amplified the coding of mvaA gene To sequence, the amplified DNA fragment was completely digested with SpeI restriction endonuclease, the reaction mixture was separated by gel electrophoresis, and the approximately 1.3 kb DNA fragment was gel extracted. The HMG1 coding sequence was removed from pAM25 by complete digestion of pAM25 with HindIII restriction endonuclease. The overhanging ends of the resulting linear DNA fragments were filled in with T4 DNA polymerase. The DNA fragment was then partially digested with SpeI restriction endonuclease, the reaction mixture was separated by gel electrophoresis, and the 4.8 kb DNA fragment was gel extracted. The isolated DNA fragment was ligated with the SpeI-digested mvaA PCR product to generate the expression plasmid pAM41. The nucleotide sequence of the atoB(opt):ERG13(opt):mvaA operon contained in pAM41 is SEQ ID NO:41. ERG13 is also known as HMGS, or HMG-CoA synthetase.

The expression plasmid pAM52 was derived from the expression plasmid pAM41 by replacing the ERG13 gene coding sequence encoding Saccharomyces cerevisiae HMG-CoA synthetase with the mvaS gene coding sequence encoding Staphylococcus aureus HMG-CoA synthetase (GenBank accession number BA000017, region: 2689180 ..2690346) to prepare. Using primers HMGS 5′SamvaS-S (SEQ ID NO: 4) and HMGS 3′SamvaS-AS (SEQ ID NO: 5), from the genomic DNA of Staphylococcus aureus subsp. aureus (ATCC 70069), PCR amplified The coding sequence of the mvaS gene was amplified, and according to the method of Geiser et al. (BioTechniques 31:88-92 (2001)), the amplified DNA fragment was used as a PCR primer to replace the HMG1 gene coding sequence in pAM41, thereby generating expression plasmid pAM52 . The nucleotide sequence of the atoB(opt):mvaS:mvaA operon contained in pAM52 is SEQ ID NO:42.

The expression plasmid pAM97 was derived from the expression plasmid pAM45, and was prepared by replacing the MevT66 operon with the (atoB(opt):mvaS:mvaA) operon of the expression plasmid pAM52. The expression plasmid pAM45 was completely digested with AsiSI and SfiI restriction enzymes, the reaction mixture was separated by gel electrophoresis, and the 8.3kb DNA fragment lacking the MevT66 operon was extracted from the gel. Using primers 19-25atoBSfiI-S (SEQ ID NO:6) and 19-25mvaA-AsiSI-AS (SEQ ID NO:7), the (atoB(opt):mvaS:mvaA) operon of pAM52 was amplified by PCR using SfiI and AsiSI restriction enzymes digested the PCR product completely, separated the reaction mixture by gel electrophoresis, gel extracted the 3.7kb DNA fragment, and ligated the separated DNA fragment to the AsiSI SfiI restriction endonuclease site of the expression plasmid pAM45 Above, the expression plasmid pAM97 was generated.

The expression plasmid pAM97-MBI was derived from the expression plasmids pAM97 and pAM45, and was prepared by replacing the MBIS operon of pAM97 with the MBI operon of pAM45. Using primers 9-70C (SEQ ID NO: 8) and 26-39B (SEQ ID NO: 9), the MBI operon was amplified by PCR from pAM45, the reaction mixture was separated by gel electrophoresis, and the DNA fragment of 4.5 kb was extracted by gel, The isolated DNA fragments were completely digested with SacI and XhoI restriction enzymes. The expression plasmid pAM97 was completely digested with SaeI and XhoI restriction endonucleases, the reaction mixture was separated by gel electrophoresis, the 7.6kb fragment was extracted from the gel, and the isolated DNA fragment was ligated with the PCR product of the MBI operon to generate the expression plasmid pAM97-MBI .

The expression plasmid pAM97-MevB was derived from the expression plasmids pAM97 and pAM45, and the MBIS operon of pAM97 was replaced by the MevB operon of pAM45. Using primers 9-70C (SEQ ID NO: 8) and 26-39A (SEQ ID NO: 10), the MevB operon was amplified by PCR from pAM45, the reaction mixture was separated by gel electrophoresis, and the DNA fragment of 3.9 kb was extracted by gel, The isolated DNA fragments were completely digested with SacI and XhoI restriction enzymes. The expression plasmid pAM97 was completely digested with SacI and XhoI restriction endonucleases, the reaction mixture was separated by gel electrophoresis, the 7.6kb fragment was gel extracted, and the isolated DNA fragment was ligated with the PCR product of the MevB operon to generate the expression plasmid pAM97-MevB .

Expression plasmid pAM128 was prepared by inserting the (atoB(opt):mvaS:mvaA) and MBIS operons of expression plasmid pAM97 into a vector containing the RK2 plasmid replication, isolation and maintenance system, which avoids antibiotic selection of host cell transformants continuous demand. The RK2 plasmid was completely digested with the PstI restriction endonuclease, the reaction mixture was separated by gel electrophoresis, the approximately 6.3 kb DNA fragment containing the complete par site was gel extracted, and the isolated DNA fragment was subcloned into the miniature RK2 replicon pRR10 (Roberts (1990) J Bacteriol.172 (11): 6204-6216) in the PstI restriction endonuclease site, the vector pAM132 was generated. The expression plasmid pAM97 was completely digested with AscI and SacI restriction enzymes, the reaction mixture was separated by gel electrophoresis, and a DNA fragment of about 9.4kb was extracted from the gel, and the separated DNA fragment was ligated to the MluI SacI restriction endonuclease site of pAM132 On point, the expression plasmid pAM128 was generated.

Example 3

This example describes a method for making an expression vector encoding an enzyme from the MEV pathway of E. faecalis arranged in an operon.

Plasmid pAM16 was inserted into pBlueScripII-KS (+ ) carrier to prepare. Using 5' phosphorylated primers 4-40mvaEF BamHI (SEQ ID NO: 11) and 4-40 mvaER HindIII (SEQ ID NO: 12), the mvaE gene encoding was amplified by PCR from Enterococcus faecalis genomic DNA (ATCC 700802) sequence. (Note that primer 4-40mvaEF BamHI changed the start codon of the mvaE gene from TTG to ATG in the amplified PCR product.) The resulting PCR product was ligated into pBlueScripII-KS(+) (Stratagene, La Jolla, CA) on the SmaI restriction endonuclease site, resulting in the expression plasmid pAM16.

Plasmid pAM18 was prepared by inserting the mvaS gene coding sequence of Enterococcus faecalis (GenBank accession number AF290092 region: 142..1293) encoding HMG-CoA synthetase (HMGS) into pBlueScripII-KS(+) vector. The coding sequence of the mvaS gene was PCR amplified from Enterococcus faecalis genomic DNA (ATCC 700802) using 5' phosphorylated primers 4-40mvaSF BgIII (SEQ ID NO: 13) and 4-39mvaSR BamHI (SEQ ID NO: 14) , the PCR product was ligated into the SmaI restriction endonuclease site of pBlueScripH-KS(+) (Stratagene, La Jolla, CA) to generate expression plasmid pAM18.

The expression plasmid pAM22 was prepared by inserting the mvaE gene coding sequence of the expression plasmid pAM16 into the pZE21- _PL-lacO1 vector. The vector pZE21-PL _-lacO1 is a derivative of the vector pZE21-MCS-1 in which the tet promoter has been replaced by the _PL-lacO1 promoter (Lutz and Bujard (1997) Nucl Acids Res. 25: 1203-1210). The expression plasmid pAM16 was completely digested with BamHI and HindIII restriction enzymes, the reaction mixture was separated by gel electrophoresis, the approximately 2.4kb DNA fragment containing the mvaE coding sequence was gel extracted, and the separated DNA fragment was inserted into pZE21-P _L-lacO1 BamHI HindIII restriction endonuclease sites, resulting in expression plasmid pAM22.

Expression plasmid pAM33 was prepared by inserting the mvaS gene coding sequence of expression plasmid pAM18 into expression plasmid pAM22. The expression plasmid pAM18 was completely digested with BglII and BamHI restriction endonucleases, the reaction mixture was separated by gel electrophoresis, the approximately 1.2kb DNA fragment containing the coding sequence of the mvaS gene was extracted from the gel, and the isolated DNA fragment was inserted into the BamHI expression plasmid pAM22 site, the expression plasmid pAM33 was generated.

Expression plasmid pAM34 was prepared by inserting the mvaS-mvaE operon of expression plasmid pAM33 into vector pAM29. The mvaS-mvaE operon was isolated by partial digestion of pAM33 with EcoRI restriction enzyme, the resulting linear DNA fragment was digested with MluI restriction endonuclease, the reaction mixture was separated by gel electrophoresis, and the approximately 3.6 kb DNA was gel extracted fragment. The vector backbone of pAM29 was obtained by completely digesting the expression vector pAM25 with MluI and EcoRI restriction enzymes, the reaction mixture was separated by gel electrophoresis, and a DNA fragment of about 2.1 kb was gel extracted. The two separated DNA fragments were ligated to generate the expression plasmid pAM34.

Example 4

This example describes the preparation of expression plasmids encoding enzymes of the DXP pathway from E. coli arranged in operons.

The expression plasmid pAM408 was made by inserting the genes encoding the enzymes of the "top" of the DXP pathway into the pAM29 vector. Enzymes at the "top" of the DXP pathway include 1-deoxy-D-xylulose-5-phosphate synthase (encoded by the dxs gene of E. coli), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (encoded by the dxr gene of Escherichia coli), 4-diphosphocytidine-2C-methyl-D-erythritol synthase (encoded by the ispD gene of Escherichia coli), and 4-diphosphocytidine-2C- Methyl-D-erythritol synthase (encoded by the ispE gene of E. coli), which together convert pyruvate and D-glyceraldehyde-3-phosphate to 4-diphosphocytidine-2C-methyl-D - Erythritol-2-phosphate. dxs (GenBank accession number U00096 region: 437539..439401), dxr (GenBank accession number U00096 Region: 193521..194717), ispD (GenBank Accession No. U00096 Region: 2869803..2870512), and ispE (GenBank Accession No. U00096 Region 1261249..1262100) gene coding sequence and add the optimal Shine Dalgarno sequence and 5′ and 3' restriction endonuclease sites to prepare a DNA fragment containing the nucleotide sequence encoding the enzyme at the "top" of the DXP pathway. PCR products were separated by gel electrophoresis, gel-extracted using a Qiagen (Valencia, CA) gel purification kit, and appropriate restriction enzymes were used (XhoI and KpnI for PCR products containing the dxs gene; KpnI and ApaI for PCR products containing the dxr gene; ApaI and NdeI for PCR products containing the ispD gene; NdeI and MluI for PCR products containing the ispE gene) were completely digested and purified using a Qiagen (Valencia, CA) PCR purification kit. Subsequently, approximately equal molar amounts of each PCR product are added to a ligation reaction to assemble the individual genes into operons. For this ligation reaction, 1 μl of the reaction mixture was used to PCR amplify two separate gene cassettes, the dxs-dxr and ispD-ispE gene cassettes. The dxs-dxr gene cassette was PCR amplified using primers 67-1A-C (SEQ ID NO: 15) and 67-1D-C (SEQ ID NO: 18), and primers 67-1E-C (SEQ ID NO: 19 ) and 67-1H-C (SEQ ID NO: 22) to PCR amplify the ispD-ispE gene cassette. The two PCR products were separated by gel electrophoresis and subjected to gel extraction. The PCR product containing the dxs-dxr gene cassette was completely digested with XhoI and ApaI restriction enzymes, the PCR product containing the ispD-ispE gene cassette was completely digested with ApaI and MluI restriction enzymes, and both PCR products were purified. Vector pAM29 was completely digested with SalI and MluI restriction enzymes, and the two digested PCR products containing the "top" DXP pathway operon were ligated into the SalI MluI restriction endonuclease site of the pAM29 vector to generate an expression plasmid pAM408 (see Figure 7 for the plasmid map).

The expression plasmid pAM409 was prepared by inserting the gene encoding the enzyme of the "bottom" of the DXP pathway into the pAM369 vector. Enzymes in the "bottom" of the DXP pathway include 2C-methyl-D-erythritol 2,4-cyclic diphosphate synthase (encoded by the ispF gene of E. coli), 1-hydroxy-2-methyl-2-( E)-butenyl-4-diphosphate synthase (encoded by the ispG gene of Escherichia coli), and isopentenyl/dimethylpropylene diphosphate synthase (encoded by the ispH gene of Escherichia coli), which together convert 4 - Conversion of cytidine diphosphate-2C-methyl-D-erythritol-2-phosphate to IPP and DMAPP. IPP is also converted to DMAPP by the activity of isopentyl diphosphate isomerase (encoded by the idi gene of E. coli). DMAPP can be further converted into FPP by the activity of farnesyl diphosphate synthase (encoded by the ispA gene of Escherichia coli). ispF (GenBank Accession No. U00096 Region: 2869323..2869802), ispG (GenBank Accession No. U00096 Region: 2638708..2639826), ispH (GenBank Accession No. U00096 Region: 26277..27227), idi (GenBank Accession No. AF119715) and ispA (GenBank Accession No. D00694 Region: 484..1383) genes and add optimal ShineDalgarno sequence and 5' and 3' restriction endonuclease sites to make operons encoding enzymes at the "bottom" of the DXP pathway as well as isopentyl diphosphate isomerase and farnesyl diphosphate synthase. The PCR products were separated by gel electrophoresis, gel extracted, and digested with appropriate restriction enzymes (BamHI and ApaI were used for PCR products containing the ispF gene; KpnI and ApaI were used for PCR products containing the ispG gene; SalI and KpnI were used for PCR products containing the ispG gene). for the PCR product containing the ispH gene; SalI and HindIII for the PCR product containing the idi gene; HindIII and NcoI for the PCR product containing the ispA gene), and purified. Roughly equal molar amounts of each PCR product are then added to a ligation reaction to assemble the individual genes into operons. For this ligation reaction, 1 μl of the reaction mixture was used to PCR amplify two separate gene cassettes, the ispF-ispG and ispH-idi-ispA gene cassettes. The ispF-ispG gene cassette was PCR amplified using primers 67-2A-C (SEQ ID NO: 23) and 67-2D-C (SEQ ID NO: 26), and primer 67-2E-C (SEQ ID NO: 27) and 67-2J-C (SEQ ID NO: 32) to PCR amplify the ispH-idi-ispA gene cassette. The two PCR products were separated by gel electrophoresis and subjected to gel extraction. The PCR product containing the ispF-ispG gene cassette was completely digested with BamHI and KpnI restriction enzymes, the PCR product containing the ispH-idi-ispA gene cassette was completely digested with KpnI and NcoI restriction enzymes, and both PCR products were purified product. lacUV5 generated by assembling the p15A origin of replication from pAM29, the ampicillin-resistant beta-lactamase gene from pZE12-luc (Lutz and Bujard (1997) Nucl Acids Res. 25:1203-1210), and oligonucleotides Promoter, construct vector pAM369. The vector pAM369 was completely digested with BamHI and NcoI restriction enzymes, and the two isolated PCR products containing the "bottom" DXP pathway operon were ligated into the BamHI NcoI restriction endonuclease site of the pAM369 vector to generate the expression plasmid pAM409 .

Expression plasmid pAM424, a derivative of expression plasmid pAM409 containing a broader host range RK2 origin of replication, was prepared by transferring the lacUV5 promoter and ispFGH-idi-ispA operon of pAM409 to the pAM257 vector. Vector pAM257 was prepared by PCR from RK2 plasmid DNA (Meyer et al. (1975) Science 190:1226-1228) using primers 9-156A (SEQ ID NO:33) and 9-156B (SEQ ID NO:34). The RK2par site was amplified, the 2.6 kb PCR product was digested completely using AatII and XhoI restriction enzymes, and the DNA fragment was ligated into a Nucl Acids Res. 25:1203-1210) in the plasmid of the chloramphenicol resistance gene, produce plasmid pAM37-par; use restriction endonuclease SacI and HindIII to digest pAM37-par completely, separate the reaction mixture by gel electrophoresis, gel extraction contains RK2 par site and DNA fragments of the chloramphenicol resistance gene, the isolated DNA fragments were ligated into the SacI HindIII site of the miniature RK2 replicon pRR10 (Roberts et al. (1990) JBacteriol.172:6204-6216) to generate the vector pAM133; pAM133 was completely digested with BglII and HindIII restriction enzymes, the reaction mixture was separated by gel electrophoresis, the approximately 6.4kb DNA fragment lacking the ampicillin resistance gene and the oriT junction origin was gel extracted, and the isolated DNA fragment was combined with a synthetic The prepared DNA fragment containing the multiple cloning site containing PciI and XhoI restriction endonuclease sites was ligated to generate vector pAM257. The expression plasmid pAM409 was completely digested with XhoI and PciI restriction enzymes, the reaction mixture was separated by gel electrophoresis, and a DNA fragment of about 4.4 kb was gel extracted. The vector pAM257 was completely digested using the restriction enzymes XhoI and PciI, and the isolated DNA fragment containing the lacUV5 promoter and the ispFGH-idi-ispA operon was ligated into the XhoI PciI restriction endonuclease site of the pAM257 vector to generate Expression plasmid pAM424 (see Figure 8 for the plasmid map).

Example 5

This example describes the preparation of expression plasmids encoding enzymes that transform FPP or GPP.

The expression plasmid pTrc99A-ADS was prepared by inserting the nucleotide sequence encoding amorpha-4,11-diene synthase ("ADS") into the vector pTrc99A. The amorpha-4,11-diene synthase sequence was prepared synthetically so that when transcribed, the amino acid sequence would be identical to that described in Merke et al. (2000) Ach.Biochem.Biophys.381:173-180, thereby encoding The nucleotide sequence of amorpha-4,11-diene synthase was optimized for expression in E. coli and is therefore flanked by 5'NcoI and 3'XmaI restriction endonuclease sites (see U.S. Patent No. 7,192,751). Use NcoI and XmaI restriction endonucleases to digest this nucleotide sequence completely, the reaction mixture is separated by gel electrophoresis, the DNA fragment of about 1.6kb is extracted by gel, and the DNA fragment of separation is inserted into pTrc99A vector (Amman et al. (1985 ) Gene 40: 183-190) at the NcoI XmaI restriction endonuclease site, an expression plasmid pTrc99A-ADS was generated (see Figure 9 for the plasmid map).

Expression plasmid pAM113 is a chloramphenicol-resistant derivative of pTrc99A-ADS. It was prepared by the following method: using 5′-phosphorylated primers 19-137cml-pAM37-AS (SEQ ID NO: 35) and 19-137cml-pAM37-S (SEQ ID NO: 36), from the vector pZA31-luc ( In Lutz and Bujard (1997) Nucl Acids Res.25:1203-1210), PCR amplifies the chloramphenicol resistance gene, and inserts the PCR product of 920bp into the FspI restriction endonuclease site of expression plasmid pTrc99A-ADS , the expression plasmid pAM113 was generated.

By inserting the Bacillus subtilis 6051 genomic DNA fragment (GenBank accession number Z99116 region: 49364..48548) comprising the nudF gene coding sequence and the upstream genome sequence into the vector pTrc99A (Amann et al. (1988) Gene 69: 301-315) To prepare the expression plasmid pC9. The expression plasmid pNudF-H was prepared by inserting the nudF gene coding sequence of Bacillus subtilis 6051 (GenBank accession number Z99116 region: 49105..48548) into the vector pTrc99A. The expression plasmid pyhfR was prepared by inserting the yhfR gene coding sequence of Bacillus subtilis 6051 (GenBank accession number Z99109 region: 97583..97002) into the vector pTrc99A.

An expression plasmid was prepared by inserting the nucleotide sequence encoding the β-farnesene synthase ("FSB") of Artemisia annua (GenBank Accession No. AY835398) codon-optimized for expression in E. coli into the pTrc99A vector pAM373. The nucleotide sequence encoding β-farnesene synthase was prepared synthetically by PCR amplification from its DNA synthesis construct using appropriate primers. To create an initial NcoI restriction endonuclease site in the PCR product containing the coding sequence for β-farnesene synthase, the codon encoding the second amino acid in the original polypeptide sequence (TCG encoding serine) was inserted in the 5'PCR The codon encoding aspartic acid (GAC) was substituted in the primer (SEQ ID NO: 37). The resulting PCR product was partially digested with NcoI restriction endonuclease and completely digested with SacI restriction endonuclease, the reaction mixture was separated by gel electrophoresis, and approximately 1.7 kb containing the β-farnesene synthase coding sequence were gel extracted For the DNA fragment, the isolated DNA fragment was connected to the NcoI SacI restriction endonuclease site of the pTrc99A vector to generate the expression plasmid pAM373 (see Figure 9 for the plasmid map).

By including the enzymes encoding α-farnesene synthase ("FSA"), γ-terpinene synthase ("GTS"), α-pinene synthase ("APS"), or terpinolene synthase ( The DNA fragment of the nucleotide sequence of "TS") was inserted into the pTrc99A vector to prepare expression plasmids pTrc99A-FSA, pTrc99A-GTS, pTrc99A-PS, pTrc99A-TS. The inserted DNA fragment is prepared synthetically, using as a template, for example, the α-farnesene synthase gene coding sequence of Picea spruce (GenBank accession number AY473627, region: 24..1766); the β-method of Artemisia annua Nesene synthase gene coding sequence (GenBank accession number AY835398); lemon gamma-terpinene synthase gene coding sequence (GenBank accession number AF514286 region: 30..1832); North American fir (GenBank accession number U87909, region: 6 ..1892) or the α-pinene synthase gene coding sequence of taeda pine (GenBank accession number AF543530 region: 1..1887); or basil (GenBank accession number AY693650), Douglas pine (GenBank accession number AY906866 region: 10. .1887), or the terpinolene synthase gene coding sequence of North American fir (GenBank accession number AF139206), all nucleotide sequences were codon-optimized for expression in Escherichia coli. A DNA fragment of FSA was amplified by PCR from its DNA synthesis construct using the primer sequences of SEQ ID NO: 39 and SEQ ID NO: 40. Use NcoI and SacI restriction endonucleases to completely digest the obtained PCR product, separate the reaction mixture by gel electrophoresis, gel extract the approximately 1.7kb DNA fragment containing the coding sequence of α-farnesene synthase, and connect the separated DNA fragment to On the NcoI SacI restriction endonuclease site of the pTrc99A vector, an expression plasmid pTrc99A-FSA was generated (see Figure 9 for the plasmid map). DNA fragments of GTS, APS, and TS were designed flanked by an initial XmaI restriction endonuclease site and a terminal XbaI restriction endonuclease site, and were cloned into cloning vectors with compatible restriction enzymes site, such as a standard pUC or pACYC derived vector, by using XbaI and XmaI restriction endonucleases to completely digest the DNA synthesis construct from which the DNA fragments can be re-released, the reaction mixture separated by gel electrophoresis, and The DNA fragment encoding the terpene synthase from 1.7 to 1.9 was gel extracted. The isolated DNA fragment was ligated into the XmaI XbaI restriction endonuclease sites of vector pTrc99A (Amman et al., Gene 40: 183-190 (1985)) to generate plasmids pTrc99A-GTS, pTrc99A-APS or pTrc99A-TS (plasmid map See Figure 9).

By inserting the nucleotide sequence encoding α-farnesene synthase ("FSA") or β-farnesene synthase ("FSB") into the pRS425-Gal1 vector (Mumberg et al. (1994) Nucl.Acids. Res.22(25):5767-5768) to prepare expression plasmids pRS425-FSA and pRS425-FSB. The inserted nucleotide sequence is prepared synthetically, and used as a template: for example, the coding sequence of α-farnesene synthase gene of Picea spruce (GenBank accession number AY473627, region: 24..1766) or β of Artemisia annua - Farnesene synthase gene coding sequence (GenBank accession number AY835398) codon-optimized for expression in Saccharomyces cerevisiae. The synthetically prepared nucleotide sequence is flanked by 5'BamHI sites and 3'XhoI sites, and thus can be cloned into compatible restriction enzyme sites in cloning vectors, such as standard pUC or pACYC source vector. Synthetically prepared nucleotide sequences were isolated by complete digestion of the DNA synthesis constructs using BamHI and XhoI restriction enzymes. The reaction mixture was separated by gel electrophoresis, and the approximately 1.7kb DNA fragment containing the coding sequence of α-farnesene synthase or β-farnesene synthase was extracted from the gel, and the separated DNA fragment was ligated to the BamHI XhoI restriction of the pRS425-Gal1 vector The expression plasmids pRS425-FSA or pRS425-FSB were generated respectively at the endonuclease site.

By encoding linalool synthase ("LLS"), limonene synthase ("LMS"), β-pinene synthase ("BPS"), β-phellandrene ("PHS"), carene synthase ("CS"), or the nucleotide sequence of sachinin synthase ("SS") was inserted into the pTrc99A vector to prepare expression plasmids pTrc99A-LLS, pTrc99A-LMS, pTrc99A-BPS, pTrc99A-PHS, pTrc99A-CS, and pTrc99A-SS. The inserted nucleotide sequence is prepared synthetically, and used as a template is: for example, the coding sequence of the linalool synthase gene of Artemisia annua (GenBank accession number AF154124, region: 13..1764), the limonene synthase gene of the North American fir Coding sequence (GenBank accession number AF006193 region: 73..1986), β-pinene synthase coding sequence of Artemisia annua (GenBank accession number AF276072 region: 1..1749), β-phellandrene synthase gene of North American fir Coding sequence (GenBank accession number AF139205 region: 34..1926), the carene synthase gene coding sequence of Salvia angustifolia (GenBank accession number AF527416 region: 78..1871), or sabinene from Medicinal Sage Synthetase gene coding sequence (GenBank accession number AF051901 region: 26..1798). Nucleotide sequences encoding β-pinene, sabinene, and β-phellandrene synthases flanked by an initial XmaI restriction endonuclease site and a terminal XbaI restriction endonuclease site encoding linalool and carene The nucleotide sequence of ene synthase is flanked by an initial NcoI restriction endonuclease site and a terminal XmaI restriction endonuclease site, while the nucleotide sequence encoding limonene synthase is flanked by an initial NcoI restriction endonuclease site. endonuclease site and terminal PstI restriction endonuclease site. Use XmaI and XbaI (vectors for β-pinene, sabinine, and β-phellandrene synthases), NcoI and XmaI restriction enzymes (vectors for linalool and carene synthases), or XbaI and PstI restriction endonuclease (vector for limonene synthase) to completely digest the DNA synthesis construct. The reaction mixture was separated by gel electrophoresis, and a DNA fragment of about 1.7 to 1.9 kb was extracted by gel, and the separated DNA fragment was ligated into the XmaIXbaI restriction endonuclease site of the pTrc99A vector (for β-pinene, juniper and β - Phellandrene synthase insertion), NcoIXmaI restriction endonuclease site (for insertion of linalool and carene synthase), or XbaI PstI restriction endonuclease site (for insertion of limonene synthase ) to produce expression plasmids pTrc99A-LLS, pTrc99A-LMS, pTrc99A-BPS, pTrc99A-PHS, pTrc99A-CS and pTrc99A-SS (see Figure 9 for the plasmid map).

Example 6

This example describes the preparation of E. coli host bacteria used in the present invention.

As detailed in Table 1, one or more expression plasmids in Examples 1 to 5 were used to chemically transform competent E. coli parental cells to construct host bacteria.

Table 1. Escherichia coli (E.coli) host bacteria

Host cell transformants were selected on Luria Bertoni (LB) agar containing antibiotics as detailed in Table 1. Single clones were transferred from LB agar to culture tubes containing 5 mL LB liquid medium and antibiotics. B003, B617, B618, B619, B650, B651, B652 and B653 host cell transformants were grown at 30°C on a shaker at 250 rpm for 30 hours. All other host cell transformants were grown at 37°C on a shaker at 250 rpm until growth reached stationary phase. The cells were adapted to the minimal medium by serial passage 4 to 5 times in M9-MOPS medium containing 0.8% glucose and antibiotics (see Table 2 for the composition of the M9-MOPS medium). Cells were maintained in 1 mL stock aliquots consisting of 400 μL sterile 50% glycerol and 600 μL liquid medium in cryovials at -80°C.

Components of Table 2-M9-MOPS medium

Element Weight (per liter) Na2HPO4 7H2O 12.8g KH2PO4 3g NaCl 0.5g NH4Cl 1g MgSO4 2mmol CaCl2 0.1mmol Thiamine 0.1ug Buffer pH7.4 100mmol (NH3)6Mo7O24 4H2O 3.7ug H3BO4 25ug CoCl2 7.1ug CuSO4 2.4ug MnCl2 16ug ZnSO4 2.9ug FeSO4 0.28mg

Example 7

This example demonstrates the stability of the expression plasmid in Escherichia coli host bacteria without antibiotics. The expression plasmid contained in the host bacteria contains the RK2 plasmid replication, isolation and maintenance system.

Host strains were established by adding stock aliquots of the strains to 125 mL bottles containing 40 mL of LM9-MOPS, 2% glucose, 0.5% yeast extract, and antibiotics as detailed in Table 1, and allowing the culture to grow overnight. Inoculum culture of B255.

The inoculum culture was inoculated at an initial _OD600 value of approximately 0.05 in two 250 mL bottles each containing 40 mL of M9-MOPS medium, 2% glucose, and 0.5% yeast extract. Culture #1 also contained 100 ug/mL carbenicillin and 34 ug/mL chloramphenicol. Culture #2 did not have any antibiotics added. Both cultures were grown at 37 °C on a shaker at 250 rpm until they reached an _OD600 value of approximately 0.2, at which point amorpha-4,11-diene was induced by adding 40 uL of 1 M IPTG to the medium. Production in host cells. At the time of induction, amorpha-4,11-diene was harvested by overlaying the culture with 8 mL of an organic overlay. During a total of 72 hours, samples were taken periodically. Production of amorpha-4,11-diene by the host strain in 2 cultures was confirmed by GC/MS as described in Example 10.

To assess plasmid stability in two cell cultures, at 72 hours, one sample of each culture was taken and streaked on LB agar plates (without antibiotics). After overnight incubation at 37°C, 50 single colonies from each culture were expressed on LB agar-(+)-antibiotic (34ug/mL chloramphenicol, 100ug/mL carbenicillin) plates and LB agar-(-) - Repeat plating on antibiotic (no antibiotic) plates. After further overnight incubation at 37°C, the LB agar-(+)-antibiotic and LB agar-(-)-antibiotic plates were found to contain approximately 50 colonies each, indicating that plasmid retention is consistent in the presence and absence of antibiotics in the medium. About 100%.

Example 8

This example demonstrates that E. faecalis HMGR exhibits increased specific activity and stability in E. coli host bacteria compared to S. cerevisiae tHMGR.

Stock aliquots of each strain were added to 125 mL bottles containing 20 mL of M9-MOPS medium, 0.8% glucose, and antibiotics as detailed in Table 5, and the cultures were grown to saturation to establish host strains B61 and Inoculum culture of B62. In 500 mL flasks, the inoculum culture was diluted 1:100 into 140 mL of fresh medium and regrown to an _OD550 value of approximately 0.1, at which time point purple was induced by adding 140 uL of 1M IPTG to each culture. Production of Sophora-4,11-diene. At 4, 12, 20, 28, 36 and 49 hours after induction, samples were taken from each culture and the cells were pelleted by centrifugation. Cell pellets were snap frozen on dry ice and then stored at -80°C.

For enzyme activity assays, cell pellets were thawed on ice and then lysed using Bugbuster (Novagen, Madison, WI), which included protease inhibitor cocktail #3 (Calbiochem, San Diego, CA), benzonase (20 μL oer 5 mL bugbuster; Novagen, Madison, WI) and lysozyme (30ug/mL). The enzyme activity of Saccharomyces cerevisiae tHMGR was tested in 50mM Tris HCl (pH7.5), 0.2mM NADPH (Sigma, St.Louis, MO) and 0.3mMDL-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) Sodium salt (Sigma, St. Louis, MO). The assay was initiated by addition of cell lysate and the disappearance of NADPH was monitored by absorbance at 340 nM. To calculate the non-specific disappearance of NADPH, the results obtained in the control test lacking HMG-CoA were subtracted from the results obtained for the test samples. The enzymatic activity of E. faecalis HMGR was similarly measured except that the assay buffer contained 100 mM potassium phosphate buffer (pH 6.5), 0.4 mM NADPH, 1.0 mM EDTA and 100 mM KCl.

Protein activity assays were performed by the method of Bradford ((1976) Anal Biochem. 72:248-254). Specific activity was calculated as Δnmol NADPH/min/mg protein.

Example 9

This example describes the calibration of _OD600 values and dry cell weight ("DCW").

To obtain the relationship between DCW and _OD600 values, representative strain B32 was grown in a high cell density procedure similar to those described in Examples 10-12. Samples were taken during the process and the _OD600 and DCW values were measured for each sample. To determine DCW values, cells were pelleted and the supernatant was discarded. Cell pellets were washed once with water and then dried in an oven at 80 °C for at least 3 days. The tubes containing the cell pellets were weighed, the weight of the tube was subtracted from the measured weight, and the remaining weight was divided by the initial volume of each sample (0.0015 L) to obtain the DCW value.

Example 10

This example demonstrates that the production of amorpha-4,11-diene is increased in E. coli host bacteria expressing Staphylococcus aureus HMGR and HMGS compared with host bacteria expressing Saccharomyces cerevisiae tHMGR and HMGS.

Stock aliquots of each strain were added to separate 125 mL bottles containing 25 mL of M9-MOPS medium, 0.8% glucose, and antibiotics as detailed in Table 1, and the cultures were allowed to grow overnight to establish host strains B32, Inoculum cultures of B153, B210, B282, B292, B86, B255 and B256.

The inoculum culture was inoculated in a separate 250 mL bottle containing 40 mL of M9-MOPS medium, 2% glucose, and antibiotics at an initial _OD600 value of approximately 0.05. The culture was grown at 30 °C on a shaker at 250 rpm until it reached an _OD600 value of approximately 0.2, at which time point amorpha-4,11-diene was induced in the host by adding 40 uL of 1 M IPTG to the medium. production in cells. Overlay the culture with 8 mL of an organic overlay such as dodecane, methyl oleate, or isopropyl myristate. For 72 hours, samples of the organic overlay and culture fluid were taken once a day. Culture broth samples were used to measure _OD600 values. The concentration of amorpha-4,11-diene was measured by transferring 5 uL of the organic overlay to a clean glass bottle containing 500 uL of ethyl acetate calibrated with β- or trans-caryophyllene as an internal standard.

By scanning only two ions, the molecular ion (204 m/z) and the 189 m/z ion, as described in Martin et al. (2001) Biotechnol. Bioeng. 75:497-503, the Organic overlay/ethyl acetate samples were analyzed on (GC/MS). To speed up the run time, the temperature program and column matrix were modified to achieve optimal peak resolution and shortest total run time. A 1 uL sample was separated on the GC using a DB-XLB column (available from Agilent Technologies, Palo Alto, CA) and a helium carrier gas. The temperature program for the analysis was as follows: 0.75 minutes at 100°C, ramp up to a temperature of 300°C at 60°C/minute, and hold at 300°C for 0.5 minutes. The decomposed samples were analyzed by a Hewlett-Packard Model 5973 mass spectrometric selective detector, monitoring 189 and 204 m/z ions. Previous mass spectrometry demonstrated that the product of amorpha-4,11-diene synthase was amorpha-4,11-diene, and using this GC method, amorpha-4,11-diene had a 3.7 min residence time. β- or trans-caryophyllene was used as internal standard for quantification. Using the ratio of the peak areas of internal standard and amorpha-4,11-diene, based on pure amorpha-4,11-diene (0.63-10 mg/L, KJF17-109-3) in caryophyllene Amorpha-4,11-diene titers were calculated from the quantified calibration curve in ethyl acetate.

Example 11

This example demonstrates the increased production of amorpha-4,11-diene by an E. coli host strain grown at a suboptimal temperature.

Add 0.5 mL of the strain stock aliquot to a 250 mL bottle containing 50 mL of M9-MOPS medium and antibiotics as detailed in Table 1 and allow the culture to grow overnight at 37 °C on a shaker at 250 rpm to establish host strain B32 inoculum cultures.

The inoculum culture was inoculated at an initial OD value of approximately _0.05 in four 250 mL bottles each containing 40 mL of fermentor batch medium (see Table 6 for medium composition), 100 mM MOPS buffer pH 7.1, and antibiotics middle. The culture was grown on a shaker at 250 rpm at 30°C or 37°C until it reached an _OD600 value of 0.18 to 0.22, at which point Amorpha-4,11- Production of dienes in host cells. At induction, amorpha-4,11-diene was harvested by overlaying the culture with 8 mL of an organic overlay. Samples were taken daily and analyzed as described in Example 10.

Example 12

This example demonstrates the increased production of amorpha-4,11-diene in Escherichia coli host bacteria grown under carbon source limited conditions.

Add 0.25 uL of the strain stock aliquot to a 250 mL bottle containing 50 mL of M9-MOPS medium and antibiotics as detailed in Table 1 and let the culture grow at 37 °C on a shaker at 250 rpm until it reaches an _OD600 Values 1 to 2, to establish an inoculum culture of host strain B32 for fermentation runs 050608-1 and 050629-1.

Add a stock aliquot of the strain to a 250 mL bottle containing 50 mL of M9-MOPS medium and antibiotics as detailed in Table 1 and let the culture grow overnight at 37 °C on a shaker at 250 rpm to set up for the fermentation protocol Inoculum culture of host strain B32 of 060403-3. Use the inoculum culture to inoculate at an initial _OD600 value of about 1 in a 250 mL bottle filled with 40 mL of M9-MOPS medium and antibiotics, and culture the culture again at 37 °C on a shaker at 250 rpm until it reaches an _OD600 value of 3 to 5.

For all fermentations, KH ₂ PO ₄ , K ₂ HPO ₄ 3H ₂ O, EDTA, citric acid, and (NH ₄ ) ₂ SO ₄ were run in bioreactors (2L Applikon Bioconsole ADI 1025s with ADI 1010 controller, Applikon Biotechnology, Foster City, CA) for heat sterilization. The remaining media components, such as stock solutions, are filter sterilized and injected through the caps. Table 3 shows the final media composition for fermentation runs 050608-1 and 050629-1. Table 4 shows the final media composition for fermentation scheme 060403-3. The starting volume for 050608-1 was 0.8 L, the starting volume for 050629-1 was 1.2 L, and the starting volume for 0)60)403-3 was 1 L. All runs were inoculated by injecting 50 mL of inoculum culture from the head.

Table 3 - Fermentation Medium Components of Fermentation Processes 050608-1 and 050629-1

Element Batch media (per liter) Feed liquid (per liter) glucose 5g 590-650g KH ₂ PO ₄ 4.2g - K ₂ HPO ₄ 3H ₂ O 15.7g - citric acid 1.7g - (NH ₄ ) ₂ SO ₄ 2g - MgSO ₄ 7H ₂ O 1.2g 12g EDTA 8.4mg 13g CoCl ₂ 6H ₂ O 0.25mg 0.4mg MnCl ₂ 4H ₂ O 1.5mg 2.35mg CuCl ₂ 2H ₂ O 0.15mg 0.25mg H ₃ BO ₄ 0.3mg 0.5mg Na ₂ MoO ₄ 2H ₂ O 0.25mg 0.4mg Zn(CH ₃ COO) ₂ 2H ₂ O 1.3mg 1.6mg Iron(III) Citrate Hydrate 10.0mg 4.0mg Thiamine Hydrochloride 4.5mg - carbenicillin 100ug 100ug tetracycline 5ug 5ug Chloramphenicol 34ug 34ug

Table 4 - Fermentation Medium Components of Fermentation Process 060403-3

Element Batch media (per liter) Feed liquid (per liter) glucose 15g 650g KH ₂ PO ₄ 4.2g - K ₂ HPO ₄ 3H ₂ O 15.7g - citric acid 1.7g - (NH ₄ ) ₂ SO ₄ 2g - MgSO ₄ 7H ₂ O 1.2g 12g EDTA 8.4mg 13mg CoCl ₂ 6H ₂ O 2.5mg 4 mg MnCl ₂ 4H ₂ O 15mg 23.5mg CuCl ₂ 2H ₂ O 1.5mg 2.5mg H ₃ BO ₄ 3mg 5mg Na ₂ MoO ₄ 2H ₂ O 2.5mg 4 mg Zn(CH ₃ COO) ₂ 2H ₂ O 13mg 16mg Iron(III) Citrate Hydrate 100mg 40mg Thiamine Hydrochloride 4.5mg - carbenicillin 100ug 100ug Tetracycline 5ug 5ug Chloramphenicol 34ug 34ug

For fermentation run 050608-1 (carbon excess), feeding was started at induction and the feeding rate was adjusted manually. For fermentation run 050629-1 (carbon limited), the feed to the fermentor was performed as shown in Table 5. For fermentation run 060403-3 (minimum carbon), dosing started automatically when the initial glucose dose (15 g) was exhausted and dissolved oxygen peaked. Up to a maximum of 27.6 g/hour, the feed rate was calculated according to the following equation:

m _s (t)=S(t ₀ )μeμ ^(t-t0)

μ = 0.12

S(t ₀ )=15g

where _t0 is the time when the initial glucose is depleted. When the maximum rate was reached, the glucose feed was limited to a rate of 9.5 g/hour and held constant at this rate for the remainder of the run.

Table 5 Feeding method of fermentation process 050629-1

Running time (hours) Glucose feeding rate (g hours) 0 0 7 0.37 10 0.74 12 1.11 14 1.48 16 2.22 18 2.96 20 3.69 twenty two 4.80 twenty four 5.91 31 7.39 33 5.54 47 3.69

Procedures 050608-1 and 050629-1 were performed at 37°C. Air flow in the bioreactor was set at 1-2 L/min; pH was maintained at 7 using ammonium hydroxide and/or sodium hydroxide; initial agitation was 500-600 rpm; antifoam B (Sigma-Aldich, St. .Louis, MO) to control foam; maintain dissolved oxygen levels above 30% using an agitation cascade. After 5-6 hours of incubation, the host cells were induced to produce amorpha-4,11-diene by adding 0.8 mL of 1 M IPTG to Procedure 050608-1 and 1.2 mL of IPTG to Procedure 050629-1. At the time of induction, the culture temperature was lowered to 30°C.

Procedure 060403-3 was performed at 30°C. Air flow in the bioreactor was set at 1-2 L/min; pH was maintained at 7 using ammonium hydroxide. Dissolved oxygen is maintained above 30% by stirring cascades and oxygen enrichment. When the _OD600 value reached about 28 (19 hours after inoculation), the host cells were induced to produce amorpha-4,11-diene by adding 1 mL of 1M IPTG.

Amorpha-4,11-diene was harvested and extracted according to two different methods. For processes 050608-1 and 050629-1, the volatile amorpha-4,11-diene present in the off-gas was collected by passing the off-gas through a gas scrubber containing 200 mL of heptanol. The heptanol was then diluted with ethyl acetate until the concentration of amorpha-4,11-diene in the sample was between 0.63 mg/L and 20 mg/L. For process 060403-3, amorpha-4,11-diene was collected in the bioreactor by adding 200 mL of an organic overlay to the fermentor upon induction. Product concentrations were obtained by combining 25 uL of culture medium plus organic overlay and 975 uL of acetonitrile, shaking the samples at maximum speed for at least 3 minutes on a Fisher Vortex Genie 2 ^TM mixer (Scientific Industries, Inc., Bohemia, NY), and centrifuging from the samples. Cells were removed and the acetonitrile solution was diluted with ethyl acetate until the concentration of amorpha-4.11-diene in the sample was between 0.63 and 20 mg/L for measurement. Ethyl acetate samples were analyzed by GC/MS as described in Example 10.

Example 13

This example demonstrates the increased production of amorpha-4,11-diene in an E. coli host strain grown under carbon source limited conditions and suboptimal temperature.

Add a stock aliquot of the strain to a 250 mL bottle containing 50 mL of M9-MOPS medium and antibiotics as detailed in Table 1 and allow the culture to grow to an OD value of _3.5 to 4.5 at 37 °C on a shaker at 250 rpm , Establish the inoculum culture of host bacteria B153.

A 2L bioreactor (Biocontroller ADI 1010 with Bioconsole ADI 1025, Applikon Biotechnology, Foster City, CA) was set up and run in the same manner as described for process 060403-3 in Example 12, except for changes in strain and induction time .

Induce the production of amorpha-4,11-diene in the host cells by adding 1 mL of 1 M IPTG to the medium. Amorpha-4,11-diene was harvested and extracted according to two different methods. In one method, volatile amorpha-4,11-diene present in the off-gas was collected by passing the off-gas through a gas scrubber containing 200 mL of heptanol. The heptanol was then diluted with ethyl acetate until the concentration of amorpha-4,11-diene in the sample reached between 0.63 and 20 mg/L. In another method, amorpha-4,11-diene was collected by adding 200 mL of an organic overlay to the fermentor upon induction.

Amorpha-4,11-diene was prepared by combining 25 uL of culture broth with 975 uL of acetonitrile and shaking the sample at maximum speed for at least 3 minutes on a Fisher Vortex Genie 2 ^TM mixer (Scientific Industries, Bohemia, NY). Cells were removed from the sample by centrifugation and the acetonitrile solution was diluted with ethyl acetate until the concentration of amorpha-4,11-diene in the sample reached between 0.63 and 20 mg/L for extraction from the medium. Ethyl acetate samples were analyzed by GC/MS as described in Example 10.

Example 14

This example demonstrates the increased production of amorpha-4,11-diene in an E. coli host strain grown under carbon and nitrogen source limited conditions and suboptimal temperature.

An inoculum culture of host strain B86 was established by adding a stock aliquot of the strain to a 250 mL bottle containing 50 mL of M9-MOPS medium and antibiotics as detailed in Table 1. Cultures were grown overnight at 37°C on a shaker at 250 rpm, subcultured the next morning in the same medium at an _OD600 of approximately 1, and grown again to an _OD600 of 3 to 5 at 37°C and 250 rpm.

Four 2L bioreactors (Biocontroller ADI 1010 with Bioconsole ADI 1025 , Applikon Biotechnology, Foster City, CA).

Exponential glucose dosing with a doubling time of 6 hours started automatically when the initial glucose dose (15 g) was exhausted and dissolved oxygen peaked. Up to a maximum of 30.4 g/hour, the feed rate was calculated according to the following equation:

m _s (t)＝S ₀ μe ^μ(t-t0)

μ＝0.12min ^-1

S ₀ =15g

where □ is the specific growth rate and _t0 is the time when the initial glucose dose is exhausted. When the maximum rate was reached, the glucose feed was reduced to a rate of 11.4 g/hour and held constant at this rate for the remainder of the run. In fermentation runs 060710-4, 060724-5, and 060619-5 (carbon and nitrogen limitation), the glucose feed was further reduced when ammonia limitation resulted in glucose accumulation in the medium.

Fermentation was carried out at a reduced temperature of 30°C. Air flow in the bioreactor was set at 1 vvm; initial agitation was 700 rpm; foam was controlled with antifoam B (Sigma-Aldich, St. Louis, MO); and agitation blades (700-1200 rpm) and oxygen enrichment were used , the dissolved oxygen pressure is controlled at 40%. In fermentation run 060327-3 (carbon limited), pH was maintained at 7 using 20% _NH4OH ; in fermentation runs 060710-4, 060724-5 and 060619-5 (carbon and nitrogen limited), initial use 20% _NH4OH , and a 50/50 mixture of 2.5N NaOH and 10N _NH4OH was used starting at 72 hours to maintain the pH at 7 to further limit the amount of ammonia entering the fermentor.

The production of amorpha-4,11-diene in the host cells was induced by adding 1 mL of 1M IPTG to the medium at an _OD600 value of approximately 30.

Amorpha-4,11-diene was harvested by covering the medium with a 10% (v/v) organic overlay. Amorpha-4,11-diene was then shaken at maximum speed on a Fisher Vortex Genie 2 ^TM mixer (Scientific Industries, Bohemia, NY) for at least 15 minutes by combining 25 uL of broth and 975 uL of methanol, Cells were removed from the samples by centrifugation and extraction was performed by adding 10 uL methanol solution to 990 uL ethyl acetate containing 10 uL/L trans-caryophyllene.

Samples were analyzed by GC/MS as described in Example 10.

Example 15

This example describes the production of amorpha-4,11-diene via the DXP pathway in E. coli host bacteria.

Stock aliquots of each strain were added to separate 125 mL bottles containing 25 mL of M9-MOPS and antibiotics as detailed in Table 1, and the cultures were allowed to grow overnight to establish host strains B003, B617, B618, and B619 inoculum cultures.

Use the inoculum culture at an initial OD ₆₀₀ value of about 0.05, inoculate in 40mL M9-MOPS medium, 45ug/mL thiamine, micronutrients, 1.00E-5mol/L FeSO ₄ , 0.1M MOPS, 0.5% yeast Extract, 20g/L D-glucose, and antibiotics in separate 250mL bottles. The culture was grown at 250 rpm in a humidified culture shaker at 30°C until it reached an _OD600 value of 0.2 to 0.3, at which time point Amorpha fruticosa- 4,11-Diene production.

At the time of induction, amorpha-4,11-diene was harvested by overlaying the culture with 8 mL of an organic overlay. Samples were taken at various time points, amorpha-4,11-diene was extracted and analyzed by GC/MS as described in Example 10. Two independent clones of each host bacterium were used for the experiment, and the average value of the results was taken. The variance between samples was found to be less than 10%.

Example 16

This example describes the production of 3-methyl-but-3-en-1-ol and 3-methyl-but-2-en-1-ol in an E. coli host.

Stock aliquots of each strain were streaked on LB agar containing antibiotics as detailed in Table 1 to establish inoculum cultures of host strains B286, B287, B288, and B291. Three independent clones were picked for each strain, and each clone was inoculated into 7 mL of LB medium containing antibiotics. Cultures were grown overnight at 37°C on a shaker at 250 rpm until late log phase. Then, the culture was inoculated into a 250 mL bottle containing 40 ml of M9-MOPS, 2% glucose, 0.5% yeast extract, and antibiotics at a concentration of _OD600 of about 0.05. The culture was grown overnight at 37°C on a shaker at 250 rpm until it reached an _OD600 value of approximately 0.2, at which point it was induced by the addition of 40 uL of 1M IPTG. Cultures were grown for 72 hours at 30°C on a shaker at 250 rpm. Once or twice a day, the _OD600 value of each culture was measured and 700 uL was sampled. To extract 3-methyl-but-3-en-1-ol and 3-methyl-but-2-en-1-ol from the culture broth, 600 uL of ethyl acetate was added to 300 uL of each withdrawn sample. The samples were then shaken for 15 minutes and 400 uL of the upper ethyl acetate phase was transferred to a clean glass vial for analysis.

Samples were analyzed on a Hewlett-Packard 6890 gas chromatograph/mass spectrometer (GC/MS). A 1 uL sample was separated on a GC using a DB-5 column (Agilent Technologies, Palo Alto, CA) with a helium carrier gas. The temperature program for the analysis was as follows: 3 minutes at 60°C, ramp up to a temperature of 300°C at 60°C/min, and hold at 300°C for 2 minutes. The total runtime is 9 minutes. Decomposed samples were analyzed by a Hewlett-Packard Model 5973 mass spectrometric selective detector. Previous mass spectra demonstrated that 3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol have a retention time of 2.067 minutes using this GC method. To focus detection on 3-methyl-but-3-en-1-ol and 3-methyl-but-2-en-1-ol, using a selective ion monitoring method, only 3-methyl-butan -3-en-1-ol and ions 56 and 68 in 3-methyl-but-2-en-1-ol.

Example 17

This example describes the production of amorpha-4,11-diene in a S. cerevisiae host.

The preparation of the host strain EPY224 is described in Ro et al. (Nature 440:940-943; 2006) and PCT Patent Publication No. WO2007/005604. By growing in YPD medium (Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, 2005 edition, ISBN 0-87969-728-8), the expression plasmid pRS425ADS was deleted in the host strain EPY224, and a single clone was used on YPD agar Plates were then spotted with individual colonies on CSM-Met His agar and CSM-Met Leu agar. Clones that grew on CSM-Met His agar but not CSM-MetLeu agar were deleted (ie had lost the plasmid pRS425ADS). One such clone was named EPY300. EPY300 was transformed with the expression plasmid pRS425-ADS-LEU2d, which is identical to pRS425-ADS except that it includes the LEU2d selection marker (Erhart and Hollenberg (1983) J. Bacteriol. 156:625-635) instead of LEU2 , to generate host strain Y185.

Y185 host cell transformants were selected on synthetic defined medium (CSM-glucose; MP Biomedicals, Solon, OH) containing 2% glucose and all amino acids except histidine, leucine, and methionine. The host strain EPY300 was auxotrophic for leucine biosynthesis (Ieu2), but the expression plasmid pRS425-ADS-LEU2d in Y185 restored leucine prototrophy (LEU2). Single clones were spotted onto selective medium (CSM-glucose-histidine, leucine, methionine) and grown for 2 days. Cells were scraped from the plate and transferred to 1 mL of 25% (v/v) glycerol in a cryovial. The suspension was mixed and then stored at -80 °C.

A stock aliquot of the strain was added to a 125 mL bottle containing 25 mL of CSM-glucose lacking leucine and methionine, and the culture was allowed to grow overnight to establish an inoculum bottle of host strain Y185. Cultures were inoculated at an initial _OD600 of approximately 0.05 in 250 mL baffled flasks containing 40 mL of synthetic defined medium lacking leucine and containing 0.2% glucose, 1.8% galactose, and 1 mM methionine. Cultures were grown at 30°C on a shaker at 200 rpm. Because the presence of glucose in the medium prevents the induction of the GAL1 promoter by galactose, the production of amorpha-4,11-diene is not induced until the cells deplete the glucose in the medium and switch to using galactose as their main carbon source. At the time of inoculation, amorpha-4,11-diene was harvested by overlaying the culture with 8 mL of an organic overlay. Samples were taken at 72 hours and 5uL of the organic solvent layer was transferred to a clean glass vial containing 500uL of ethyl acetate containing known concentrations of β- or trans-caryophyllene as an internal standard.

Organic overlay/ethyl acetate samples were analyzed as described in Example 10 on a Hewlett-Packard 6890 Gas Chromatograph/Mass Spectrometer (GC/MS).

After 72 hours of growth, the three yeast cultures were found to produce 60.68, 54.48 and 59.25 mg/L of amorpha-4,11-diene.

Example 19

This example describes the production of amorpha-4,11-diene in a Saccharomyces cerevisiae host comprising a native mevalonate pathway and exogenous The mevalonate pathway.

Yeast strains CEN.PK2-1C(Y002) (MATA; ura3-52; trp1-289; leu2-3,112; his3Δ1; MAL2-8C; SUC2) and CEN.PK2-1D(Y003) (MATα; ura3-52 ; trp1-289; Ieu2-3,112; his3Δ1; MAL2-8C; SUC2) (J.P. van Dijken et al., Enzyme MicrobTechnol26, 706 (June 1, 2000)), in standard rich nutrient medium (YPD) or Cultivated in a synthetic medium with defined components (.D.Rose, F.Winston, P.Heiter, Methods in yeast genetics: a laboratory course manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1990)), and lack of appropriate nutrients to screen for integrated transformants, plasmid retention, and meiotic progeny.

DNA-mediated transformation into Saccharomyces cerevisiae was performed according to the lithium acetate method as described in R.H. Schiestl, R.D. Gietz, Curr Genet 16, 339 (December 1989). All gene disruptions and substitutions were confirmed by phenotypic analysis, polymerase chain reaction ("PCR") of colonies, and sequencing of amplified genomic DNA. Plasmids pAM489-pAM498 were constructed using pCR2.1 (Invitrogen, Carlsbad CA) and are illustrated by Figures 7A-C and Table 6. The HISMX marker sequence is described in M.S. Longtine et al., Yeast 14, 953 (July 1998). Propagation of plasmid DNA was performed in E. coli strain DH5α.

Table 6

Strains 5'HR Gene #1 Crick promoter Watson promoter Gene #2 genetic markers 3'HR pAM489 TRP1 tHMGR GAL1 GAL10 ERG20 TRP1 TRP1 pAM490 TRP1 tHMGR CUP1 CUP1 ERG20 TRP1 TRP1 pAN491 URA3 tHMGR GAL1 GAL10 ERG13 URA3 URA3 pAM492 URA3 IDI1 CUP1 CUP1 tHMGR URA3 URA3 pAM493 ADE1 tHMGR GAL1 GAL10 IDI1 ADE1 URA3 pAM494 ADE1 tHMGR CUP1 CUP1 IDI1 ADE1 ADE1 pAM495 HIS3 ERG12 GAL1 GAL10 ERG10 HISMX HIS3 pM496 HIS3 ERG12 CUP1 CUP1 ERG10 HISMX HIS3 pAM497 LEU2 ERG19 GAL1 GAL1 ERG8 HISMX LEU2 pAM498 LEU2 ERG19 CUP1 CUP1 ERG8 HISMX LEU2

Saccharomyces cerevisiae strains Y002 and Y003 were prepared for introduction of inducible mevalonate pathway genes as follows. Using primers 50-56-pw100-G (SEQ ID NO: 44) and 50-56-pw1o1-G (SEQ ID NO: 45) containing 45 base pairs of homology to the native ERG9 promoter, from pAM328 ( The KanMX-PMET3 region was amplified by PCR in SEQ ID NO: 43), and the ERG9 promoter was replaced with the MET3 promoter of Saccharomyces cerevisiae. Adopt 40% w/w polyethylene glycol 3350 (Sigma-Aldrich, St.Louis, MO), 100mM lithium acetate (Sigma Company), 10 μ g salmon sperm DNA (Invitrogen Company) to incubate at 30 ℃ for 30 minutes, then at 42 ℃ After heat shock for 30 minutes (as described by Schiestl & Gietz, Curr. Genet. 16:339 (1989)), 10 μg of the resulting PCR product was transformed into logarithmic growing Y002 and Y003 strains. Positive recombinants were identified by their ability to grow on rich media containing 0.5 μg/ml Geneticin (Invitrogen, Carlsbad, CA) and confirmed by PCR identification. The obtained clones were named Y93(MATA) and Y94(MATα). Then, the ADE1 open reading frame was replaced by the Candida glabrata LEU2 gene (CgLEU2). Using primers 61-67-CPK066-G (SEQ ID NO: 46) and 61-67-CPK067-G (SEQ ID NO: 47) containing 50 base pairs of homology to the sides of the ADE1 open reading frame (ORF), From C. glabrata genomic DNA (ATCC, Manassas, VA), a 3.5 KB CgLEU2 genomic locus was amplified. 10 μg of the resulting PCR product was transformed into logarithmic growing Y93 and Y94 as described above, and the ade1-strain was screened for growth on supplemented medium lacking leucine and confirmed by PCR identification. The resulting clones were named Y176(MAT A) and Y177(MATα).

To prepare Saccharomyces cerevisiae strain Y188, 2 μg of plasmid DNA from pAM491 (SEQ ID NO: 48) and pAM495 (SEQ ID NO: 49), respectively, were digested overnight with PmeI (New England Biolabs, Beverly, MA) and incubated as above. introduced into logarithmically grown Y176 as described. Positive recombinants were screened for by growth on media lacking uracil and histidine. Integration into the correct genomic site was confirmed by PCR identification.

To prepare Saccharomyces cerevisiae strain Y189, 2 μg of plasmid DNA from pAM489 (SEQ ID NO:50) and pAM497 (SEQ ID NO:51), respectively, were digested overnight with PmeI and introduced into a logarithmic growing Y177. Positive recombinants were screened for by growth on media lacking tryptophan and histidine. Integration into the correct genomic site was confirmed by PCR identification.

Approximately 1 x ¹⁰⁷ cells from Y188 and Y189 were mixed on YPD medium plates for 6 h at room temperature and allowed to hybridize. The mixed cell cultures were then plated on media lacking histidine, uracil, and tryptophan to screen for growth of diploid cells. 2 μg of plasmid DNA from pAM493 (SEQ ID NO: 52) was digested overnight with PmeI and introduced into logarithmically growing diploid cells as described above. Positive recombinants were screened by growth on media lacking adenine. Integration into the correct genomic site was confirmed by PCR identification. The resulting strain was named Y238.

To prepare haploid strains containing fully complementary introduced genes, Y238 was sporulated in a liquid medium containing 2% potassium acetate and 0.02% raffinose. Using a Singer Instruments MSM300 series micromanipulator (Singer Instruments Co, LTD, Somerset, UK), approximately 200 genetic tetraspores (tetraspores are the meiotic products of tetraspores) were isolated. Independent genetic isolates containing the appropriate complement of the introduced genetic material were identified by their ability to grow in the absence of adenine, histidine, uracil and tryptophan. Integration of all introduced DNA was confirmed by PCR identification. The resulting strains were named Y210(MAT A) and Y211(MATα).

2 μg of plasmid DNA from pAM426 (SEQ ID NO:53) containing the S. cerevisiae codon-optimized amorphadiene synthase (ADS) expressed from the S. cerevisiae GAL1 promoter was prepared as described above. , imported into logarithmically grown Y210 and Y211. S. cerevisiae strains containing the pAM426 plasmid were screened for their ability to grow on supplemented medium lacking leucine. The resulting strains were named Y225(MAT A) and Y227(MATα).

2 μg of plasmid DNA from pAM322 (SEQ ID NO:54) containing S. cerevisiae codon-optimized amorphadiene synthase (ADS) and cytochrome P450 monooxygenase expressed by S. cerevisiae GAL1 (AMO), and cytochrome P450 oxidoreductase (CPR) expressed from the S. cerevisiae GAL10 promoter, were introduced into logarithmic growing Y210 and Y211 as described above. S. cerevisiae strains containing the pAM322 plasmid were screened for their ability to grow on supplemented medium lacking leucine. The resulting strains were named Y222(MAT A) and Y224(MATα).

Example 19

This example describes the production of α-farnesene or β-farnesene in an E. coli host.

A stock aliquot of each strain was added to a 125 mL bottle containing 25 mL of LM9-MOPS, 0.8% glucose, 0.5% yeast extract, and antibiotics as detailed in Table 1, and the culture was allowed to grow overnight to establish host strain B552 and B592 inoculum cultures.

The inoculum culture was inoculated at an initial _OD600 value of approximately 0.05 in a 250 mL bottle containing 40 mL of M9-MOPS, 2% glucose, 0.5% yeast extract, and antibiotics. The culture was grown at 30°C on a shaker at 250 rpm until it reached an _OD600 value of approximately 0.2, at which point production of α-farnesene or β-farnesene in the host cells was induced by adding 40 uL of 1 M IPTG. At the time of induction, harvest α-farnesene by overlaying the culture with 8 mL of organic overlay. Samples were taken every 24 hours, transferring 2 uL to 10 uL of the organic overlay to clean glass vials containing 1 mL of ethyl acetate calibrated with trans-caryophyllene as internal standard, until 120 hours (total of 5 time points). Alternatively, 1 mL of the aliquoted culture was spun down, the cell pellet was resuspended in 250 uL of sterile water, and the cell suspension was transferred to a glass vial containing 1 mL of ethyl acetate calibrated with trans-caryophyllene as an internal standard. Separately, a 0.5 mL aliquot of the whole culture broth was added to a glass vial containing 1 mL of ethyl acetate calibrated with trans-caryophyllene as an internal standard. Whole broth samples were extracted by ethyl acetate by shaking the glass vial for 10 minutes, after which time 600 uL of the ethyl acetate extract was transferred to a clean glass vial.

Whole cultures of organic overlay/ethyl acetate samples and ethyl acetate extracts were analyzed in full scan mode (50-500 m/z) on an Agilent 6890N gas chromatograph (GC/MS) equipped with an Agilent 5975 mass spectrometer liquid sample. To speed up the run time, the temperature program and column matrix were modified to achieve optimal peak resolution and shortest total run time. A 1 uL sample was separated using an HP-5MS column (Agilent Technologies Inc., Palo Alto, CA) and a helium carrier gas. The temperature program for the analysis was as follows: hold at 150°C for 3 minutes, ramp at 25°C/min to a temperature of 200°C, ramp at 60°C/min to a temperature of 300°C, and hold at 300°C for 1 minute. Previous mass spectrometry demonstrated that the product of β-farnesene synthase was β-farnesene, and using this GC method, β-farnesene had a retention time of 4.33 minutes. Farnesene titers were calculated using the generated peak areas compared to a quantitative calibration curve of pure β-farnesene (Sigma-Aldrich Chemical Company, St. Louis, MO) in trans-caryophyllene-standardized ethyl acetate .

At 120 hours, host strain B592 produced about 400 mg/L of α-farnesene (average over 3 independent clones), and the maximum specific productivity was about 46 mg/L/OD ₆₀₀ . At 120 hours, host strain B552 produced approximately 1.1 g/L of β-farnesene (average over 3 independent clones), and the maximum specific productivity was approximately 96 mg/L/ _OD600 (1 representative clone).

Example 20

This example describes the production of β-farnesene in E. coli host bacteria via the DXP pathway.

Stock aliquots of each strain were added to separate 125 mL vials containing 25 mL of M9-MOPS and antibiotics as detailed in Table 1, and the cultures were allowed to grow overnight to establish the Inoculate the culture.

The inoculum culture was inoculated at an initial _OD600 value of about 0.05 in a medium containing 40mL M9-MOPS minimal medium, 45ug/mL thiamine, micronutrients, 1.00E-5mol/LFeSO ₄ , 0.1M MOPS, 0.5% yeast Extract, 20g/L D-glucose, and antibiotics in separate 250mL bottles. Cultures were grown at 30 °C in a humidified culture shaker at 250 rpm until they reached an _OD600 value of 0.2 to 0.3, at which point β-actin was induced in the host cells by adding 40 uL of 1M IPTG to the medium. Nescene. At the time of induction, β-farnesene was harvested by overlaying the culture with 8 mL of an organic overlay. Samples were taken at various time points, transferring 100 uL samples of the upper organic overlay to clean tubes. The tubes were centrifuged to separate any residual cells or medium, and a 10 uL sample of the organic overlay was transferred to a clean glass GC vial containing 500 uL ethyl acetate calibrated with β- or trans-caryophyllene as an internal standard. The mixture was shaken for 30 seconds and then analyzed as described in Example 18. Escherichia coli host strain B653 produced about 7 mg/g DCW of β-farnesene.

Example 21

This example describes the production of α-farnesene or β-farnesene in a Saccharomyces cerevisiae host.

Strain EPY300 was prepared by removing the expression plasmid from S. cerevisiae strain EPY224 (Ro et al. (2006) Nature 440:940-943; PCT Patent Publication No. WO2007/005604) by culturing in nutrient-rich medium. Then the expression plasmid pRS425-FSA or pR425-FSB was used to transform the strain EPY300 to produce host bacteria Y166 and Y164, respectively.

Host cell transformants were selected on synthetic defined medium (SM-glu) containing 2% glucose and all amino acids except leucine. The host strain EPY300 was auxotrophic for leucine biosynthesis (Ieu2), but the expression plasmid pRS425-FSA or pRS425-FSB restored leucine prototrophy (LEU2). Single clones were transferred to culture flasks containing 5 mL of liquid SM-glu lacking leucine. Cultures were grown at 30°C with shaking until growth reached stationary phase. Cells were maintained in 1 mL stock aliquots consisting of 400 μL sterile 50% glycerol and 600 μL liquid medium in cryovials at -80°C.

The stock aliquot was added to a 125 mL bottle containing 25 mL of leucine-deficient SM-glu and the culture was grown overnight to establish an inoculum culture. The inoculum culture was inoculated at an initial _OD600 value of approximately 0.05 in 250 mL baffled flasks containing 40 mL of synthetic defined medium lacking leucine, 0.2% glucose, and 1.8% galactose. Cultures were grown at 30°C on a shaker at 200 rpm. Because the presence of glucose in the medium prevented the induction of the Gal1 promoter by galactose, farnesene production was not induced until the cells depleted the glucose in the medium and switched to using galactose as their primary carbon source. The culture was overlaid with 8 mL of methyl oleate or isopropyl myristate. Samples were taken every 24 hours, and 2-10 uL of the organic solvent layer was transferred to a clean glass vial containing 500 uL of ethyl acetate containing a known concentration of β- or trans-caryophyllene as an internal standard. In addition, 0.5 mL aliquots of the whole broth were added to glass vials containing 1 mL ethyl acetate calibrated with trans-caryophyllene as internal standard. The entire broth sample was extracted with ethyl acetate by shaking the glass vial for 10 minutes, after which time 600 uL of the ethyl acetate extract was transferred to a clean glass vial.

At 120 hours, host strain Y166 produced approximately 9.8 mg/L of α-farnesene (average over 3 independent clones), and the maximum specific productivity was approximately 3 mg/L/ _OD600 (1 representative clone). At 120 hours, host strain Y164 produced approximately 56 mg/L of β-farnesene (average over 3 independent clones), and the maximum specific productivity was approximately 20 mg/L/ _OD600 (1 representative clone).

Example 22

This example describes the production of gamma-terpinene, alpha-pinene and terpinolene in an E. coli host.

Stock aliquots of each strain were added to separate 125 mL bottles containing 25 mL of LM9-MOPS, 2% glucose, 0.5% yeast extract, and antibiotics as detailed in Table 1, and the cultures were grown overnight to In the late stages, the production of γ-terpinene (Escherichia coli DH1-Tlr[pMevT, pMevB-Gpps, pAM445]), α-pinene (Escherichia coli DH1-Tlr[pMevT, pMevB-Gpps, pAM443 or pAM442]) was established , or terpinolene (Escherichia coli DH1-Tlr [pMevT, pMevB-Gpps, pAM444] host bacteria inoculation culture.

The inoculum culture was inoculated at an initial _OD600 value of approximately 0.05 in a 250 mL bottle containing 40 mL of M9-MOPS, 2% glucose, 0.5% yeast extract, and antibiotics. At the time of inoculation, the culture was also overlaid with 4 mL of hexadecane. The culture was grown at 30°C on a shaker at 200-250 rpm until it reached an _OD600 value of approximately 0.2, at which point production of the compound of interest to the host cell was induced in the host cell by adding 40 uL of 1M IPTG. For 96 hours, samples were taken once a day, and 200 uL of the hexadecane layer was transferred to a 0.6 mL microcentrifuge tube. For analysis, the hexadecane overlay was diluted 1:1 or 1:10 in 1.8 mL GC vials with ethyl acetate standardized with trans-caryophyllene as internal standard. Additionally, a 1 mL aliquot of the culture was spun down, the pellet was resuspended in 250 uL sterile water, and the cell suspension was transferred to a glass vial containing 1 mL ethyl acetate calibrated with trans-caryophyllene as an internal standard. The cell pellet was extracted with ethyl acetate by shaking the vial for 15 minutes, followed by transferring 500 uL of the ethyl acetate extract to a clean glass vial.

Analysis of hexadecane/ethyl acetate samples and ethyl acetate extracted cell pellets in full scan mode (50-500m/z) on an Agilent 6890N gas chromatograph (GC/MS) equipped with an Agilent 5975 mass spectrometer sample. To speed up the run time, the temperature program and column matrix were modified to achieve optimal peak resolution and shortest total run time. 1 μL of the sample was split (split ratio selected between 1:2 and 1:50 based on sample concentration) and then separated using an HP-5MS column (Agilent Technologies, Palo Alto, CA) with a helium carrier gas. The temperature program for the analysis was as follows: hold at 75°C for 3 minutes, ramp at 20°C/min to a temperature of 115°C, ramp at 60°C/min to a temperature of 300°C, and hold at 300°C for 0.5 minutes. The various products, γ-terpinene, α-pinene, and terpinolene, were observed at 5.4, 4.1, 5.4, and 5.9 minutes, respectively. Titers were calculated using the resulting peak areas against a quantitative calibration curve of pure standards in trans-caryophyllene spiked ethyl acetate.

Example 23

This example describes the production of linalool, limonene, β-pinene, β-phellandrene, carene, or juniper in an E. coli host.

Stock aliquots of each strain were added to separate 125 mL bottles containing 25 mL of M9-MOPS, 0.5% yeast extract, 2% glucose, and antibiotics as detailed in Table 1, and the cultures were allowed to grow overnight to establish inoculum. Cultures.

The inoculum culture was inoculated in a 250 mL baffled bottle containing 40 mL of M9-MOPS, 0.5% yeast extract, 2% glucose, and antibiotics at an initial _OD600 value of approximately 0.05. The culture was grown at 30° C. on a shaker at 250 rpm until it reached an OD ₆₀₀ value of approximately 0.2, at which point production of the compound of interest in the host cells was induced by adding 40 uL of 1 M IPTG to the medium. Isolate the compound of interest from the medium by solvent-solvent extraction or, if the titer of the compound of interest is large enough to saturate the medium and form a second phase, by precipitation and decantation .

sequence listing

SEQ ID NO: 1

MevT66 operon

GAATTCAAAGGAGGAAAATAAAATGAAGAACTGTGTGATTGTTTCTGCGGTCCG

CACGGCGATCGGCAGCTTTAACGGCTCTTTAGCGAGCACCTCTGCAATCGATCTG

GGTGCGACGGTCATTAAGGCCGCCATTGAACGCGCCAAAATCGACAGCCAGCAC

GTTGATGAGGTGATCATGGGCAATGTGTTACAAGCCGGCCTGGGTCAAAACCCA

GCGCGTCAAGCACTGTTAAAATCTGGTCTGGCCGAGACCGTGTGTGGCTTCACCG

TCAATAAGGTTTGCGGCTCTGGCCTGAAGAGCGTGGCCCTGGCAGCACAAGCGA

TTCAAGCCGGTCAGGCACAAAGCATCGTTGCGGGTGGCATGGAGAACATGTCTC

TGGCGCCGTACTTATTAGATGCCAAAGCCCGCAGCGGTTATCGCCTGGGCGATGG

TCAGGTGTACGACGTCATCTTACGCGATGGCTTAATGTGCGCGACCCACGGTTAC

CACATGGGTATTACGGCCGAAAACGTGGCGAAAGAATACGGCATTACGCGCGAG

ATGCAGGATGAATTAGCACTGCACTCTCAGCGCAAAGCAGCAGCCGCGATCGAG

TCTGGTGCGTTTACGGCGGAAATCGTGCCAGTTAACGTGGTCACGCGCAAGAAG

ACGTTCGTTTTTCAGCCAGGACGAGTTCCCGAAGGCAAACAGCACCGCGGAGGCC

TTAGGTGCCTTACGCCCAGCCTTTGACAAAGCGGGCACGGTCACCGCCGGTAATG

CGAGCGGCATCAATGATGGTGCAGCGGCACTGGTCATCATGGAAGAGAGCGCCG

CATTAGCAGCGGGTCTGACCCCATTAGCGCGCATTAAATCTTATGCCAGCGGCGG

CGTCCCACCAGCCCTGATGGGCATGGGTCCGGTCCCAGCCACGCAAAAAGCCCT

GCAATTAGCGGGCCTGCAACTGGCCGACATTGATCTGATCGAGGCGAACGAGGC

GTTTGCAGCGCAGTTCCTGGCGGTGGGTAAGAATCTGGGCTTCGACAGCGAGAA

AGTCAATGTGAACGGTGGCGCGATTGCGTTAGGCCATCCGATTGGTGCAAGCGG

CGCACGCATCTTAGTGACGTTACTGCACGCCATGCAGGCACGCGACAAGACCTTA

GGCCTGGCGACCTTATGTATTGGTGGCGGTCAAGGTATCGCCATGGTGATCGAAC

GCCTGAACTGAAGATCTAGGAGGAAAGGAAAATGAAACTGAGCACCAAGCTGTG

CTGGTGTGGCATCAAGGGTCGCCTGCGCCCACAAAAGCAGCAACAGCTGCACAA

CACGAACCTGCAAATGACCGAGCTGAAAAAGCAGAAGACGGCCGAGCAAAAGA

CCCGCCCGCAGAACGTTGGCATCAAGGGCATCCAGATTTATATCCCGACGCAGTG

TGTCAACCAATCTGAGCTGGAGAAATTCGATGGCGTCAGCCAGGGTAAGTACAC

CATCGGCCTGGGCCAGACCAACATGAGCTTCGTGAACGACCGTGAGGACATCTA

TTCTATGAGCCTGACGGTGCTGTCTAAGCTGATCAAAGAGCTACAACATCGACACG

AATAAGATCGGTCGTCTGGAGGTGGGTACGGAGACGCTGATTGACAAGAGCAAA

AGCGTGAAGTCTGTCTTAATGCAGCTGTTCGGCGAGAACACGGATGTCGAGGGT

ATCGACACCCTGAACGCGTGTTACGGCGGCACCAACGCACTGTTCAATAGCCTGA

ACTGGATTGAGAGCAACGCCTGGGATGGCCGCGATGCGATCGTCGTGTGCGGCG

ATATCGCCATCTATGACAAGGGTGCGGCACGTCCGACCGGCGGTGCAGGCACCG

TTGCGATGTGGATTGGCCCGGACGCACCAATTGTCTTCGATTCTGTCCGCGCGTC

TTACATGGAGCACGCCTACGACTTTTACAAGCCGGACTTCACGAGCGAATACCCG

TACGTGGACGGCCACTTCTCTGACCTGCTATGTGAAGGCGCTGGACCAGGTTT

ATAAGTCTTATAGCAAAAAGGCGATTTCTAAGGGCCTGGTCAGCGACCCGGCAG

GCAGCGACGCCCTGAACGTGCTGAAGTATTTCGACTACAACGTGTTCCATGTCCC

GACCTGCAAATTAGTGACCAAATCTTATGGCCGCCTGTTATATAATGATTTCCGT

GCCAACCCGCAGCTGTTCCCGGAGGTTGACGCCGAGCTGGCGACGCGTGATTAC

GACGAGAGCCTGACCGACAAGAACATCGAGAAGACCTTCGTCAACGTCGCGAAG

CCGTTCCACAAAGAGCGTGTGGCCCAAAAGCCTGATCGTCCCGACCAACACGGGC

AACATGTATACCGCGTCTGTCTACGCGGCATTCGCGAGCCTGCTGAATTACGTCG

GTTCTGACGACCTGCAGGGCAAGCGCGTTGGCCTGTTCAGCTACGGTAGCGGCTT

AGCGGCCAGCCTGTATAGCTGCAAAATTGTCGGCGACGTCCAGCACATCATCAA

GGAGCTGGACATCACCAACAAGCTGGCGAAGCGCATCACCGAGACGCCGAAAG

ATTACGAGGCAGCGATCGAGTTACGCGAGAATGCGCATCTGAAGAAGAACTTCA

AGCCGCAAGGTAGCATCGAGCACCTGCAGAGCGGCGTCTACTACCTGACGAACA

TTGACGACAAGTTCCGCCGTTCTTATGACGTCAAAAAGTAACTAGTAGGAGGAA

AACATCATGGTGCTGACGAACAAAACCGTCATTAGCGGCAGCAAGGTGAAGTCT

CTGAGCAGCGCCCAAAGCTCTAGCAGCGGCCCGTCTAGCAGCAGCGAGGAGGAC

GACAGCCGTGACATTGAGTCTCTGGACAAGAAGATCCGCCCGCTGGAGGAGTTA

GAGGCCCTGCTGAGCAGCGGCAACACCAAGCAGCTGAAGAACAAGGAAGTTGC

AGCGCTGGTGATCCACGGTAAGCTGCCACTGTATGCGCTGGAAAAGAAACTGGG

CGATACGACGCGTGCGGTCGCGGTGCGTCGCAAAGCCTTAAGCATCTTAGCGGA

GGCCCCGGTGTTAGCCAGCGACCGCCTGCCGTACAAGAACTACGACTACGACCG

CGTGTTTGGCGCGTGCTGCGAGAATGTCATTGGCTACATGCCGTTACCGGTTGGT

GTGATCGGCCCGCTGGTCATTGATGGCACGAGCTATCACATTCCAATGGCGACCA

CGGAAGGTTGCTTAGTCGCCAGCGCCATGCGTGGCTGTAAGGCGATTAACGCCG

GCGGTGGCGCGACGACCGTGTTAACCAAGGATGGTATGACGCGCGGTCGGGTCG

TCCGCTTCCCAACGCTGAAGCGCAGCGGCGCGTGTAAGATTTGGCTGGATTCTGA

GGAGGGCCAAAACGCGATCAAGAAAGCCTTCAACTCTACGAGCCGTTTCGCGCG

TTTACAGCATATCCAGACCTGCCTGGCCGGCGACCTGCTGTTCATGCGCTTCCGC

ACCACCACGGGCGATGCGATGGGCATGAACATGATCAGCAAGGGCGTCGAATAT

AGCCTGAAACAAATGGTGGAAGAATATGGCTGGGAGGACATGGAGGTTGTCTCT

GTGAGCGGCAACTATTGCACCGACAAGAAGCCGGCAGCCATTAACTGGATTGAG

GGTCGCGGCAAAAGCGTCGTGGCAGAAGCGACCATCCCAGGCGACGTGGTCCGT

AAGGTTCTGAAGAGCGACGTCAGCGCCCTGGTTGAGTTAAATATCGCGAAAAAC

CTGGTCGGCAGCGCGATGGCGGGCAGCGTGGGTGGCTTTAACGCACATGCAGCG

AATCTGGTTACGGCGGTTTTTCTTAGCCTTAGGTCAGGACCCAGCCCAAAATGTCG

AGAGCAGCAACTGCATTACCTTAATGAAAGAGGTTGACGGTGACCTGCGCATCA

GCGTTTCTATGCCGTCTATCGAGGTCGGCACGATCGGCGGCGGCACCGTTTTAGA

ACCGCAAGGTGCGATGCTGGATCTGCTGGGCGTGCGCGGCCCACATGCAACGGC

CCCAGGCACCAATGCCCGCCAACTGGCCCGTATCGTGGCCTGCGCGGTTCTGGCG

GGTGAGCTGAGCCTGTGCGCCGCATTAGCCGCGGGCCATTTAGTTCAATCTCACA

TGACCCACAACCGCAAGCCGGCAGAACCAACCAAGCCAAATAACCTGGACGCAA

CCGACATTAACCGTCTGAAGGATGGCAGCGTCACGTGCATTAAAAGCTGAGCAT

GCTACTAAGCTT

SEQ ID NO: 2

Primer 4-49 mvaA SpeI

5'-GCTACTAGTAGGAGGAAAACATCATGCAAAGTTAGATAAGAATTTCCG-3'

SEQ ID NO: 3

Primer 4-49 mvaAR XbaI

5'-GCTTCTAGACTATTGTTGTCTAATTTCTTGTAAAATGCG-3'

SEQ ID NO: 4

Primer HMGS 5′SamvaS-S

5'-

GAACTGAAGATCTAGGAGGAAAGCAAAATGACAATAGGTATCGACAAAATAAACT-3’

SEQ ID NO: 5

Primer HMGS 3′SamvaS-AS

5'-TTGCATGATGTTTTCCTCCTACTAGTTACTCTGGTCTGTGATATTCGCGAAC-3'

SEQ ID NO: 6

Primer 1 9-25 atoB SfiI-S

5'-GCTAGGCCATCCTGGCCATGAAGAACTGTGTGATTGTTTCTG-3'

SEQ ID NO: 7

Primer 1 9-25 mvaA-AsiSI-AS

5'-GCTTGCGATCGCCGGCGGATTTGTCCTACTCAG-3'

SEQ ID NO: 8

Primer 9-70C

5'-CCACCTCGAGATGTCATTACCGTTCTTAACTTCTG-3'

SEQ ID NO: 9

Primer 26-39B

5'-TGGTGGAGCCTCTTATTTAAGCTGGGTAAATGCAGATAATCG-3'

SEQ ID NO: 10

Primer 26-39A

5'-TTCTTGAGCTCTTATTCCTTTGGTAGACCAGTCTTTGCG-3'

SEQ ID NO: 11

Primer 4-40 mvaEF BamHI

5'-TATGGATCCTAAGGAGGATATTTAGATGAAAACAGTAGTTATTATTGATGC-3'

SEQ ID NO: 12

Primer 4-40 mvaER HindIII

5'-AGCTAAGCTTTTATTGTTTTTCTTAAATCATTTAAAATAGC-3'

SEQ ID NO: 13

Primer 4-40 mvaSF BglII

5'-TATAGATCTTAAGGAGGATATTTAGATGACAATTGGGATTGATAAAATTAG-3'

SEQ ID NO: 14

Primer 4-39mvaSRBamHI

5'-TTTGGATCCTTAGTTTCGATAAGAGCGAACGG-3'

SEQ ID NO: 15

Primers 67-1A-C for PCR amplification of the coding sequence of the dxs gene

5′-ACA CTC GAG GAG GAA TAA ATG AGT TTT GAT ATT GCC AAA TAC CCG-3’

SEQ ID NO: 16

Primer 67-1B-C for PCR amplification of the coding sequence of the dxs gene

5’-TGA TGG TAC CTT ATG CCA GCC AGG CCT TGA TTT TGG C-3’

SEQ ID NO: 17

Primer 67-1 C-C for PCR amplification of the coding sequence of the dxr gene

5’-ACT AGG TAC CAG GAG GAA TAA ATG AAG CAA CTC ACC ATT CTG GGC -3′

SEQ ID NO: 18

Primer 67-1D-C for PCR amplification of the coding sequence of the dxr gene

5′-AATTGA TGG GCC CTC AGC TTG CGA GAC GCA TCA CCT C-3’

SEQ ID NO: 19

Primer 67-1E-C for PCR amplification of the coding sequence of the ispD gene

5′-CAT AAA GGG CCC AGG AGG AAT AAA TGG CAA CCA CTC ATT TGG ATG -3’

SEQ ID NO: 20

Primer 67-1F-C for PCR amplification of the coding sequence of the ispD gene

5’-TAT TGT TCA TAT GTT ATG TAT TCT CCT GAT GGA TGG TTC G-3’

SEQ ID NO: 21

Primer 67-1G-C for PCR amplification of the coding sequence of the ispE gene

5’-AAC TAA CAC ATA TGA GGA GGA ATA AAT GCG GAC ACA GTG GCC CTC -3’

SEQ ID NO: 22

Primer 67-1H-C for PCR amplification of the coding sequence of the ispE gene

5’-TGT TAG TTA CGC GTT TAA AGC ATG GCT CTG TGC AAT GG -3’

SEQ ID NO: 23

Primers 67-2A-C for PCR amplification of the coding sequence of the ispF gene

5’-ACG GGA TCC AGG AGG AAT AAA TGC GAA TTG GAC ACG GTT TTG ACG-3′

SEQ ID NO: 24

Primer 67-2B-C for PCR amplification of the coding sequence of the ispF gene

5′-TTT AGT TGG GCC CTC ATT TTG TTG CCT TAA TGA GTA GCG CC-3’

SEQ ID NO: 25

Primer 67-2C-C for PCR amplification of the coding sequence of the ispG gene

5′-TAC TAA GGG CCC AGG AGG AAA TAA TGC ATA ACC AGG CTC CAA TTCAAC G-3’

SEQ ID NO: 26

Primer 67-2D-C for PCR amplification of the coding sequence of the ispG gene

5’-TCC GGG TAC CTT ATT TTT CAA CCT GCT GAA CGT CAA TTC G -3′

SEQ ID NO: 27

Primer 67-2E-C for PCR amplification of the coding sequence of the ispH gene

5’-AAC AGG TAC CAG GAG GAA ATA ATG CAG ATC CTG TTG GCC AAC C-3′

SEQ ID NO: 28

Primer 67-2F-C for PCR amplification of the coding sequence of the ispH gene

5′-TGG ATG AAG TCG ACT TAA TCG ACT TCA CGA ATA TCG ACA CGC AGC -3’

SEQ ID NO: 29

Primer 67-2G-C for PCR amplification of the coding sequence of the idi gene

5′-CAT CAA GTC GAC AGG AGG AAA TAA TGC AAA CGG AAC ACG TCA TTTTAT TG-3’

SEQ ID NO: 30

Primer 67-2H-C for PCR amplification of the coding sequence of the idi gene

5′-TAA TGC AAG CTT ATT TAA GCT GGG TAA ATG CAG ATA ATC G -3’

SEQ ID NO: 31

Primer 67-2I-C for PCR amplification of the coding sequence of the ispA gene

5′-CAG TAA AGC TTA GGA GGA AAT AAT GGA CTT TCC GCA GCA ACT CG-3′

SEQ ID NO: 32

Primer 67-2J-C for PCR amplification of the coding sequence of the ispA gene

5′-TAG TTC CAT GGT TAT TTA TTA CGC TGG ATG ATG TAG TCC GC-3’

SEQ ID NO: 33

Primer 9-156A for PCR amplification of RK2 par site

5'-ACATAGACGTCGGGAAAGCGAGGATCTAGGTAGGG-3'

SEQ ID NO: 34

Primer 9-156B for PCR amplification of RK2 par site

5'-TTCCCGCTCGAGGTGGCGGACCATAGGCAGATCAG-3'

SEQ ID NO: 35

Primer 19-137cml-pAM37-AS

5'-GACGTCGATATCTGGCGAAAATG-3'

SEQ ID NO: 36

Primer 19-137cml-pAM37-S

5'-TACTAGTGCTTGGATTCTCACC-3'

SEQ ID NO: 37

PCR Amplification Primer of Nucleotide Sequence Encoding β-Farnesene Synthase

5'-CCATG GAC ACTCTGCCGATCTCTTCCGTAAGC -3'

SEQ ID NO: 38

PCR Amplification Primer of Nucleotide Sequence Encoding β-Farnesene Synthase

5'-GAGCTCTCATACGACCATAGGGTGTACG-3'

SEQ ID NO: 39

Primers for PCR amplification of nucleotide sequence encoding α-farnesene synthase

5'-CCATGGACCTGGCAGTAGAAATTGC-3'

SEQ ID NO: 40

Primers for PCR amplification of nucleotide sequence encoding α-farnesene synthase

5'-GAGCTCTTACATCGGTACCGGCTCCAG-3'

SEQ ID NO: 41

atoB(opt): HMGS(opt): mvaA operon

ATGAAGAACTGTGTGATTGTTTCTGCGGTCCGCACGGCGATCGGCAGCTTTAACG

GCTCTTTAGCGAGCACCTCTGCAATCGATCTGGGTGCGACGGTCATTAAGGCCGC

CATTGAACGCGCCAAAATCGACAGCCAGCACGTTGATGAGGTGATCATGGGCAA

TGTGTTACAAGCCGGCCTGGGTCAAAAACCCAGCGCGTCAAGCACTGTTAAAATCT

GGTCTGGCCGAGACCGTGTGTGGCTTCACCGTCAATAAGGTTTGCGGCTCTGGCC

TGAAGAGCGTGGCCCTGGCAGCACAAGCGATTCAAGCCGGTCAGGCACAAAGCA

TCGTTGCGGGTGGCATGGAGAACATGTCTCTGGCGCCGTACTTATTAGATGCCAA

AGCCCGCAGCGGTTATCGCCTGGGCGATGGTCAGGTGTACGACGTCATCTTACGC

GATGGCTTAATGTGCGCGACCCACGGTTACCACATGGGTATTACGGCCGAAAAC

GTGGCGAAAGAATACGGCATTACGCGCGAGATGCAGGATGAATTAGCACTGCAC

TCTCAGCGCAAAGCAGCAGCCGCGATCGAGTCTGGTGCGTTTACGGCGGAAATC

GTGCCAGTTAACGTGGTCACGCGCAAGAAGACGTTCGTTTTTCAGCCAGGACGAG

TTCCCGAAGGCAAACAGCACCGCGGAGGCCTTAGGTGCCTTACGCCCAGCCTTTG

ACAAAGCGGGCACGGTCACCGCCGGTAATGCGAGCGGCATCAATGATGGTGCAG

CGGCACTGGTCATCATGGAAGAGAGCGCCGCATTAGCAGCGGGTCTGACCCCAT

TAGCGCGCATTAAATCTTATGCCAGCGGCGGCGTCCCACCAGCCCTGATGGGCAT

GGGTCCGGTCCCAGCCACGCAAAAAGCCCTGCAATTAGCGGGCCTGCAACTGGC

CGACATTGATCTGATCGAGGCGAACGAGGCGTTTGCAGCGCAGTTCCTGGCGGT

GGGTAAGAATCTGGGCTTCGACAGCGAGAAAGTCAATGTGAACGGTGGCGCGAT

TGCGTTAGGCCATCCGATTGGTGCAAGCGGCGCACGCATCTTAGTGACGTTACTG

CACGCCATGCAGGCACGCGACAAGACCTTAGGCCTGGCGACCTTATGTATTGGTG

GCGGTCAAGGTATCGCCATGGTGATCGAACGCCTGAACTGAAGATCTAGGAGGA

AAGCAAAATGAAACTGAGCACCAAGCTGTGCTGGTGTGGCATCAAGGGTCGCCT

GCGCCCACAAAAGCAGCAACAGCTCACAACACGAACCTGCAAATGACCGAGCT

GAAAAAGCAGAAGACGGCCGAGCAAAAGACCCGCCCGCAGAACGTTGGCATCA

AGGGCATCCAGATTTATATCCCGACGCAGTGTGTCAACCAATCTGAGCTGGAGA

AATTCGATGGCGTCAGCCAGGGTAAGTACCACCATCGGCCTGGGCCAGACCAACA

TGAGCTTCGTGAAGGACCGTGAGGACATCTATTCTATGAGCCTGACGGTGCTGTC

TAAGCTGATCAAGAGCTACAACATCGACACGAATAAGATCGGTCGTCTGGAGGT

GGGTACGGAGACGCTGATTGACAAGAGCAAAAGCGTGAAGTCTGTCTTAATGCA

GCTGTTCGGCGAGAACACGGATGTCGAGGGTATCGACACCCTGAACGCGTGTTA

CGGCGGCACCAACGCACTGTTCAATAGCCTGAACTGGATTGAGAGCAACGCCTG

GGATGGCCGCGATGCGATCGTCGTGTGCGGCGATATCGCCATCTATGACAAGGG

TGCGGCACGTCCGACCGGCGGTGCAGGCACCGTTGCGATGTGGATTGGCCCGGA

CGCACCAATTGTCTTCGATTCTGTCCGCGCGTCTTACATGGAGCACGCCTACGAC

TTTTACAAGCCGGACTTCACGAGCGAATACCCGTACGTGGACGGCCACTTCTCTC

TGACCTGCTATGTGAAGGCGCTGGACCAGGTTTATAAGTCTTATAGCAAAAAGGC

GATTTCTAAGGGCCTGGTCAGCGACCCGGCAGGCAGCGACGCCCTGAACGTGCT

GAAGTATTTCGACTACAACGTGTTCCATGTCCCGACCTGCAAATTAGTGACCAAAA

TCTTATGGCCGCCTGTTATATAATGATTTCCGTGCCAACCCGCAGCTGTTCCCGGA

GGTTGACGCCGAGCTGGCGACGCGTGATTACGACGAGAGCCTGACCGACAAGAA

CATCGAGAAGACCTTCGTCAACGTCGCGAAGCCGTTCCACAAAGAGCGTGTGGC

CCAAAGCCTGATCGTCCCGACCAACACGGGCAACATGTATACCGCGTCTGTCTAC

GCGGCATTCGCGAGCCTGCTGAATTACGTCGGTTCTGACGACCTGCAGGGCAAGC

GCGTTGGCCTGTTCAGCTACGGTAGCGGCTTAGCGGCCAGCCTGTATAGCTGCAA

AATTGTCGGCGACGTCCAGCACATCATCAAGGAGCTGGACATCACCAACAAGCT

GGCGAAGCGCATCACCGAGACGCCGAAAGATTACGAGGCAGCGATCGAGTTACG

CGAGAATGCGCATCTGAAGAAGAACTTCAAGCCGCAAGGTAGCATCGAGCACCT

GCAGAGCGGCGTCTACTACCTGACGAACATTGACGACAAGTTCCGCCGTTCTTAT

GACGTCAAAAAGTAACTAGTAGGAGGAAAACATCATGCAAAGTTTAGATAAGAA

TTTCCGACATTTATCTCGTCAACAAAAGTTACAACAATTGGTAGATAAGCAATGG

TTATCAGAAGATCAATTCGACATTTTATTGAATCATCCATTAATTGATGAGGAAG

TAGCAAATAGTTTAATTGAAAATGTCATCGCGCAAGGTGCATACCCGTTGGATT

ATTACCGAATATCATTGTGGACGATAAGGCATATGTTGTACCTATGATGGTGGAA

GAGCCTTCAGTTGTCGCTGCAGCTAGTTATGGTGCAAAGCTAGTGAATCAGACTG

GCGGATTTAAAACGGTATCTTCTGAACGTATTATGATAGGTCAAATCGTCTTGA

TGGCGTTGACGATACTGAAAAATTATCAGCAGACATTAAAGCTTTAGAAAAGCA

AATTCATAAAATTGCGGATGAGGCATATCTTCTATTAAAGCGCGTGGTGGTGGT

TACCAACGTATAGCTATTGATACATTTCCTGAGCAACAGTTACTATTCTTTAAAAG

TATTTGTTGATACGAAAGATGCTATGGGCGCTAATATGCTTAATACGATTTTAGA

GGCCATAACTGCATTTTTTAAAAAATGAATCTCCACAAAGCGACATTTTAATGAGT

ATTTTATTCCAATCATGCAACAGCGTCCGTTGTTAAAGTTCAAGGCGAAATTGACG

TTAAAGATTTAGCAAGGGGCGAGAGAACTGGAGAAGAGGTTGCCAAACGAATG

GAACGTGCTTCTGTATTGGCACAAGTTGATATTCATCGTGCTGCAACACATAATA

AAGGTGTTATGAATGGCATACATGCCGTTGTTTTAGCAACAGGAAATGATACGCG

TGGTGCAGAAGCAAGTGCGCATGCATACGCGAGTCGTGACGGACAGTATCGTGG

TATTGCAACATGGAGATACGATCAAAAACGTCAACGTTTAATTGGTACAATAGA

AGTGCCTATGACATTGGCAATCGTTGGCGGTGGTACAAAAGTATTACCAATTGCT

AAAGCTTCTTTAGAATTGCTAAATGTAGATTCAGCACAAGAATTAGGTCATGTAG

TTGCTGCCGTTGGTTTAGCACAGAACTTTGCAGCATGTCGCGCGCTCGTTTCCGA

AGGTATCCAGCAAGGCCATATGAGCTTGCAATATAAATCTTTAGCTATTGTTGTA

GGTGCAAAAGGTGATGAAATTGCGCAAGTAGCTGAAGCATTGAAGCAAGAACCC

CGTGCGAATACACAAGTAGCTGAACGCATTTTACAAGAAATTAGACAACAATAG

SEQ ID NO: 42

atoB(opt):mvaS(opt):mvaA operon

ATGAAGAACTGTGTGATTGTTTCTGCGGTCCGCACGGCGATCGGCAGCTTTAACG

GCTCTTTAGCGAGCACCTCTGCAATCGATCTGGGTGCGACGGTCATTAAGGCCGC

CATTGAACGCGCCAAAATCGACAGCCAGCACGTTGATGAGGTGATCATGGGCAA

TGTGTTACAAGCCGGCCTGGGTCAAAAACCCAGCGCGTCAAGCACTGTTAAAATCT

GGTCTGGCCGAGACCGTGTGTGGCTTCACCGTCAATAAGGTTTGCGGCTCTGGCC

TGAAGAGCGTGGCCCTGGCAGCACAAGCGATTCAAGCCGGTCAGGCACAAAGCA

TCGTTGCGGGTGGCATGGAGAACATGTCTCTGGCGCCGTACTTATTAGATGCCAA

AGCCCGCAGCGGTTATCGCCTGGGCGATGGTCAGGTGTACGACGTCATCTTACGC

GATGGCTTAATGTGCGCGACCCACGGTTACCACATGGGTATTACGGCCGAAAAC

GTGGCGAAAGAATACGGCATTACGCGCGAGATGCAGGATGAATTAGCACTGCAC

TCTCAGCGCAAAGCAGCAGCCGCGATCGAGTCTGGTGCGTTTACGGCGGAAATC

GTGCCAGTTAACGTGGTCACGCGCAAGAAGACGTTCGTTTTTCAGCCAGGACGAG

TTCCCGAAGGCAAACAGCACCGCGGAGGCCTTAGGTGCCTTACGCCCAGCCTTTG

ACAAAGCGGGCACGGTCACCGCCGGTAATGCGAGCGGCATCAATGATGGTGCAG

CGGCACTGGTCATCATGGAAGAGAGCGCCGCATTAGCAGCGGGTCTGACCCCAT

TAGCGCGCATTAAATCTTATGCCAGCGGCGGCGTCCCACCAGCCCTGATGGGCAT

GGGTCCGGTCCCAGCCACGCAAAAAGCCCTGCAATTAGCGGGCCTGCAACTGGC

CGACATTGATCTGATCGAGGCGAACGAGGCGTTTGCAGCGCAGTTCCTGGCGGT

GGGTAAGAATCTGGGCTTCGACAGCGAGAAAGTCAATGTGAACGGTGGCGCGAT

TGCGTTAGGCCATCCGATTGGTGCAAGCGGCGCACGCATCTTAGTGACGTTACTG

CACGCCATGCAGGCACGCGACAAGACCTTAGGCCTGGCGACCTTATGTATTGGTG

GCGGTCAAGGTATCGCCATGGTGATCGAACGCCTGAACTGAAGATCTAGGAGGA

AAGCAAAATGACAATAGGTATCGACAAAATAAACTTTTACGTTCCAAAGTACTA

TGTAGACATGGCTAAATTAGCAGAAGCACGCCAAGTAGACCCAAACAAATTTTT

AATTGGAATTGGTCAAACTGAAATGGCTGTTAGTCCTGTAAACCAAGACATCGTT

TCAATGGGCGCTAACGCTGCTAAGGACATTATAACAGACGAAGATAAAAAGAAA

ATTGGTATGGTAATTGTGGCAACTGAATCAGCAGTTGATGCTGCTAAAGCAGCCG

CTGTTCAAATTCACAACTTATTAGGTATTCAACCTTTTGCACGTTGCTTTGAAATG

AAAGAAGCTTGTTATGCTGCAACACCAGCAATTCAATTAGCTAAAGATTATTTAG

CAACTAGACCGAATGAAAAAGTATTAGTTATTGCTACAGATACAGCACGTTATG

GATTGAATTCAGGCGGCGAGCCAACACAAGGTGCTGGCGCAGTTGCGATGGTTA

TTGCACATAATCCAAGCATTTTGGCATTAAATGAAGATGCTGTTGCTTACACTGA

AGACGTTATGATTTCTGGCGTCCAACTGGACATAAATATCCATTAGTTGATGGT

GCATTATCTAAAGATGCTTATATCCGCTCATTCCAACAAAGCTGGAATGAATACG

CAAAACGTCAAGGTAAGTCGCTAGCTGACTTCGCATCTCTATGCTTCCATGTTCC

ATTTACAAAAATGGGTAAAAAGGCATTAGAGTCAATCATTGATAACGCTGATGA

AACAACTCAAAGAGCGTTTACGTTCAGGATATGAAGATGCTGTAGATTATAACCGT

TATGTCGGTAATATTTTACTGGATCATTATATTTAAGCCTAATATCATTACTTGA

AAATCGTGATTTACAAGCTGGTGAAACAATCGGTTTTATTCAGTTATGGCTCAGGT

TCAGTTGGTGAATTTATAGTGCGACATTAGTTGAAGGCTACAAAGATCATTTAG

ATCAAGCTGCACATAAAAGCATTATTAAATAACCGTACTGAAGTATCTGTTGATGC

ATATGAAACATTCTTCAAACGTTTTGATGACGTTGAATTTGACGAAGAACAAGAT

GCTGTTCATGAAGATCGTCATATTTTCTACTTATCAAATATTGAAAATAACGTTCG

CGAATATCACAGACCAGAGTAACTAGTAGGAGGAAAACATCATGCAAAGTTTAG

ATAAGAATTTCCGACATTTATCTCGTCAAACAAAGTTACAAATTGGTAGATAA

GCAATGGTTATCAGAAGATCAATTCGACATTTATTGAATCATCCATTAATTGAT

GAGGAAGTAGCAAATAGTTTAATTGAAAATGTCATCGCGCAAGGTGCATTACCC

GTTGGATTATTACCGAATATCATTGTGGACGATAAGGCATATGTTGTACCTATGA

TGGTGGAAGAGCCTTCAGTTGTCGCTGCAGCTAGTTATGGTGCAAAGCTAGTGAA

TCAGACTGGCGGATTTAAAACGGTATCTTCTGAACGTATTATGATAGGTCAAATC

GTCTTTGATGGCGTTGACGATACTGAAAAATTATCAGCAGACATTAAAGCTTTAG

AAAAGCAAATTCATAAAATTGCGGATGAGGCATATCCTTTCTATTAAAGCGCGTG

GTGGTGGTTACCAACGTATAGCTATTGATACATTTCCTGAGCAACAGTTACTATC

TTTAAAAGTATTTGTTGATACGAAAGATGCTATGGGCGCTAATATGCTTAATACG

ATTTTAGAGGCCATAACTGCATTTTTTAAAAAATGAATCTCCACAAAGCGACATTT

TAATGAGTATTTTTATCCAATCATGCAACAGCGTCCGTTGTTAAAGTTCAAGGCGA

AATTGACGTTAAAGATTTAGCAAGGGGCGAGAGAACTGGAGAAGAGGTTGCCAA

ACGAATGGAACGTGCTTCTGTATTGGCACAAGTTGATATTCATCGTGCTGCAACA

CATAATAAAGGTGTTATGAATGGCATACATGCCGTTGTTTTAGCAACAGGAAATG

ATACGCGTGGTGCAGAAGCAAGTGCGCATGCATACGCGAGTCGTGACGGACAGT

ATCGTGGTATTGCAACATGGAGATACGATCAAAAACGTCAACGTTTAATTGGTAC

AATAGAAGTGCCTATGACATTGGCAATCGTTGGCGGTGGTACAAAAGTATTACC

AATTGCTAAAGCTCTTTAGAATTGCTAAATGTAGATTCAGCACAAGAATTAGGT

CATGTAGTTGCTGCCGTTGGTTTAGCACAGAACTTTGCAGCATGTCGCGCGCTCG

TTTCCGAAGGTATCCAGCAAGGCCATATGAGCTTGCAATATAAATCTTTAGCTAT

TGTTGTAGGTGCAAAAGGTGATGAAATTGCGCAAGTAGCTGAAGCATTGAAGCA

AGAACCCCGTGCGAATACACAAGTAGCTGAACGCATTTTACAAGAAATTAGACA

ACAATAG

SEQ ID NO: 43

pAM328-ERG9-KANMX-MET3 Promoter-ERG9 (vector backbone not included)

CAATACCGACTTACCATCCCTATTTGCTTTGCCCTTTTTTCTTTCCACTGCATGGCG

GCGTTAGTATCGAATGGATGGCGGCGTTAGTATCGAATCGACAGCAGTATAGCG

ACCAGCATTCACATACGATTGACGCATGATATTACTTTCTGCGCACTTAACTTCG

CATCTGGGCAGATGATGTCGAGGCGAAAAAAAATATAAATCACGCTAACATTTG

ATTAAAATAGAACAACTACAATATAAAAAAAACTATACAAATGACAAGTTCTTGA

AAACAAGAATCTTTTTATTGTCAGTACTGATTAGAAAAACTCATCGAGCATCAAA

TGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTGAAAAAGCC

GTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGAT

CCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTC

CCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAA

TCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCC

AGCCATTACGCTCGTCATCAAAAATCACTCGCATCAACCAAACCGTTATTCATTCG

TGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACA

AACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATT

TTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTGCCGGGGATC

GCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTC

GGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACAT

CATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTT

CCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCAT

TTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAAACGT

GAGTCTTTTCCTTACCCATGGTTGTTTATGTTCGGATGTGATGTGAGAACTGTATC

CTAGCAAGATTTTAAAAGGAAGTATATGAAAGAAGAACCTCAGTGGCAAATCCT

AACCTTTTATATTTCTCTACAAGGGGCGCGGCGTGGGGACAATTCAACGCGTCTGT

GAGGGGAGCGTTTTCCCTGCTCGCAGGTCTGCAGCGAGGAGCCGTAATTTTTGCTT

CGCGCCGTGCGGCCATCAAAATGTATGGATGCAAATGATTATACATGGGGATGT

ATGGGCTAAATGTACGGGCGACAGTCACATCATGCCCCTGAGCTGCGCACGTCA

AGACTGTCAAGGAGGGTATTCTGGGCCTCCATGTCGCTGGCCGGGTGACCCGGC

GGGGACGAGGCAAGCTAAACAGATCTGATCTTGAAACTGAGTAAGATGCTCAGA

ATACCCGTCAAGATAAGAGTATAATGTAGAGTAATATACCAAGTATTCAGCATAT

TCTCCTCTTCTTTTGTATAAATCACGGAAGGGATGATTTATAAGAAAAATGAATA

CTATTACACTTCATTTACCACCCTCTGATCTAGATTTTCCAACGATATGTACGTAG

TGGTATAAGGTGAGGGGGTCCACAGATATAACATCGTTTAATTTTAGTACTAACAG

AGACTTTTGTCACAACTACATATAAGTGTACAAATATAGTACAGATATGACACAC

TTGTAGCGCCAACGCGCATCCTACGGATTGCTGACAGAAAAAAAAGGTCACGTGA

CCAGAAAAGTCACGTGTAATTTTGTAACTCACCGCATTCTAGCGGTCCCTGTCGT

GCACACTGCACTCAACACCATAAACCTTAGCAACCTCCAAAGGAAATCACCGTA

TAACAAAGCCACAGTTTTTACAACTTAGTCTCTTATGAAGTTACTTACCAATGAGA

AATAGAGGCTCTTTCTCGAGAAATATGAATATGGATATATATATATATATATA

TATATATATATATATGTAAACTTGGTTCTTTTTTAGCTTGTGATCTCTAGCTTGGG

TCCTCTCTCTGTCGTAACAGTTGTGATATCGGCTGCCTTCATCTCGACCGGATGCAA

TGCCAATTGTAATAGCTTTCCCATGTTAATTATACTTTATTCTT

SEQ ID NO: 44

GAGTGAACCTGCTGCCTGGCGTGCTCTGACTCAGTACATTTCATAGTGGATGGCG

GCGTTAGTATC

SEQ ID NO: 45

CGTGTATACGTTTTCCGCTTCTGCTCTTCGTCTTTTCTCTTCTTCCGATATCACAAC

TGTT ACGA

SEQ ID NO: 46

GGTAAGACGGTTGGGTTTTTATCTTTTGCAGTTGGTACTATTAAGAACAATCACAG

GAAACAGCTATGACC

SEQ TD NO: 47

TTGCGTTTTGTACTTTGGTTCGCTCAATTTTGCAGGTAGATAATCGAAAAGTTGTA

AAACGACGGCCAGT

SEQ ID NO: 48

pAM491 sequence (excluding vector backbone)

GTTTAAACTTGCTAAATTCGAGTGAAACACAGGAAGACCAGAAAATCCTCATTTC

ATCCATATTAACAATAATTTCAAATGTTTTATTTGCATTATTTGAAACTAGGGAAG

ACAAGCAACGAAACGTTTTGAAAATTTTGAGTATTTTCAATAAATTTGTAGAGGA

CTCAGATATTGAAAAAAAGCTACAGCAATTAATACTTGATAAGAAGAGTATTGA

GAAGGGCAACGGTTCATCATCTCATGGATCTGCACATGAACAAACACCAGAGTC

AAACGACGTTGAAATTGAGGCTACTGCGCCAATTGATGACAATACAGACGATGA

TAACAAACCGAAGTTATCTGATGTAGAAAAGGATTAAAGATGCTAAGAGATAGT

GATGATATTTCATAAATAATGTAATTCTATATGTTAATTACCTTTTTTGCGAGG

CATATTTATGGTGAAGGATAAGTTTTGACCATCAAAGAAGGTTAATGTGGCTGTG

GTTTCAGGGTCCATACCCGGGAGTTATGACAATTACAACAACAGAATTCTTTCTA

TATATGCACGAACTTGTAATATGGAAGAAATTATGACGTACAAACTATAAAGTA

AATATTTTACGTAACACATGGTGCTGTTGTGCTTCTTTTTCAAGAGAATACCAATG

ACGTATGACTAAGTTTAGGATTTAATGCAGGTGACGGACCCATCTTTCAAACGAT

TTATATCAGTGGCGTCCAAATTGTTAGGTTTTGTTGGTTCAGCAGGTTTCCTGTTG

TGGGTCATATGACTTTGAACCAAATGGCCGGCTGCTAGGGCAGCACATAAGGAT

AATTCACCTGCCAAGACGGCACAGGCAACTATTCTTGCTAATTGACGTGCGTTGG

TACCAGGAGCGGTAGCATGTGGGCCTCTTACACCTAATAAGTCCAACATGGCACC

TTGTGGTTCTAGAACAGTACCACCACCGATGGTACCCTACTTCGATGGATGGCATG

GATACGGAAATTCTCAAATCACCGTCCACTTCTTTCATCAATGTTATACAGTTGG

AACTTTCGACATTTTGTGCAGGATCTTGTCCTAATGCCAAGAAAACAGCTGTCAC

TAAATTAGCTGCATGTGCGTTAAAATCCACCAACAGACCCAGCCATTGCAGATCCA

ACCAAATTCTAGCAATGTTCAACTCAACCAATGCGGAAACATCACTTTTTAACA

CTTTTCTGACAACATCACCAGGAATAGTAGCTTCTGCGACGACACTCTTACCACG

ACCTTCGATCCAGTTGATGGCAGCTGGTTTTTTGTCGGTACAGTAGTTACCAGAA

ACGGAGACAACCTCCATATCTTCCCAGCCATACTCTTCTACCATTTGCTTTAATGA

GTATTCGACACCCTTAGAAATCATATTCATACCCATTGCGTCACCAGTAGTTGTTC

TAAATCTCATGAAGAGTAAATCTCCTGCTAGACAAGTTTGAATATGTTGCAGACG

TGCAAATCTTGATGTAGAGTTAAAAGCTTTTTTAATTGCGTTTTGTCCCTCTTCTG

AGTCTAACCATATCTTACAGGCACCAGATCTTTTCAAAGTTGGGAAACGGACTAC

TGGGCCTCTTGTCATACCATCCTTAGTTAAAAACAGTTGTTGCACCACCGCCAGCA

TTGATTGCCTTACAGCCACGCATGGCAGAAGCTACCAAACAACCCTCTGTAGTTG

CCATTGGTATATGATAAGATGTACCATCGATAACCAAGGGGCCTATAACACCAA

CGGGCAAAGGCATGTAACCTATAACATTTTCACAACAAGCGCCAAATACGCGGT

CGTAGTCATAATTTTTATATGGTAAACGATCAGATGCTAATACAGGAGCTTCTGC

CAAAATTGAAAGAGCCTTCCTACGTACCGCAACCGCTCTCGTAGTATCACCTAAT

TTTTTCTCCAAAGCGTACAAAGGTAACTTACCGTGAATAACCAAGGCAGCGACCT

CTTTGTTCCTTCAATTGTTTTGTATTTCACTACTTAATAATGCTTCTAATTCTTCTA

AAGGACGTATTTTTCTTATCCAAGCTTTCAATATCGCGGGAATCATCTTCCTCACTA

GATGATGAAGGTCCTGATGAGCTCGATTGCGCAGATGATAAACTTTTGACTTTCG

ATCCAGAAATGACTGTTTTTATTGGTTAAAACTGGTGTAGAAGCCTTTTGTACAGG

AGCAGTAAAAGACTTCTTGGTGACTTCAGTCTTCACCAATTGGTCTGCAGCCATT

ATAGTTTTTTCTCCTTGACGTTAAAGTATAGAGGTATATTAACAATTTTTTGTTGA

TACTTTTTATGACATTTGAATAAGAAGTAATACAAACCGAAAATGTTGAAAGTATT

AGTTAAAGTGGTTATGCAGCTTTTGCATTTATATATCTGTTAATAGATCAAAAAT

CATCGCTTCGCTGATTAATTACCCCAGAAATAAGGCTAAAAAACTAAATCGCATTA

TTATCCTATGGTTGTTAATTTGATTCGTTGATTTGAAGGTTTGTGGGGCCAGGTTA

CTGCCAATTTTTCCTCTTTCATAACCATAAAAGCTAGTATTGTAGAATCCTTTATTGT

TCGGAGCAGTGCGGCGCGAGGCACATCTGCGTTTCAGGAACGCGACCGGTGAAG

ACCAGGACGCACGGAGGAGAGTCTTCCGTCGGAGGGCTGTCGCCCGCTCGGCGG

CTTCTAATCCGTACTTCAATATAGCAATGAGCAGTTAAGCGTATTACTGAAAGTT

CCAAAGAGAAGGTTTTTTTAGGCTAAGATAATGGGGCTCTTTACATTTCCACAAC

ATATAAGTAAGATTAGATAGGATATGTATATGGTGGTATTGCCATGTAATATGA

TTATTAAACTTCTTTGCGTCCATCCAAAAAAAAAAGTAAGAATTTTTGAAAATTCA

ATATAAATGAAACTCTCAACTAAACTTTGTTGGTGTGGTATTAAAGGAAGACTTA

GGCCGCAAAAGCAACAACAATTACACAATACAAACTTGCAAATGACTGAACTAA

AAAAACAAAAGACCGCTGAACAAAAAACCAGACCTCAAAATGTCGGTATTAAAG

GTATCCAAATTTACATCCCAACTCAATGTGTCAACCAATCTGAGCTAGAGAAATT

TGATGGCGTTTCTCAAGGTAAATACACAATTGGTCTGGGCCAAACCAACATGTCT

TTTGTCAATGACAGAGAAGATATCTACTCGATGTCCCTAACTGTTTTGTCTAAGTT

GATCAAGAGTTACAACATCGACACCAACAAAATTGGTAGATTAGAAGTCGGTAC

TGAAACTCTGATTGACAAGTCCAAGTCTGTCAAAGTCTGTCTTGATGCAATTGTTT

GGTGAAAACACTGACGTCGAAGGTATTGACACGCTTAATGCCTGTTACGGTGGTA

CCAACGCGTTGTTCAACTCTTTGAACTGGATTGAATCTAACGCATGGGATGGTAG

AGACGCCATTGTAGTTTGCGGTGATATTGCCATCTACGATAAGGGTGCCGCAAGA

CCAACCGGTGGTGCCGGTACTGTTGCTATGTGGATCGGTCCTGATGCTCCAATTG

TATTTGACTCTGTAAGAGCTTCTTACATGGAACACGCCTACGATTTTTACAAGCC

AGATTTCACCAGCGAATATCCTTACGTCGATGGTCATTTTTTCATTAACTTGTTACG

TCAAGGCTCTTGATCAAGTTTACAAAGAGTTATTTCCAAGAAGGCTATTTTCTAAAGG

GTTGGTTAGCGATCCCGCTGGTTCGGATGCTTTGAACGTTTTGAAATATTTCGACT

ACAACGTTTTCCATGTTCCAACCTGTAAATTGGTCACAAAATCATACGGTAGATT

ACTATATAACGATTTCAGAGCCAATCCTCAATTGTTCCCAGAAGTTGACGCCGAA

TTAGCTACTCGCGATTATGACGAATCTTTAACCGATAAGAACATTGAAAAAACTT

TTGTTAATGTTGCTAAGCCATTCCACAAAGAGAGAGTTGCCCAATCTTTGATTGT

TCCAACAAACACAGGTAACATGTACACCGCATCTGTTTATGCCGCCTTTGCATCT

CTATTAAACTATGTTGGATCTGACGACTTACAAGGCAAGCGTGTTGGTTTATTTTC

TTACGGTTCCGGTTTAGCTGCATCTCTATATTCTTGCAAAATTGTTGGTGACGTCC

AACATATTATCAAGGAATTAGATATTACTAACAAATTAGCCAAGAGAATCACCG

AAACTCCAAAGGATTACGAAGCTGCCATCGAATTGAGAGAAAATGCCCATTTGA

AGAAGAACTTCAAACCTCAAGGTTCCATTGAGCATTTGCAAAGTGGTGTTTACTA

CTTGACCAACATCGATGACAAATTTAGAAGATCTTACGATGTTAAAAAATAATCT

TCCCCCATCGATTGCATCTTGCTGAACCCCCTTTCATAAATGCTTTATTTTTTTGGC

AGCCTGCTTTTTTTAGCTCTCATTTAATAGAGTAGTTTTTTTAATCTATATACTAGG

AAAACTCTTTTATTTAATAACAATGATATATATACCCGGGAAGCTTTTCAATTC

ATCTTTTTTTTTTTTGTTCTTTTTTTTGATTCCGGTTTCTTTGAAATTTTTTTGATTC

GGTAATCTCCGAGCAGAAGGAAGAACGAAGGAAGGAGCACAGACTTAGATTGG

TATATATACGCATATGTGGTGTTGAAGAAACATGAAATTGCCCAGTATTCTTAAC

CCAACTGCACAGAACAAAAACCTGCAGGAAACGAAGATAAATCATGTCGAAAGC

TACATATAAGGAACGTGCTGCTACTCATCCTAGTCCTGTTGCTGCCAAGCTATTT

AATATCATGCACGAAAAGCAAACAAACTTGTGTGCTTCATTGGATGTTCGTACCA

CCAAGGAATTACTGGAGTTAGTTGAAGCATTAGGTCCCAAAAATTTGTTTACTAAAA

AACACATGTGGATATCTTGACTGATTTTTCCATGGAGGGCACAGTTAAGCCGCTA

AAGGCATTTATCCGCCAAGTACAATTTTTACTCTTCGAAGACAGAAAATTTGCTG

ACATTGGTAATACAGTCAAATTGCAGTACTCTGCGGGTGTATACAGAATAGCAG

AATGGGCAGACATTACGAATGCACACGGTGTGGTGGGCCCAGGTATTGTTAGCG

GTTTGAAGCAGGCGGCGGAAGAAGTAACAAAGGAACCTAGAGGCCTTTTGATGT

TAGCAGAATTGTCATGCAAGGGCTCCCTAGCTACTGGAGAATATACTAAGGGTA

CTGTTGACATTGCGAAGAGCGACAAAGATTTTGTTATCGGCTTTATTGCTCAAAG

AGACATGGGTGGAAGAGATGAAGGTTACGATTGGTTGATTATGACACCCGGTGT

GGGTTTAGATGACAAGGGAGACGCATTGGGTCAACAGTATAGAACCGTGGATGA

TGTGGTCTCTACAAGGATCTGACATTATTATTGTTGGGTTTAAAC

SEQ ID NO: 49

pAM492 sequence (excluding vector backbone)

GTTTAAACTTGCTAAATTCGAGTGAAACACAGGAAGACCAGAAAATCCTCATTTC

ATCCATATTAACAATAATTTCAAATGTTTTATTTGCATTATTTGAAACTAGGGAAG

ACAAGCAACGAAACGTTTTTTGAAAATTTGAGTATTTCAATAAATTTGTAGAGG

ACTCAGATATTGAAAAAAAGCTACAGCAATTAATACTTGATAAGAAGAGTATTG

AGAAGGGCAACGGTTCATCATCTCATGGATCTGCACATGAACAAACACCAGAGT

CAAACGACGTTGAAATTGAGGCTACTGCGCCAATTGATGACAATACAGACGATG

ATAACAAACCGAAGTTATCTGATGTAGAAAAGGATTAAAGATGCTAAGAGATAG

TGATGATATTTCATAAATAATGTAATTCTATATGTTAATTACCTTTTTTGCGAG

GCATATTATGGTGAAGGATAAGTTTTGACCATCAAAGAAGGTTAATGTGGCTGT

GGTTTCAGGGTCCATACCCGGGTATATATATCATTGTTATTAAATAAAGAGTT

TTCCTAGTATAGATTAAAAAACTACTCTATTAAATGAGAGCTAAAAAAAAGCA

GGCTGCCAAAAAAATAAAGCATTTATGAAGGGGGTTCAGCAAGATGCAATCGAT

GGGGGAAGATTATTTTTTAACATCGTAAGATTCTTCTAAATTTGTCATCGATGTTG

GTCAAGTAGTAAACCACTTTGCAAATGCTCAATGGAACCTTGAGGTTTGAAGT

TCTTCTTCAAATGGGCATTTTCCTCAATTCGATGGCAGCTTCGTAATCCTTTGGA

GTTTTCGGTGATTCTCTTGGCTAATTTGTTAGTAATATCTAATTCCTTGATAATATG

TTGGACGTCACCAACAAATTTTGCAAGAATATAGAGATGCAGCTAAACCGGAACC

GTAAGAAAATAAACCAACACGCTTGCCTTGTAAGTCGTCAGATCCAACATAGTTT

AATAGAGATGCAAAGGCGGCATAAACAGATGCGGTGTACATGTTACCTGTGTTT

GTTGGAACAATCAAAGATTGGGCAACTCTCTCTTTGTGGAATGGCTTAGCAACAT

TAACAAAAGTTTTTTCAATGTTCTTATCGGTTAAAGATTCGTCATAATCGCGAGT

AGCTAATTCGGCGTCAACTTCTGGGAACAATTGAGGATTGGCTCTGAAATCGTTA

TATAGTAATTCTACCGTATGATTTTGTGACCAATTTACAGGTTGGAACATGGAAAA

CGTTGTAGTCGAAATATTTCAAAACGTTCAAAAGCATCCGAACCAGCGGGATCGCT

AACCAACCCTTTAGAAATAGCCTTCTTGGAATAACTCTTGTAAACTTGATCAAGA

GCCTTGACGTAACAAGTTAATGAAAAATGACCATCGACGTAAGGATATTCGCTG

GTGAAATCTGGCTTGTAAAAATCGTAGGCGTGTTCCATGTAAGAAGCTCTTACAG

AGTCAAATACAATTGGAGCATCAGGACCGATCCACATAGCAACAGTACCGGCAC

CACCGGTTGGTCTTGCGGCACCCTATCGTAGATGGCAATATCACCGCAAACTAC

AATGGCGTCTCTCACCATCCCATGCGTTAGATTCAATCCAGTTCAAAGAGTTGAAC

AACGCGTTGGTACCACCGTAACAGGCATTAAGCGTGTCAATACCTTCGACGTCAG

TGTTTTCACCAAACAATTGCATCAAGACAGACTTGACAGACTTGGACTTGTCAAT

CAGAGTTTCAGTACCGACTTCTAATCTACCAATTTTGTTGGTGTCGATGTTGTAAC

TCTTGATCAACTTAGACAAAACAGTTAGGGACATCGAGTAGATATCTTCTCTGTC

ATTGACAAAAGACATGTTGGTTTGGCCCAGACCAATTGTGTATTTACCTTGAGAA

ACGCCATCAAATTTCTCTAGCTCAGATTGGTTGACACATTGAGTTGGGATGTAAA

TTTGGATACCTTTAATACCGACATTTTGAGGTCTGGTTTTTTGTTCAGCGGTCTTT

TGTTTTTTTAGTTCAGTCATTTGCAAGTTTTGTATTGTGTAATTGTTGTTGCTTTTGC

GGCCTAAGTCTTCCTTTAATACCACACCAACAAAGTTTAGTTGAGAGTTTCATTTTT

ATGTGATGATTGATTGATTGATTGTACAGTTTGTTTTTCTTAATATCTATTTCGAT

GACTTCTATATGATATTGCACTAACAAGAAGATATTATAATGCAATTGATACAAG

ACAAGGAGTTATTTGCTTCTCTTTTATATGATTCTGACAATCCATATTGCGTTGGT

AGTCTTTTTTGCTGGAACGGTTCAGCGGAAAAGACGCATCGCTCTTTTTGCTTCTA

GAAGAAATGCCAGCAAAAGAATCTCTTGACAGTGACTGACAGCAAAAATGTCTT

TTTCTAACTAGTAACAAGGCTAAGATATCAGCCTGAAATAAAGGGTGGTGAAGT

AATAATTAAATCATCCGTATAAAACCTATACACATATATGAGGAAAAATAATACA

AAAGTGTTTTAAATACAGATACATACATGAACATATGCACGTATAGCGCCCAAAT

GTCGGTAATGGGATCGGCTTACTAATTATAAAATGCATCATAGAAATCGTTGAAG

TTGACGCAGCGACTCGAGATCCATAGGAGCAACTCATGTCTGAACTTCAACGATT

TCTATGATGCATTTTAATTAGTAAGCCGATCCCATTACCGACATTTGGGCGCT

ATACGTGCATATGTTCATGTATGTATCTGTATTTAAAACACTTTTGTATTATTTTT

CCTCATATATGTGTATAGGTTTATACGGATGATTTAATTATTACTTCACCACCCTT

TATTTCAGGCTGATATCTTAGCCTTGTTACTAGTTAGAAAAAGACATTTTTGCTGT

CAGTCACTGTCAAGAGATTTCTTTTGCTGGCATTTCTTCTAGAAGCAAAAAGAGCG

ATGCGTCTTTTCCGCTGAACCGTTCCAGCAAAAAAGACTACCAACGCAATATGGA

TTGTCCAGAATCATATAAAAGAGAAGCAAATAACTCCTTGTCTTGTATCAATTGCA

TTATAATATCTTCTTGTTAGTGCAATATCATATAGAAGTCATCGAAATAGATATT

AAGAAAAACAAAACTGTACAATCAATCAATCAATCATCACATAAAATGGCTGCAG

ACCAATTGGTGAAGACTGAAGTCACCAAGAGTCTTTTACTGCTCCTGTACAAAAA

GGCTTCTACACCAGTTTTTAACCAATAAAACAGTCATTTCTGGATCGAAAGTCAAAA

AGTTTATCATCTGCGCAATCGAGCTCATCAGGACCTTTCATCATCTAGTGAGGAAG

ATGATTCCCGCGATATTGAAAGCTTGGATAAGAAAATACGTCCTTTAGAAGAATT

AGAAGCATTATTAAGTAGTGGAAATACAAAACAATTGAAGAACAAAGAGGTCGC

TGCCTTGGTTATTCACGGTAAGTTACCTTTGTACGCTTTGGAGAAAAAATTAGGT

GATACTACGAGAGCGGTTGCGGTACGTAGGAAGGCTCTTTCAATTTTGGCAGAA

GCTCCTGTATTAGCATCTGATCGTTTACCATATAAAAATTATGACTACGACCGCG

TATTTGGCGCTTGTTGTGAAAATGTTATAGGTTACATGCCTTTGCCCGTTGGTGTT

ATAGGCCCCTTGGTTATCGATGGTACATCTTTATCATATACCAATGGCAACTACAG

AGGGTTGTTTGGTAGCTTCTGCCATGCGTGGCTGTAAGGCAATCAATGCTGGCGG

TGGTGCAACAACTGTTTTAACTAAGGATGGTATGACAAGAGGCCCAGTAGTCCGT

TTCCCAACTTTGAAAAGATCTGGTGCCTGTAAGATATGGTTAGACTCAGAAGAGG

GACAAAACGCAATTAAAAAAGCTTTTAACTCTACATCAAGATTTGCACGTCTGCA

ACATATTCAAACTTGTCTAGCAGGAGATTTACTCTTCATGAGATTTAGAACAACT

ACTGGTGACGCAATGGGTATGAATATGATTTCTAAGGGTGTCGAATACTCATTAA

AGCAAATGGTAGAAGAGTATGGCTGGGAAGATATGGAGGTTGTCTCCGTTTCTG

GTAACTACTGTACCGACAAAAAACCAGCTGCCATCAACTGGATCGAAGGTCGTG

GTAAGAGTGTCGTCGCAGAAGTAGTATTCCTGGTGATGTTGTCAGAAAAGTGTT

AAAAAGTGATGTTTCCGCATTGGTTGAGTTGAACATTGCTAAGAATTTGGTTGGA

TCTGCAATGGCTGGGTCTGTTGGTGGATTTAACGCACATGCAGCTAATTTAGTGA

CAGCTGTTTTCTTGGCATTAGGACAAGATGCTGCACAAAATGTCGAAAGTTCCAA

CTGTATAACATTGATGAAAGAAGTGGACGGTGATTTGAGAATTTCCGTATCCATG

CCATCCATCGAAGTAGGTACCATCGGTGGTGGTACTGTTCTAGAACCACAAGGTG

CCATGTTGGACTTATTAGGTGTAAGAGGCCCACATGCTACCGCTCCTGGTACCAA

CGCACGTCAATTAGCAAGAATAGTTGCCTGTGCCGTCTTGGCAGGTGAATTATCC

TTATGTGCTGCCCTAGCAGCCGGCCATTTGGTTCAAAGTCATATGACCCACAACA

GGAAACCTGCTGAACCAACAAAACCTAACAATTTGGACGCCACTGATATAAATC

GTTTGAAAGATGGGTCCGTCACCTGCATTAAATCCTAAACTTAGTCATACGTCAT

TGGTATTTCTCTTGAAAAAGAAGCACAACAGCACCATGTGTTACGTAAAATATTTA

CTTTATAGTTTGTACGTCATAATTTCTTCCATATTACAAGTTCGTGCATATATAGA

AAGAATTCTGTTGTTGTAATTGTCATAACTCCCGGGAAGCTTTTCAATTCATCTTT

TTTTTTTTTGTTCTTTTTTTTGATTCCGGTTTCTTTGAAATTTTTTTGATTCGGTAAT

CTCCGAGCAGAAGGAAGAACGAAGGAAGGAGCACAGACTTAGATTGGTATATAT

ACGCATATGTGGTGTTGAAGAAACATGAAATTGCCCAGTATTCTTAACCCAACTG

CACAGAACAAAAACCTGCAGGAAACGAAGATAAATCATGTCGAAAGCTACATAT

AAGGAACGTGCTGCTACTCATCCTAGTCCTGTTGCTGCCAAGCTATTTAATATCA

TGCACGAAAAGCAAACAAACTTGTGTGCTTCATTGGATGTTCGTACCACCAAGGA

ATTACTGGAGTTAGTTGAAGCATTAGGTCCCAAAAATTTGTTTACTAAAAACACAT

GTGGATATCTTGACTGATTTTTCCATGGAGGGCACAGTTAAGCCGCTAAAGGCAT

TATCCGCCAAGTACAATTTTTACTCTTCGAAGACAGAAAATTTGCTGACATTGG

TAATACAGTCAAATTGCAGTACTCTGCGGGTGTATACAGAATAGCAGAATGGGC

AGACATTACGAATGCACACGGTGTGGTGGGCCCAGGTATTGTTAGCGGTTTGAA

GCAGGCGGCGGAAGAAGTAACAAAGGAACCTAGAGGCCTTTTGATGTTAGCAGA

ATTGTCATGCAAGGGCTCCCTAGCTACTGGAGAATATACTAAGGGTACTGTTGAC

ATTGCGAAGAGCGACAAAGATTTTGTTATCGGCTTTATTGCTCAAAGAGACATGG

GTGGAAGAGATGAAGGTTACGATTGGTTGATTATGACACCCGGTGTGGGTTTAG

ATGACAAGGGAGACGCATTGGGTCAACAGTATAGAACCGTGGATGATGTGGTCT

CTACAGGATCTGACATTATTATTGTTGGGTTTAAAC

SEQ ID NO: 50

pAM489 sequence (excluding vector backbone)

GTTTAAACTACTATTAGCTGAATTGCCACTGCTATCGTTGTTAGTGGCGTTAGTGC

TTGCATTCAAAGACATGGAGGGCGTTATTACGCCGGAGCTCCTCGACAGCAGATC

TGATGACTGGTCAATATTTTTGCATTGAGGCTCTGTTTGGAATTATATTTTGAG

ATGACCCATCTAATGTACTGGTATCACCAGATTTCATGTCGTTTTTTAAAAGCGGCT

GCTTGAGTCTTAGCAATAGCGTCACCATCTGGTGAATCCTTTGAAGGAACCACTG

ACGAAGGTTTGGACAGTGACGAAGAGGATCTTTCCTGCTTTGAATTAGTCGCGCT

GGGAGCAGATGACGAGTTGGTGGAGCTGGGGGCAGGATTGCTGGCCGTCGTGGG

TCCTGAATGGGTCCTTGGCTGGTCCATCTCTATTCTGAAAACGGAAGAGGAGTAG

GGAATATTACTGGCTGAAAATAAGTCTTGAATGAACGTATACGCGTATATTTCTA

CCAATCTCTCAACACTGAGTAATGGTAGTTATAAGAAAGAGACCGAGTTAGGGA

CAGTTAGAGGCGGTGGAGATATTCCTTATGGCATGTCTGGCGATGATAAAACTTT

TCAAACGGCAGCCCCGATCTAAAAGAGCTGACACCCGGGAGTTATGACAATTAC

AACAACAGAATTCTTTCTATATGCACGAACTTGTAATATGGAAGAAATTATGA

CGTACAAACTATAAAGTAAATATTTTACGTAACACATGGTGCTGTTGTGCTTCTTT

TTCAAGAGAATACCAATGACGTATGACTAAGTTTAGGATTTAATGCAGGTGACG

GACCCATTCTTTCAAACGATTTATATCAGTGGCGTCCAAATTGTTAGGTTTTGTTGG

TTCAGCAGGTTTCCTGTTGTGGGTCATATGACTTTGAACCAAATGGCCGGCTGCT

AGGGCAGCACATAAGGATAATTCACCTGCCAAGACGGCACAGGCAACTATTCTT

GCTAATTGACGTGCGTTGGTACCAGGAGCGGTAGCATGTGGGCCTCTTACACCTA

ATAAGTCCAACATGGCACCTTGTGGTTCTAGAACAGTACCACCACCGATGGTACC

TACTTCGATGGATGGCATGGATACGGAAATTCTCAAATCACCGTCCACTTCTTTC

ATCAATGTTATACAGTTGGAACTTTCGACATTTTGTGCAGGATCTTGTCCTAATGC

CAAGAAAACAGCTGTCACTAAATTAGCTGCATGTGCGTTAAAATCCACCAACAGA

CCCAGCCATTGCAGATCCAACCAAATTCTTAGCAATGTTCAACTCAACCAATGCG

GAAACATCACTTTTTTAACACTTTTCTGACAACATCACCAGGAATAGTAGCTTCTG

CGACGACACTCTTACCACGACCTTCGATCCAGTTGATGGCAGCTGGTTTTTTGTC

GGTACAGTAGTTACCAGAAACGGAGACAACCTCCATATCTCCCAGCCATACTCT

TCTACCATTTGCTTTAATGAGTATTCGACACCCTTAGAAATCATATTCATACCCAT

TGCGTCACCAGTAGTTGTTCTAAATCTCATGAAGAGTAAATCTCCTGCTAGACAA

GTTTGAATATGTTGCAGACGTGCAAATCTTGATGTAGAGTTAAAAGCTTTTTAA

TTGCGTTTTGTCCCTCTTCTGAGTCTAACCATATCTTACAGGCACCAGATCTTTTC

AAAGTTGGGAAACGGACTACTGGGCCTCTTGTCATACCATCCTTAGTTAAAACAG

TTGTTGCACCACCGCCAGCATTGATTGCCTTACAGCCACGCATGGCAGAAGCTAC

CAAACAACCCCTCTGTAGTTGCCATTGGTATATGATAAGATGTACCATCGATAACC

AAGGGGCCTATAACACCAACGGGCAAAGGCATGTAACCTATAACATTTTCACAA

CAAGCGCCAAATACGCGGTCGTAGTCATAATTTTTATATGGTAAACGATCAGATG

CTAATACAGGAGCTTCTGCCAAAATTGAAAGAGCCTTCCTACGTACCGCAACCGC

TCTCGTAGTATCACCTAATTTTTTCTCCAAAGCGTACAAAGGTAACTTACCGTGA

ATAACCAAGGCAGCGACCTCTTTGTTTCTTCAATTGTTTTGTATTTCCACTACTTAA

TAATGCTTCTAATTCTTCTAAAGGACGTATTTTTCTTATCCAAGCTTTCAATATCGC

GGGAATCATCTTTCCTCACTAGATGATGAAGGTCCTGATGAGCTCGATTGCGCAGA

TGATAAACTTTTGACTTTCGATCCAGAAATGACTGTTTTTATTGGTTAAAACTGGTG

TAGAAGCCTTTTGTACAGGAGCAGTAAAAGACTTCTTGGTGACTTCAGTCTTCAC

CAATTGGTCTGCAGCCATTATAGTTTTTTTCTCTTGACGTTAAAGTATAGAGGTAT

ATTAACAATTTTTTGTTGATACTTTTTATGACATTTGAATAAGAAGTAATACAAAC

CGAAAATGTTGAAAGTATTAGTTAAAGTGGTTATGCAGCTTTTGCATTTATATAT

CTGTTAATAGATCAAAAATCATCGCTTCGCTGATTAATTACCCCAGAAATAAGGC

TAAAAAACTAATCGCATTTATTATCCTATGGTTGTTAATTTGATTCGTTGATTTGAA

GGTTTGTGGGGCCAGGTTACTGCCAATTTTTCCTCTTTCATAACCATAAAAGCTAG

TATTGTAGAATCTTTATTGTTCGGAGCAGTGCGGCGCGAGGCACATCTGCGTTTC

AGGAACGCGACCGGTGAAGACCAGGACGCACGGAGGAGAGTCTTCCGTCGGAG

GGCTGTCGCCCGCTCGGCGGCTTCTAATCCGTACTTCAATATAGCAATGAGCAGT

TAAGCGTATTACTGAAAGTTCCAAGAGAAGGTTTTTTTAGGCTAAGATAATGGG

GCTCTTTACATTTCCACAACATATAAGTAAGATTAGATATGGATATGTATATGGT

GGTATTGCCATGTAATATGATTATTAAACTTCTTTGCGTCCATCCAAAAAAAAAG

TAAGAATTTTTGAAAATTCAATATAAATGGCTTCAGAAAAAGAAATTAGGAGAG

AGAGATTCTTGAACGTTTTTCCCTAAATTAGTAGAGGAATTGAACGCATCGCTTTT

GGCTTACGGTATGCCTAAGGAAGCATGTGACTGGTATGCCCACTCATTGAACTAC

AACACTCCAGGCGGTAAGCTAAATAGAGGTTTGTCCGTTGTGGACACGTATGCTA

TTCTCTCCAACAAGACCGTTGAACAATTGGGGCAAGAAGAATACGAAAAGGTTG

CCATTCTAGGTTGGTGCATTGAGTTGTTGCAGGCTTACTTCTTGGTCGCCGATGAT

ATGATGGACAAGTCCATTACCAGAAGAGGCCAACCATGTTGGTACAAGGTTCCT

GAAGTTGGGGAAATTGCCATCAATGACGCATTCATGTTAGAGGCTGCTATCTACA

AGCTTTTGAAATCTCACTTCAGAAACGAAAAATACTACATAGATATCACCGAATT

GTTCCATGAGGTCACCTTCCAAACCGAATTGGGCCAATTGATGGACTTAATCACT

GCACCTGAAGACAAAGTCGACTTGAGTAAGTTCTCCCTAAAGAAGCACTCCTTCA

TAGTTACTTTCAAGACTGCTTACTATTCTTTTCTACTTGCCTGTCGCATTGGCCATG

TACGTTGCCGGTATCACGGATGAAAAGGATTTGAAACAAGCCAGAGATGTCTTG

ATTCCATTGGGTGAATACTTCCAAAATTCAAGATGACTACTTAGACTGCTTCGGTA

CCCCAGAACAGATCGGTAAGATCGGTACAGATATCCAAGATAACAAATGTTCTT

GGGTAATCAACAAGGCATTGGAACTTGCTTCCGCAGAACAAAGAAAGACTTTAG

ACGAAAATTACGGTAAGAAGGACTCAGTCGCAGAAGCCAAATGCAAAAAGATTT

TCAATGACTTGAAAATTGAACAGCTATACCACGAATATGAAGAGTCTATTGCCAA

GGATTTGAAGGCCAAAATTTCTCAGGTCGATGAGTCTCGTGGCTTCAAAGCTGAT

GTCTTAACTGCGTTCTTGAACAAAGTTTACAAGAGAAGCAAATAGAACTAACGCT

AATCGATAAAACATTAGATTTCAAACTAGATAAGGACCATGTATAAGAACTATA

TACTTCCAATATAATATAGTATAAGCTTTAAGATAGTATCTCTCGATCTACCGTTC

CACGTGACTAGTCCAAGGATTTTTTTTAACCCGGGATATATGTGTACTTTGCAGTT

ATGACGCCAGATGGCAGTAGTGGAAGATATTTCTTTATTGAAAAATAGCTTGTCAC

CTTACGTACAATCTTGATCCGGAGCTTTTCTTTTTTTGCCGATTAAGAATTCGGTC

GAAAAAAGAAAAGGAGAGGGCCAAGAGGGAGGGCATTGGTGACTATTGAGCAC

GTGAGTATACGTGATTAAGCACACAAAGGCAGCTTGGAGTATGTCTGTTATTAAT

TTCACAGGTAGTTCTGGTCCATTGGTGAAAGTTTGCGGCTTGCAGAGCACAGAGG

CCGCAGAATGTGCTCTAGATTCCGATGCTGACTTGCTGGGTATTATATGTGTGCC

CAATAGAAAGAGAACAATTGACCCGGTTATTGCAAGGAAAATTTCAAGTCTTGT

AAAAGCATATAAAAATAGTTCAGGCACTCCGAAATACTTGGTTGGCGTGTTTCGT

AATCAACCTAAGGAGGATGTTTTGGCTCTGGTCAATGATTACGGCATTGATATCG

TCCAACTGCATGGAGATGAGTCGTGGCAAGAATACCAAGAGTTCCTCGGTTTGCC

AGTTATTAAAAGACTCGTATTTCCAAAAGACTGCAACATACTACTCAGTGCAGCT

TCACAGAAACCTCATTCGTTTATTTCCCTTGTTTGATTCAGAAGCAGGTGGGACAG

GTGAACTTTTGGATTGGAACTCGATTTCTGACTGGGTTGGAAGGCAAGAGAGCCC

CGAAAGCTTACATTTTATGTTAGCTGGTGGACTGACGCCGTTTAAAC

SEQ ID NO: 51

pAM497 sequence (excluding vector backbone)

GTTTAAACTTTTCCAATAGGTGGTTAGCAATCGTCTTACTTTCTAACTTTTCTTAC

CTTTACATTTTCAGCAATATATATATATATTTCAAGGATATACCATTCTAATGT

CTGCCCCTAAGAAGATCGTCGTTTTGCCAGGTGACCACGTTGGTCAAGAAATCAC

AGCCGAAGCCATTAAGGTTCTTAAAAGCTATTTCTGATGTTCGTTCCAATGTCAAG

TTCGATTTCGAAAATCATTTAATTGGTGGTGCTGCTATCGATGCTACAGGTGTTCC

ACTTCCAGATGAGGCGCTGGAAGCCTCCAAGAAGGCTGATGCCGTTTTGTTAGGT

GCTGTGGGTGGTCCTAAATGGGGTACCGGTAGTGTTAGACCTGAACAAGGTTTAC

TAAAAATCCGTAAAGAACTTCAATTGTACGCCAACTTAAGACCATGTAACTTTGC

ATCCGACTCTCTTTTAGACTATCTCCAATCAAGCCACAATTTGCTAAAGGTACTG

ACTTCGTTGTTGTCAGAGAATTACTGGGAGGTATTTACTTTGGTAAGAGAAAGGA

AGACGTTTAGCTTGCCTCGTCCCCGCCGGGTCACCCGGCCAGCGACATGGAGGCC

CAGAATACCTCTCTTGACAGTCTTGACGTGCGCAGCTCAGGGGCATGATGTGACT

GTCGCCCGTACATTTAGCCCATACATCCCCATGTATAATCATTTGCATCCATACAT

TTTGATGGCCGCACGGCGCGAAGCAAAAATTACGGCTCCTCGCTGCAGACCTGC

GAGCAGGGAAACGCTCCCCTCACAGACGCGTTGAATTGTCCCCCACGCCGCGCCC

CTGTAGAGAAATATAAAAGGTTAGGATTTGCCACTGAGGTTCTTCTTTCATATAC

TTCCTTTTAAAATCTTGCTAGGATACAGTTTCTCACATCACATCCGAACATAAACA

ACCATGGCAGAACCAGCCCAAAAAAAGCAAAAACAAACTGTTCAGGAGCGCAA

GGCGTTTATCTCCCGTATCACTAATGAAACTAAAATTCAAATCGCTATTTCGCTG

AATGGTGGTTATATTCAAATAAAAGATTCGATTCTTCCTGCAAAGAAGGATGACG

ATGTAGCTTCCCAAGCTACTCAGTCACAGGTCATCGATATTCACACAGGTGTTGG

CTTTTTGGATCATATGATCCATGCGTTGGCAAAACACTCTGGTTGGTCTCTTATTG

TTGAATGTATTGGTGACCTGCACATTGACGATCACCATACTACCGAAGATTGCGG

TATCGCATTAGGGCAAGCGTTCAAAGAAGCAATGGGTGCTGTCCGTGGTGTAAA

AAGATTCGGTACTGGGTTCGCACCATTGGATGAGGCGCTATCACGTGCCGTAGTC

GATTTATCTAGTAGACCATTTGCTGTAATCGACCTTGGATTGAAGAGAGAGATGA

TTGGTGATTTATCCACTGAAATGATTCCACACTTTTTGGAAAGTTTCGCGGAGGC

GGCCAGAATTACTTTGCATGTTGATTGTCTGAGAGGTTTCAACGATCACCACAGA

AGTGAGAGTGCGTTCAAGGCTTTGGCTGTTGCCATAAGAGAAGCTATTTTCTAGCA

ATGGCACCAATGACGTTCCCTCAACCAAAGGTGTTTTGATGTGAAGTACTGACAA

TAAAAAGATTCTTGTTTTTCAAGAACTTGTCATTTGTATAGTTTTTTATATTGTAG

TTGTTCTATTTTTAATCAAATGTTAGCGTGATTTATATTTTTTTTTCGCCTCGACATCA

TCTGCCCAGATGCGAAGTTAAGTGCGCAGAAAGTAATATCATGCGTCAATCGTAT

GTGAATGCTGGTCGCTATACTGCTGTCGATTCGATACTAACGCCGCCATCCACCC

GGGTTTCTCATTCAAGTGGTAACTGCTGTTAAAATTAAGATATTTTATAAATTGAA

GCTTGGTCGTTCCGACCAATACCGTAGGGAAACGTAAATTAGCTATTGTAAAAAAA

AGGAAAAGAAAAGAAAAGAAAAATGTTACATATCGAATTGATCTTATTCCTTTG

GTAGACCAGTCTTTGCGTCAATCAAAGATTCGTTTGTTTCTTGTGGGCCTGAACC

GACTTGAGTTAAAATCACTCTGGCAACATCCTTTTGCAACTCAAGATCCAATTCA

CGTGCAGTAAAGTTAGATGATTCAAATTGATGGTTGAAAGCCTCAAGCTGCTCAG

TAGTAAATTTCTTGTCCCATCCAGGAACAGAGCCAAACAATTTATAGATAAATGC

AAAGAGTTTCGACTCATTTTCAGCTAAGTAGTACAACACAGCATTTGGACCTGCA

TCAAACGTGTATGCAACGATTGTTTCTCCGTAAAACTGATTAATGGTGTGGCACC

AACTGATGATACGCTTGGAAGTGTCATTCATGTAGAATATTGGAGGGAAAGAGT

CCAAACATGTGGCATGGAAAGAGTTGGAATCCATCATTGTTTCCTTTGCAAAGGT

GGCGAAATCTTTTTCAACAATGGCTTTACGCATGACTTCAAATCTCTTTGGTACG

ACATGTTCAATTCTTTCTTTAAATAGTTCGGAGGTTGCCACGGTCAATTGCATACC

CTGAGTGGAACTCACATCCTTTTTAATATCGCTGACAACTAGGACACAAGCTTTC

ATCTGAGGCCAGTCAGAGCTGTCTGCGATTTGTACTGCCATGGAATCATGACCAT

CTTCAGCTTTTCCCATTTCCCAGGCCACGTATCCGCCAAACAACGATCTACAAGC

TGAACCAGACCCCTTTCTTGCTATTCTAGATATTTCTGAAGTTGACTGTGGTAATT

GGTATAACTTAGCAATTGCAGAGACCAATGCAGCAAAGCCAGCAGCGGAGGAAG

CTAAACCAGCTGCTGTAGGAAAGTTATTTTCGGAGACAATGTGGAGTTTCCATTG

AGATAATGTGGGCAATGAGGCGTCCTTCGATTCCATTTCCTTTCTTAATTGGCGTA

GGTCGCGCAGACAATTTTGAGTTCTTTCATTGTCGATGCTGTGTGGTTCTCCATTT

AACCACAAAGTGTCGCGTTCAAACTCAGGTGCAGTAGCCGCAGAGGTCAACGTT

CTGAGGTCATCTTGCGATAAAGTCACTGATATGGACGAATTGGTGGGCAGATTCA

ACTTCGTGTCCCTTTTTCCCCCAATACTTAAGGGTTGCGATGTTGACGGGTGCGGT

AACGGATGCTGTGTAAACGGTCATTATAGTTTTTTCTCCTTGACGTTAAAGTATA

GAGGTATATTAACAATTTTTTGTTGATACTTTTTATGACATTTGAATAAGAAGTAAT

ACAAACCGAAAATGTTGAAAGTATTAGTTAAAGTGGTTATGCAGCTTTTGCATTT

ATATATCTGTTAATAGATCAAAAATCATCGCTTCGCTGATTAATTACCCCAGAAA

TAAGGCTAAAAAACTAATCGCATTATATCCTATGGTTGTTAATTTGATTCGTTGA

TTTGAAGGTTTGTGGGGCCAGGTTACTGCCAATTTTTCCTCTTTCATAACCATAAAA

GCTAGTATTGTAGAATCTTTTATTGTTCGGAGCAGTGCGGCGCGAGGCACATCTGC

GTTTCAGGAACGCGACCGGTGAAGACCAGGACGCACGGAGGAGAGTCTTCCGTC

GGAGGGCTGTCGCCCGCTCGGCGGCTTCTAATCCGTACTTCAATATAGCAATGAG

CAGTTAAGCGTATTACTGAAAGTTCCAAAAGAGAAGGTTTTTTTTAGGCTAAGATAA

TGGGGCTCTTTACATTTCCACAACATATAAGTAAGATTAGATATGGATATGTATA

TGGTGGTATTGCCATGTAATATGATTATTAAACTTCTTTGCGTCCATCCAAAAAA

AAAGTAAGAATTTTTGAAAATTCAATATAAATGTCAGAGTTGAGAGCCTTCAGTG

CCCCAGGGAAAGCGTTACTAGCTGGTGGATATTTAGTTTTAGATCCGAAATATGA

AGCATTTGTAGTCGGATTATCGGCAAGAATGCATGCTGTAGCCCATCCTTACGGT

TCATTGCAAGAGTCTGATAAGTTTGAAGTGCGTGTGAAAAGTAAACAATTTAAA

GATGGGGAGTGGCTGTACCATATAAGTCCTAAAACTGGCTTCATTCCTGTTTCGA

TAGGCGGATCTAAGAACCCTTTCATTGAAAAAGTTATCGCTAACGTATTTAGCTA

CTTTAAGCCTAACATGGACGACTACTGCAATAGAAACTTGTTCGTTATTGATATT

TTCTCTGATGATGCCTACCATTCTCAGGAGGACAGCGTTACCGAACATCGTGGCA

ACAGAAGATTGAGTTTTCATTCGCACAGAATTGAAGAAGTTCCCAAAACAGGGC

TGGGCTCCTCGGCAGGTTTAGTCACAGTTTTAACTACAGCTTTGGCCTCCTTTTTT

GTATCGGACCTGGAAAATAATGTAGACAAATATAGAGAAGTTATTCATAATTTAT

CACAAGTTGCTCATTGTCAAGCTCAGGGTAAAATTGGAAGCGGGTTTGATGTAGC

GGCGGCAGCATATGGATCTATCAGATATAGAAGATTCCCACCCGCATTAATCTCT

AATTTGCCAGATATTGGAAGTGCTACTTACGGCAGTAAACTGGCGCATTTGGTTA

ATGAAGAAGACTGGAATATAACGATTAAAAGTAACCATTTACCTTCGGGATTAA

CTTTATGGATGGGCGATATTAAGAATGGTTCAGAAACAGTAAAACTGGTCCAGA

AGGTAAAAAAATTGGTATGATTCGCATATGCCGGAAAGCTTGAAAATATATACAG

AACTCGATCATGCAAATTCTAGATTTATGGATGGACTATCTAAACTAGATCGCTT

ACACGAGACTCATGACGATTACAGCGATCAGATATTTGAGTCTCTTGAGAGGAAT

GACTGTACCTGTCAAAAAGTATCCTGAGATCACACAGAAGTTAGAGATGCAGTTGCC

ACAATTAGACGTTCCTTTAGAAAAATAACTAAAGAATCTGGTGCCGATATCGAAC

CTCCCGTACAAACTAGCTTATTGGATGATTGCCAGACCTTAAAAGGAGTTCTTAC

TTGCTTAATACCTGGTGCTGGTGGTTATGACGCCATTGCAGTGATTGCTAAGCAA

GATGTTGATCTTAGGGCTCAAACCGCTGATGACAAAAGATTTTCTAAGGTTCAAT

GGCTGGATGTAACTCAGGCTGACTGGGGTGTTAGGAAAGAAAAAAGATCCGGAAA

CTTATCTTGATAAATAACTTAAGGTAGATAATAGTGGTCCATGTGACATCTTTAT

AAATGTGAAGTTTGAAGTGACCGCGCTTAACATCTAACCATTCATCTTCCGATAG

TACTTGAAATTGTTCCTTTCGGCGGCATGATAAAATTCTTTTAATGGGTACAAGCT

ACCCGGGAAAGATTCTCTTTTTTATGATATTTGTACATAAACTTTATAAATGAAA

TTCATAATAGAAACGACACGAAATTACAAAATGGAATATGTTCATAGGGTAGAC

GAAACTATATACGCAATCTACATACATTTATCAAGAAGGAGAAAAAAGGAGGATG

TAAAGGAATACAGGTAAGCAAATTGATACTAATGGCTCAACGTGATAAGGAAAA

AGAATTGCACTTTAACATTAATATTGACAAGGAGGAGGGCACCACAAAAAAGT

TAGGTGTAACAGAAAATCATGAAACTATGATTCCTAATTTATATATTGGAGGATT

TTCTCTAAAAAAAAAAAATACAACAAATAAAAAACACTCAATGACCTGACCAT

TTGATGGAGTTTAAGTCAATACCTTCTTGAACCATTTCCCATAATGGTGAAAGTT

CCCTCAAGAATTTACTCTGTCAGAAACGGCCTTAACGACGTAGTCGACCTCCTC

TTCAGTACTAAATCTACCAATACCAAATCTGATGGAAGAATGGGCTAATGCATCA

TCCTTACCCAGCGCATGTAAAACATAAGAAGGTTCTAGGGAAGCAGATGTACAG

GCTGAACCCGAGGATAATGCGATATCCCTAGTGCCATCAATAAAGATTCTCCTT

CCACGTAGGCGAAAGAAACGTTAACACGTTTAAAC

SEQ ID NO: 52

pAM493 sequence (excluding vector backbone)

GTTTAAACTACTCAGTATATTAAGTTTCGAATTGAAGGGCGAACTCTTTATTCGAA

GTCGGAGTCACCACAACACTTCCGCCCATACTCTCCGAATCCTCGTTTCCTAAAG

TAAGTTTACTTCCACTTGTAGGCCTATTATTAATGATATCTGAATAATCCTCTATT

AGGGTTGGATCATTCAGTAGCGCGTGCGATTGAAAGGAGTCCATGCCCGACGTC

GACGTGATTAGCGAAGGCGCGTAACCATTGTCATGTCTAGCAGCTATAGAACTA

ACCTCCTTGACACCACTTGCGGAAGTCTCATCAACATGCTCTTCCTTATTACTCAT

TCTCTTACCAAGCAGAGAATGTTATTCTAAAAACTACGTGTATTTCACCTCTTTCTC

GACTTGAACACGTCCAACTCCTTAAGTACTACCACAGCCAGGAAAGAATGGATC

CAGTTCTACACGATAGCAAAGCAGAAAACACAACCAGCGTACCCCCTGTAGAAGC

TTCTTTGTTTACAGCACTTGATCCATGTAGCCATACTCGAAATTTCAACTCATCTG

AAACTTTTCCTGAAGGTTGAAAAAGAATGCCATAAGGGTCACCCGAAGCTTATTC

ACGCCCGGGAGTTATGACAATTACAACAACAGAATTCTTTTCTATATATGCACGAA

CTTGTAATATGGAAGAAATTATGACGTACAAACTATAAAGTAAATATTTTACGTA

ACACATGGTGCTGTTGTGCTTCTTTTTCAAGAGAATACCAATGACGTATGACTAA

GTTTAGGATTTAATGCAGGTGACGGACCCATCTTTCAAACGATTTATATCAGTGG

CGTCCAAATTGTTAGGTTTTGTTGGTTCAGCAGGTTTCCTGTTGTGGGTCATATGA

CTTTGAACCAAATGGCCGGCTGCTAGGGCAGCACATAAGGATAATTCACCTGCC

AAGACGGCACAGGCAACTATTCTTGCTAATTGACGTGCGTTGGTACCAGGAGCG

GTAGCATGTGGGCCTCTTACACCTAATAAGTCCAACATGGCACCTTGTGGTTCTA

GAACAGTACCACCACCGATGGTACCCTACTTCGATGGATGGCATGGATACGGAAA

TTCTCAAATCACCGTCCACTTCTTTCATCAATGTTATACAGTTGGAACTTTCGACA

TTTTGTGCAGGATCTTGTCCTAATGCCAAGAAAACAGCTGTCACTAAATTAGCTG

CATGTGCGTTAAATCCACCAACAGACCCAGCCATTGCAGATCCAACCAAAT

TCTTAGCAATGTTCAACTCAACCAATGCGGAAACATCACTTTTTTAACACTTTTCTG

ACAACATCACCAGGAATAGTAGCTTCTGCGACGACACTCTTACCACGACCTTCGA

TCCAGTTGATGGCAGCTGGTTTTTTGTCGGTACAGTAGTTACCAGAAACGGAGAC

AACCTCCATATCTTCCCAGCCATACTCTTCTACCATTTGCTTTAATGAGTATTCGA

CACCCTTAGAAATCATATTCATACCCATTGCGTCACCAGTAGTTGTTCTAAATCTC

ATGAAGAGTAAATCTCCTGCTAGACAAGTTTGAATATGTTGCAGACGTGCAAATC

TTGATGTAGAGTTAAAAGCTTTTTTAATTGCGTTTTGTCCCTCTTCTGAGTCTAAC

CATATCTTACAGGCACCAGATCTTTTCAAAGTTGGGAAACGGACTACTGGGCCTC

TTGTCATACCATCCTTAGTTAAAACAGTTGTTGCACCACCGCCAGCATTGATTGC

CTTACAGCCACGCATGGCAGAAGCTACCAAACAACCCTCTGTAGTTGCCATTGGT

ATATGATAAGATGTACCATCGATAACCAAGGGGCCTATAACACCAACGGGCAAA

GGCATGTAACCTATAACATTTTCACAACAAGCGCCAAATACGCGGTCGTAGTCAT

AATTTTTATATGGTAAACGATCAGATGCTAATACAGGAGCTTCTGCCAAAATTGA

AAGAGCTTCCTACGTACCGCAACCGCTCTCGTAGTATCACCTAATTTTTTCTCCA

AAGCGTACAAAGGTAACTTACCGTGAATAACCAAGGCAGCGACCCTCTTTGTTCTT

CAATTGTTTTGTATTTCCACTACTTAATAATGCTTCTAATTCTTCTAAAGGACGTA

TTTTCTTATCCAAGCTTTCAATATCGCGGGAATCATCTCTCCTCACTAGATGATGAA

GGTCCTGATGAGCTCGATTGCGCAGATGATAAACTTTTGACTTTCGATCCAGAAA

TGACTGTTTTTATTGGTTAAAACTGGTGTAGAAGCCTTTTGTACAGGAGCAGTAAA

AGACTTCTTGGTGACTTCAGTCTTCACCAATTGGTCTGCAGCCATTATAGTTTTTT

CTCCTTGACGTTAAAGTATAGAGGTATATTAACAATTTTTTGTTGATACTTTTATG

ACATTTGAATAAGAAGTAATACAAACCGAAAATGTTGAAAGTATTAGTTAAAGT

GGTTATGCAGCTTTTGCATTTATATCTGTTAATAGATCAAAAATCATCGCTTCG

CTGATTAATTACCCCAGAAATAAGGCTAAAAAACTAATCGCATTATTATTCCTATG

GTTGTTAATTTGATTCGTTGATTTGAAGGTTTGTGGGGCCAGGTTACTGCCAATTT

TTCCTTTTCATAACCATAAAAGCTAGTATTGTAGAATCCTTTATTGTTCGGAGCAGT

GCGGCGCGAGGCACATCTGCGTTTCAGGAACGCGACCGGTGAAGACCAGGACGC

ACGGAGGAGAGTCTTCCGTCGGAGGGCTGTCGCCCGCTCGGCGGCTTCTAATCCG

TACTTCAATATAGCAATGAGCAGTTAAGCGTATTACTGAAAGTTCCAAAAGAGAA

GGTTTTTTTTAGGCTAAGATAATGGGGCTCTTTACATTTCCACAACATATAAGTAA

GATTAGATATGGATATGTATATGGTGGTATTGCCATGTAATATGATTATTAAACT

TCTTTGCGTCCATCCAAAAAAAAAAGTAAGAATTTTTGAAAATTCAATATAAATGA

CTGCCGACAACAATAGTATGCCCCATGGTGCAGTATCTAGTTACGCCAAAATTAGT

GCAAAACCAAACACCTGAAGACATTTTGGAAGAGTTTCCTGAAATTATTCCATTA

CAACAAAAGACCTAATACCCGATCTAGTGAGACGTCAAATGACGAAAGCGGAGAA

ACATGTTTTTCTGGTCATGATGAGGAGCAAATTAAGTTAATGAATGAAAATTGTA

TTGTTTTGGATTGGGACGATAATGCTATTGGTGCCGGTACCAAGAAAGTTTGTCA

TTTAATGGAAAATATTGAAAAGGGTTTACTACATCGTGCATTCTCCGTCTTTATTT

TCAATGAACAAGGTGAATTACTTTTACAACAAAGAGCCACTGAAAAAATAACTT

TCCCTGATCTTTGGACTAACACATGCTGCTCTCATCCACTATGTATTGATGACGAA

TTAGGTTTGAAGGGTAAGCTAGACGATAAGATTAAGGGCGCCTATTACTGCGGCG

GTGAGAAAACTAGATCATGAATTAGGTATTCCAGAAGATGAAACTAAGACAAGG

GGTAAGTTTCACTTTTTAAACAGAAATCCATTACATGGCACCAAGCAATGAACCAT

GGGGTGAACATGAAATTGATTACATCCTATTTTATAAGATCAACGCTAAAGAAA

ACTTGACTGTCAACCCAAACGTCAATGAAGTTAGAGACTTCAAATGGGTTTCACC

AAATGATTTGAAAACTATGTTTGCTGACCCAAGTTACAAGTTTACGCCTTGGTTT

AAGATTATTTGCGAGAATTACTTATTCAACTGGTGGGAGCAATTAGATGACCTTT

CTGAAGTGGAAAATGACAGGCAAATTCATAGAATGCTATAACAACGCGTCAATA

ATATAGGCTACATAAAAAATCATAATAACTTTGTTATCATAGCAAAATGTGATATA

AAACGTTTCATTTCACCTGAAAAATAGTAAAAATAGGCGACAAAAATCCTTAGT

AATATGTAAACTTTTATTTTCTTTATTTACCCGGGAGTCAGTCTGACTCTTGCGAGA

GATGAGGATGTAATAATACTAATCTCGAAGATGCCATCTAATACATATAGACATA

CATATATATATATATACATTCTATATATTCTTACCCAGATTCTTTGAGGTAAGACG

GTTGGGTTTTATCTTTTGCAGTTGGTACTATTAAGAACAATCGAATCATAAGCATT

GCTTACAAAGAATACACATACGAAATATTAACGATAATGTCAATTACGAAGACT

GAACTGGACGGTATATTGCCATTGGTGGCCAGAGGTAAAGTTAGAGACATATAT

GAGGTAGACGCTGGTACGTTGCTGTTTGTTGCTACGGATCGTATCTCTGCATATG

ACGTTATTATGGAAAACAGCATTCCTGAAAAGGGGATCCTATTGACCAAACTGTC

AGAGTTCTGGTTCAAGTTCCTGTCCAACGATGTTCGTAATCATTTGGTCGACATC

GCCCCAGGTAAGACTATTTTCGATTATCTACCTGCAAAATTGAGCGAACCAAAGT

ACAAAACGCAACTAGAAGACCGCTCTCTATTGGTTCACAAACATAAACTAATTCC

ATTGGAAGTAATTGTCAGAGGCTACATCACCGGATCTGCTTGGAAAGAGTACGT

AAAAACAGGTACTGTGCATGGTTTGAAACAACCTCAAGGACTTAAAGAATCTCA

AGAGTTCCCAGAACCAATCTTCACCCCATCGACCAAGGCTGAACAAGGTGAACA

TGACGAAAACATCTCTCCTGCCCAGGCCGCTGAGCTGGTGGGTGAAGATTTGTCA

CGTAGAGTGGCAGAACTGGCTGTAAAACTGTACTCCAAGTGCAAAGATTATGCT

AAGGAGAAGGGCATCATCATCGCAGACACTAAATTGTTTAAAC

SEQ ID NO: 53

pAM426 sequence

TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGAC

GGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGC

GTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCA

GATTGTACTGAGAGTGCACCATATCGACTACGTCGTAAGGCCGTTTCTGACAGAG

TAAAATTCTTGAGGGAACTTTCACCATTATGGGAAATGCTTCAAGAAGGTATTGA

CTTAAACTCCATCAAATGGTCAGGTCATTGAGTGTTTTTTATTTGTTGTATTTTT

TTTTTTAGAGAAAATCCTCCAATATCAAATTAGGAATCGTAGTTTCATGATTTTCT

GTTACACCTAACTTTTTGTGTGGTGCCCTGCTCCTTGTCAATATTAATGTTAAAGT

GCAATTCTTTTTCCTTATCACGTTGAGCCATTAGTATCAATTTGCTTACCTGTATT

CCTTTACTATCCCTCCTTTTTCTCCTTCTTGATAAATGTATGTAGATTGCGTATATAG

TTTCGTCTACCCTATGAACATATTCCATTTTGTAATTTCGTGTCGTTTCTATTATGA

ATTTCATTTATAAAGTTTTATGTACAAATATCATAAAAAAAGAGAATCTTTTTAAG

CAAGGATTTTCTTAACTTCTTCGGCGACAGCATCACCGACTTCGGTGGTACTGTT

GGAACCACCTAAATCACCAGTTCTGATACCTGCATCCAAAAACCTTTTAACTGCA

TCTTCAATGGCCTTACCTTCTTCAGGCAAGTTCAATGACAATTTCAACATCATTGC

AGCAGACAAGATAGTGGCGATAGGGTCAAACCTTATTCTTTGGCAAATCTGGAGC

AGAACCGTGGCATGGTTCGTACAAAACCAAATGCGGTGTTCTTGTCTGGCAAAGA

GGCCAAGGACGCAGATGGCAACAAACCCAAGGAACCTGGGATAACGGAGGCTT

CATCGGAGATGATATCACCAAACATGTTGCTGGTGATTATAATACCATTTAGGTG

GGTTGGGTTCTTAACTAGGATCATGGCGGCAGAATCAATCAATTGATGTTGAACC

TTCAATGTAGGGAATTCGTTCTTGATGGTTTCCTCCACAGTTTTTCTCATAATCT

TGAAGAGGCCAAAAGATTAGCTTTATCCAAGGACCAAATAGGCAATGGTGGCTC

ATGTTGTAGGGCCATGAAAGCGGCCATTCTTGTGATTCTTTGCACTTCTGGAACG

GTGTATTGTTCACTATCCCCAAGCGACACCATCACCATCGTCTTTCCTTTCTCTTACC

AAAGTAAATACCTCCCACTAATTCTCTGACAACAACGAAGTCAGTACCTTTAGCA

AATTGTGGCTTGATTGGAGATAAGTCTAAAAGAGAGTCGGATGCAAAGTTACAT

GGTCTTAAGTTGGCGTACAATTGAAGTTCTTTACGGATTTTTAGTAAACCTTGTTC

AGGTCTAACACTACCGGTACCCCATTTAGGACCAGCCACAGCACCTAACAAAAC

GGCATCAACCTTCTTGGAGGCTTCCAGCGCCTCATCTGGAAGTGGGACACCTGTA

GCATCGATAGCAGCACCACCAATTAAATGATTTTCGAAATCGAACTTGACATTGG

AACGAACATCAGAAATAGCTTTAAGAACCTTAATGGCTTCGGCTGTGATTTCTTG

ACCAACGTGGTCACCTGGCAAAACGACGATCTTCTTAGGGGCAGACATTACAAT

GGTATATCCTTGAAATATATATAAAAAAAAGGCGCCTTAGACCGCTCGGCCAAAC

AACCAATTACTTGTTGAGAAATAGAGTATAATTATCCTATAAATATAACGTTTTT

GAACACACATGAACAAGGAAGTACAGGACAATTGATTTTGAAGAGAATGTGGAT

TTTGATGTAATTGTTGGGATTCCATTTTTAATAAGGCAATAATATTAGGTATGTGG

ATATACTAGAAGTTCTCCTCGACCGTCGATATGCGGTGTGAAATACCGCACAGAT

GCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAA

ATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCG

GCAAAATCCCTTAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTC

CAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGC

GAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGGAAGATCCGAGGCCTAGC

TTTAACGAACGCAGAATTTTCGAGTTATTAAACTTAAAATACGCTGAACCCGAAC

ATAGAAATATCGAATGGGAAAAAAAACTGCATAAAGGCATTAAAAGAGGAGC

GAATTTTTTTTTTAATAAAAAATCTTAATAATCATTAAAAGATAAATAATAGTCTAT

ATATACGTATATAAATAAAAAATATTCAAAAAATAAAATAAACTATTTATTTTAGC

GTAAAGGATGGGGAAAGAGAAAAGAAAAAAATTGATCTATCGATTTCAATTCAA

TTCAATTTATTTCTTTTCGGATAAGAAAGCAACACCTGGCAATTCCTTACCTTCCA

ATAATTCCAAAGAAGCACCACCACCAGTAGAGACATGGGAGACCCGGGCCATGG

TTAGATAGACATAGGGTAAACTAGCAATGATTTGATCAAATGCTTGTATTCATCT

CCCATTCTCGTAAAATTGTCTTTACCTGCATATTGGACCTCTAAAAATTGGCAAA

GATATATAACAGCCATAAGTAAAGGTCTTGGGATATTCTTTGTTGTTAAATACTC

TCTGTTTATGTCTTTTCCAAACGTCCTCCACTTCCTTAAATCAGTGTCTGAGCAT

ATTCTTCGTTGACATTGTATTCCCTTCATGTAAGATTCTAAAGAGCTTGAACTATGT

TTTCTCTCCTGTTCCGCTTTATGAGTCATCAGGTCATTTAATCTCCTACCCAGAAT

ACCACTGTAACGGAATAAAGGCGGAGCAGATACAGCCCACTCAACTGATTCCTT

AGTGAAAATATCGCTCATTCCTAGATAACAGGTAGTTGTTAGCAAGTTTGCACCA

CCAGTGATAATAACTACGGGATCGTGCTCTTCAGTTGTCGGTATGTGTCCTTCATT

AGCCCATTTCGCTTCTACCATTAGATTCCTTACGAATTCTTTAACGAACTCCTTCC

CACAGTTGAATAAATCAGTTTCTACCTTCTTTGGCCAGAAACTCCTCCATTTCTGTG

TAGGTATCCATGAATAATTTGTAAATAGGCTTCATGTATTCCGGCAACGTGTCTA

AGCAGGTGATCGACCATCTTTCCACGGCTTCAGTGAAAATCTTTAACTCCTCGTA

AGTTCCATATGCGTCATACGTGTCATCAATAAGTGTTATCACAGCAACTGCCTTA

GTGAAAAAAAACTCTAGCTCTTGAATACTGGGGTTCGTAACCAGAACCTAAACCCC

AAAAATAGCATTCAACGATACGATCTCTCAGACATGGGGCATTTTTCTTAATATC

AAATGCCTTCCACCACTTGCATACGTGACTCAACTCTTTCCTTATGTAGGCTCTGCA

ATAGATTGAACTCCAGTTTAGCTAACTTTAGCAGAGTTTTTATTATGGGAGTCTTGT

TGCTGATAGAAGGGTATGTACTGGGCGGCCTCGATCCTTGGCAAATCTCTTCCACA

ATGGTTGCTTTAAAGCTCTCTGGATTTCAGTGAATAAAGCGGGGTTTGTACTAAAA

CGCGTCCTTTGTCATAATCGATAGCCTTGATCTTGTGAATCCCAGGGCATCTTCAA

GAATTATTTCGCCCGGAACTCTCATGGACGTAGCCTCATATAATTCCAAACAATCC

TTCAACATCATTCGCTAACGATTGTTTAAAAGCACCATTCTTGTCTTTATAGTTAT

TAAACACATCACACGTGACATAGTATCCTTGTTTACGCATCAGCCTAAACCATAA

GCTAGACCTGTCGCCATTCCAATTATCACCATAGGTCTCGTAAATACATTGCAAT

GCATGATCAATTTCACGTTCAAAATGATACGGAATACCTAAACGTTGAATCTCGT

CAATCAGCTTCAACAAATTTGCATGTTTCATAGGAATATCCAATGCTTCCTTTAAC

AACTGTCTTACTTCCTTCTTTAGATCGTTTACTATTTGCTCCACACCCTGTTCAACT

TGTTTCTCATAAATCAAAAATTGATCGCCCCAAATAGAAGGTGGGAAATTTGCAA

TTGGCCTTATAGGTTTCCTTCAGTCAAGGCCATTGTTTTCTGCAGATCCGGGGTT

TTTTCTCCTTGACGTTAAAGTATAGAGGTATATTAACAATTTTTTGTTGATACTTT

TATTACATTTGAATAAGAAGTAATACAAACCGAAAATGTTGAAAGTATTAGTTAA

AGTGGTTATGCAGTTTTTGCATTTATATCTGTTAATAGATCAAAAATCATCGCT

TCGCTGATTAATTACCCCAGAAATAAGGCTAAAAAACTAATCGCATTATCATCCCT

ATGGTTGTTAATTTGATTCGTTCATTTGAAGGTTTGTGGGGCCAGGTTACTGCCAA

TTTTTCTCTTTCATAACCATAAAAGCTAGTATTGTAGAATCCTTTATTGTTCGGAGC

AGTGCGGCGCGAGGCACATCTGCGTTTCAGGAACGCGACCGGTGAAGACGAGGA

CGCACGGAGGAGAGTCTTCCTTCGGAGGGCTGTCACCCGCTCGGCGGCTTCTAAT

CCGTACTAAGATCTGCTTTAATTTGGCCGGCGAACGTGGCGAGAAAGGAAGGGA

AGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTG

CGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCGC

CATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTT

CGCTATTACGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTT

TGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTT

CGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATTCCACA

GAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGC

CAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCT

GACGAGCATCACAAAAATCGACGCTCCAAGTCAGAGGTGGCGAAACCCGACAGG

ACTATAAAGATACCAGGCGTTTCCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTT

CCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGG

CGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCC

AAGCTGGGCTGTGTGCACGAACCCCCGTTCAGCCCGACCGCTGCGCCTTATCCG

GTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGC

AGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTT

CTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGC

GCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCA

AACAAACCACCGCTGGTAGCGGTGGTTTTTTTTGTTTGCAAGCAGCAGATTACGCG

CAGAAAAAAAAGGATCTCAAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCT

CAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGG

ATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTAAATCAATCTAAAGTA

TATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTAT

CTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGA

TAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCG

AGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAG

GGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAAT

TGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTG

TTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTC

AGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAA

AAGCGGTTAGTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGT

GTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCG

TAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTG

TATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCA

CATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAA

CTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCAC

CCAACTGATCTTCAGCATCTTTTACTTTTCACCAGCGTTTCTGGGTGAGCAAAAAC

AGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAA

TACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTC

ATGAGCGGATACATATTTGAATGTATTTAGAAAAAATAAACAAATAGGGGTTCCG

CGCACATTTCCCCGAAAAGTGCCACCTGAACGAAGCATCTGTGCTTCATTTTGTA

GAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGC

ATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGAATC

TGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTCAAAC

AAAGAATCTGAGCTGCATTTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATT

TTACCAACAAAGAATCTATACTTCTTTTTTGTTCTCAAAAAATGCATCCCGAGAG

CGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTTCTCCTTTGTGCGCTCTAT

AATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGG

CTACTTTGGTGTCTATTTTTCCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGT

TTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCG

ATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGC

GTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTTCTATTTTGTCTCT

ATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATG

AATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAA

AAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGT

AGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCA

ATGTTTGTGGAAGCGGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCG

TTTTTGGTTTTTTGAAAGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCT

GAAGTTCCCTATACTTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGT

TTCCGAAAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCA

CTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATATATATATACATGAGAA

GAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGTCTATTTATGT

AGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGGGGTA

TCGTATGCTTCCTTCAGCACTACCCCTTTAGCTGTTCTATATGCTGCCACTCCTCAA

TTGGATTAGTCTCATCCTTCAATGCTATCATTTTCCTTTGATATTGGATCATACTAA

GAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCT

TTCGTC

SEQ TD NO: 54

pAM322 sequence

TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGAC

GGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGC

GTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCA

GATTGTACTGAGAGTGCACCATATCGACTACGTCGTAAGGCCGTTTCTGACAGAG

TAAAATTCTTGAGGGAACTTTCACCATTATGGGAAATGCTTCAAGAAGGTATTGA

CTTAAACTCCATCAAATGGTCAGGTCATTGAGTGTTTTTTATTTGTTGTATTTTTTTT

TTTTTTAGAGAAAATCCTCCAATATCAAATTAGGAATCGTAGTTTCATGATTTTCT

GTTACACCTAACTTTTTGTGTGGTGCCCTCCTCCTTGTCAATATTAATGTTAAAGT

GCAATTCTTTTTCCTTATCACGTTGAGCCATTAGTATCAATTTGCTTACCTGTATT

CCTTTACTATCCCTCCTTTTTCTCCTTCTTGATAAATGTATGTAGATTGCGTATATAG

TTTCGTCTACCCTATGAACATATTCCATTTTGTAATTTCGTGTCGTTTCTATTATGA

ATTTCATTTATAAAGTTTTATGTACAAATATCATAAAAAAAGAGAATCTTTTTAAG

CAAGGATTTTCTTAACTTCTTCGGCGACAGCATCACCGACTTCGGTGGTACTGTT

GGAACCACCTAAATCACCAGTTCTGATACCTGCATCCAAAAACCTTTTAACTGCA

TCTTCAATGGCCTTACCTTCTTCAGGCAAGTTCAATGACAATTTCAACATCATTGC

AGCAGACAAGATAGTGGCGATAGGGTCAAACCTTATTCTTTGGCAAATCTGGAGC

AGAACCGTGGCATGGTTCGTACAAAACCAAATGCGGTGTTCTTGTCTGGCAAAGA

GGCCAAGGACGCAGATGGCAACAAACCCAAGGAACCTGGGATAACGGAGGCTT

CATCGGAGATGATATCACCAAACATGTTGCTGGTGATTATAATACCATTTAGGTG

GGTTGGGTTCTTAACTAGGATCATGGCGGCAGAATCAATCAATTGATGTTGAACC

TTCAATGTAGGGAATTCGTTCTTGATGGTTTCCTCCACAGTTTTTCTCATAATCT

TGAAGAGGCCAAAAGATAGCTTTATCCAAGGACCAAATAGGCAATGGTGGCTC

ATGTTGTAGGGCCATGAAAGCGGCCATTCTTGTGATTCTTTGCACTTCTGGAACG

GTGTATTGTTCACTATCCCCAAGCGACACCATCACCATCGTCTTTCCTTTCTCTTACC

AAAGTAAATACCTCCCACTAATTCTCTGACAACAACGAAGTCAGTACCTTTAGCA

AATTGTGGCTTGATTGGAGATAAGTCTAAAAGAGAGTCGGATGCAAAGTTACAT

GGTCTTAAGTTGGCGTACAATTGAAGTTCTTTACGGATTTTAGTAAACCTTGTTC

AGGTCTAACACTACCGGTACCCCATTTAGGACCAGCCACAGCACCTAACAAAAC

GGCATCAACCTTCTTGGAGGCTTCCAGCGCCTCATCTGGAAGTGGGACACCTGTA

GCATCGATAGCAGCACCACCAATTAAATGATTTTCGAAATCGAACTTGACATTGG

AACGAACATCAGAAATAGCTTTAAGAACCTTAATGGCTTCGGCTGTGATTTCTTG

ACCAACGTGGTCACCTGGCAAAACGACGATCTTCTTAGGGGCAGACATTACAAT

GGTATATCCTTGAAATATATATAAAAAAAAGGCGCCTTAGACCGCTCGGCCAAAC

AACCAATTACTTGTTGAGAAATAGAGTATAATTATCCTATAAATATAACGTTTTT

GAACACACATGAACAAGGAAGTACAGGACAATTGATTTTGAAGAGAATGTGGAT

TTTGATGTAATTGTTGGGATTCCATTTTTAATAAGGCAATAATATTAGGTATGTGG

ATATACTAGAAGTTCTCCTCGACCGTCGATATGCGGTGTGAAATACCGCACAGAT

GCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAA

ATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCG

GCAAAATCCCTTAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTC

CAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGC

GAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGGAAGATCCGAGGCCTAGC

TTTAACGAACGCAGAATTTTCGAGTTATTAAACTTAAAATACGCTGAACCCGAAC

ATAGAAATATCGAATGGGAAAAAAAACTGCATAAAGGCATTAAAAGAGGAGC

GAATTTTTTTTTTAATAAAAAATCTTAATAATCATTAAAAGATAAATAATAGTCTAT

ATATACGTATATAAATAAAAAATATTCAAAAAATAAAATAAACTATTTATTTTAGC

GTAAAGGATGGGGAAAGAGAAAAGAAAAAAATTGATCTATCGATTTCAATTCAA

TTCAATTTATTTCTTTTCGGATAAGAAAGCAACACCTGGCAATTCCTTACCTTCCA

ATAATTCCAAAGAAGCACCACCACCAGTAGAGACATGGGAGACCCGGGCCATGG

TTAGATAGACATAGGGTAAACTAGCAATGATTTGATCAAATGCTTGTATTCATCT

CCCATTCTCGTAAAATTGTCTTTACCTGCATATTGGACCTCTAAAAATTGGCAAA

GATATATAACAGCCATAAGTAAAGGTCTTGGGATATTCTTTGTTGTTAAATACTC

TCTGTTTATGTCTTTTCCAAACGTCCTCCACTTCCTTAAATCAGTGTCTGAGCAT

ATTCTTCGTTGACATTGTATTCCCTTCATGTAAGATTCTAAAGAGCTTGAACTATGT

TTTCTCTCCTGTTCCGCTTTATGAGTCATCAGGTCATTTAATCTCCTACCCAGAAT

ACCACTGTAACGGAATAAAGGCGGAGCAGATACAGCCCACTCAACTGATTCCTT

AGTGAAAATATCGCTCATTCCTAGATAACAGGTAGTTGTTAGCAAGTTTGCACCA

CCAGTGATAATAACTACGGGATCGTGCTCTTCAGTTGTCGGTATGTGTCCTTCATT

AGCCCATTTCGCTTCTACCATTAGATTCCTTACGAATTCTTTAACGAACTCCTTCC

CACAGTTGAATAAATCAGTTTCTACCTTCTTTGGCCAGAAACTCCTCCATTTCTGTG

TAGGTATCCATGAATAATTTGTAAATAGGCTTCATGTATTCCGGCAACGTGTCTA

AGCAGGTGATCGACCATCTTTCCACGGCTTCAGTGAAAATCTTTAACTCCTCGTA

AGTTCCATATGCGTCATACGTGTCATCAATAAGTGTTATCACAGCAACTGCCTTA

GTGAAAAAAAACTCTAGCTCTTGAATACTGGGGTTCGTAACCAGAACCTAAACCCC

AAAAATAGCATTCAACGATACGATCTCTCAGACATGGGGCATTTTTCTTAATATC

AAATGCCTTCCACCACTTGCATACGTGACTCAACTCTTTCCTTATGTAGGCTCTGCA

ATAGATTGAACTCCAGTTTAGCTAACTTTAGCAGAGTTTTTATTATGGGAGTCTTGT

TGCTGATAGAAGGGTATGTACTGGGCGGCCTCGATCCTTGGCAAATCTCTTCCACA

ATGGTTGCTTTAAAGCTCTCTGGATTTCAGTGAATAAAGCGGGGTTTGTACTAAAA

CGCGTCCTTTGTCATAATCGATAGCCTTGATCTTGTGAATCCCAGGGCATCTTCAA

GAATTATTTCGCCCGGAACTCTCATGGACGTAGCCTCATATAATTCCAAACAATCC

TTCAACATCATTCGCTAACGATTGTTTAAAAGCACCATTCTTGTCTTTATAGTTAT

TAAACACATCACACGTGACATAGTATCCTTGTTTACGCATCAGCCTAAACCATAA

GCTAGACCTGTCGCCATTCCAATTATCACCATAGGTCTCGTAAATACATTGCAAT

GCATGATCAATTTCACGTTCAAAATGATACGGAATACCTAAACGTTGAATCTCGT

CAATCAGCTTCAACAAATTTGCATGTTTCATAGGAATATCCAATGCTTCCTTTAAC

AACTGTCTTACTTCCTTCTTTAGATCGTTTACTATTTGCTCCACACCCTGTTCAACT

TGTTTCTCATAAATCAAAAATTGATCGCCCCAAATAGAAGGTGGGAAATTTGCAA

TTGGCCTTATAGGTTTCCTTCAGTCAAGGCCATTGTTTTCTGCAGATCCGGGGTT

TTTTCTCCTTGACGTTAAAGTATAGAGGTATATTAACAATTTTTTGTTGATACTTT

TATTACATTTGAATAAGAAGTAATACAAACCGAAAATGTTGAAAGTATTAGTTAA

AGTGGTTATGCAGTTTTTGCATTTATATCTGTTAATAGATCAAAAATCATCGCT

TCGCTGATTAATTACCCCAGAAATAAGGCTAAAAAACTAATCGCATTATCATCCCT

ATGGTTGTTAATTTGATTCGTTCATTTGAAGGTTTGTGGGGCCAGGTTACTGCCAA

TTTTTCTCTTTCATAACCATAAAAGCTAGTATTGTAGAATCCTTTATTGTTCGGAGC

AGTGCGGCGCGAGGCACATCTGCGTTTCAGGAACGCGACCGGTGAAGACGAGGA

CGCACGGAGGAGAGTCTTCCTTCGGAGGGCTGTCACCCGCTCGGCGGCTTCTAAT

CCGTACTAAGATCTGCTTTAATTTGGCCGGCGAACGTGGCGAGAAAGGAAGGGA

AGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTG

CGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCGC

CATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTT

CGCTATTACGCCAGCTGAATTGGAGCGACCTCATGCTATACCTGAGAAAGCAACC

TGACCTACAGGAAAGAGTTACTCAAGAATAAGAATTTTCGTTTTAAAAACCTAAGA

GTCACTTTAAAAATTTGTATACACTTTATTTTTTTTAACTTATTTAATAATAAAAA

TCATAAATCATAAGAAATTCGCTTATTTGAAGTGTCAACAACGTATCTACCAAC

GATTTGACCCTTTTCCATCTTTTTCGTAAATTTCTGGCAAGGTAGACAAGCCGACA

ACCTTGATTGGAGACTTGACCAAACCTCTGGCGAAGAATTGTTAATTAAGAGTCA

GTCGACTTAAAAACTAGGGACCAATAGCAATTCTGTTTTACGTTGCATTGTTGCA

CCTGAACTTTCCGTCATGTCAATTTGATCATATGAAACTTCCATTGGGCAACTTCCA

GTTGAAATGATAAAGAATGTTGGCTAGTGGCAGTTGAACATTGGCCAAACCTAA

CGCAGCGCCAGGACACATACGACGTCCAGCCCCAAATGGTAAATATTCATATTC

GGCGCCCATCACTGTTGCCGAAGAGTTTTCAAATCTTTCAGGTATAAACGCTTCT

GCATCCTTCCAGTATTCAGGATCTCTATTGATCGCAAACACATTAACGATTAATTT

CGTTTTGTTAGGGATATTATAACCAGCCAAGTTTACTGGCTGACGACATTCTCTA

GGTAGCACTAACGGCAAGGGTGGGTGTAGTCTAAGAGTCTCTTTGATGACCATAT

TCAAGTAGGACAATTCTTGTATATCTTCTTCATGTATTTTTTTCTTTCCCATTCAAG

GCCTTACGTAATTCAGCCTGAACCTTTTCCATTGCTTTCGGACATTTTATTAGCTC

GCTTATAGCCCCATTCTATGGTAGAACTTGAAGTGTCGGTCCCTGCACCGAACATG

TCCAAAATTATTGCTTGATATTATCCGAAGTCAGAGGAAACTCAGCAGAATCCT

TTAATCTAAGTAATACATCTAATAGGGTTTCGTTGGTTTTGGATGACGTATTTACG

GTATGTTCAGCTACCAAATTGTCAATTAAGTTATCAATCTTTTTACGTAGGCTAGT

TAATCTTGCCTCTCTTACCGCTCAAGTGATGCAAGAACTTTTTAGATGGGAAAATA

TCGGCAACATCGAAACCGCCTGTTTGTCTCAGTATTTCTTAACAATTTCAGTAAG

TTCCTTTTGATCTTTAATTCCCTTACCAAACGCAGCACGGGATAGTATAGTGGCA

ATTAGTTTAAAAAACGTTTTTCACTTAAATTTACTGGTCTACCACTACCTGAAGCCTT

TATTTCCTGGACTAAATTCCAACATTCTTCTTCCCTCAACGATTGAAATGACTTAA

CCTTTTTTACAGACAACAATTCAAGAGTACAAAATCTTCCTTAATTGTCTCCAGTAT

TCCCCATATGGAGCAAGGACAACATCAGTGTTATGATATAAAACTATTTTCCCAG

TTAAAGTTTCGGGTCTATTAGCGAAAGTAATATCGTAGGTTGTAAGAATTTCCTT

AGCCCACTTAGGACTCGACACGACTATTGTGGGTACCTCTCCCAATTGAAGGTGC

ATTAGCGAACCATATTTTCTCGCTAAATCCCTTACACCCCTGTGTGGTGTGGTTCC

GATCAAATGGTGCATGTGACCAATGATGGGTAGCCTCCAAGGTTCCGGCAAGGA

CTTTTTAGTTGACTTACTTCTAGTGGCAAATTTGTACACGAACAACAAAATAGTT

GCTAAAGCAATTGATGTAGTTAAAGATAGTGCCATAGCCTTTAAAATTGACTTCA

TTGTTTCCTAGGCCTTTAGTGAGGGTTGAATTCGAATTTTCAAAAATTCTTACTT

TTTTTTTGGATGGACGCCAAAGAAGTTTAATAATCATATTACATGGCATTACCACC

ATATACATATCCATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATG

TAAAGAGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAA

TACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGAAGCCGCCGAGCGGG

TGACAGCCCTCCGAAGGAAGACTTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGC

GTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCT

ACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCA

CAAACCTTCAAATGAACGAATCAAATTAACAACCATAGGATGATAATGCGATTA

GTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTA

TTAACAGATATATAAATGCAAAAACTGCATAACCACTTTAACTAATACTTTCAAC

ATTTTCGGTTTGTATTACTTCTTATTCAAATGTAATAAAAGTATCAACAAAAAATT

GTTAATATACCCTCTATACTTTAACGTCAAGGAGAAAAAACCCCAAGCTTCCCGGG

AAAACAATGCAATCGACAACTTCCGTTAAACTATCACCTTTCGATCTTATGACTG

CCTTGTTAAATGGTAAAGTTAGTTTCGACACGTCCAATACTTCCGATACAAATAT

ACCACTGGCGGTTTTCATGGAAAACAGGGAATTGCTTATGATATTAACAACCAGT

GTGGCCGTTTTAATTGGTTGTGTGGTTGTATTGGTATGGAGAAGATCATCAAGTG

CCGCTAAGAAGGCCGCCGAATCACCAGTCATTGTCGTCCCAAAAGAAAGTCACTG

AAGATGAGGTTGATGACGGCAGAAAGAAAGTTACTGTATTTTTCGGGACACAAA

CGGGGACTGCGGAAGGTTTTGCGAAAGCTCTAGTTGAAGAAGCCAAGGCAAGGT

ACGAAAAAGCAGTATTCAAAGTTATTGATTTAGATGACTACGCCGCAGAAGATG

ATGAATACGAAGAAAAGCTAAAGAAAGAATCTTTGGCATTCTTCTTTTTAGCTAC

CTATGGTGACGGAGAACCAACAGATAACGCCGCTAGATTCTATAAATGGTTTACT

GAAGGAGAAGAAAAAGGTGAGTGGTTAGATAAGTTACAATACGCTGTCTTTGGA

TTGGGAAATCGTCAATATGAACACTTCAATAAGATTGCAAAAGTGGTCGATGAA

AAATTAGTTGAGCAGGGGGCTAAAAGGTTAGTGCCTGTCGGTATGGGTGATGAC

GATCAATGTATCGAAGATGATTTTACTGCTTGGAAGGAATTGGTTTGGCCAGAAT

TAGATCAGCTATTGAGGGACGAAGATGACACAAGTGTCGCTACTCCGTACACCG

CCGCTGTTGGCGAATATCGTGTTGTTTTTCACGATAAACCTGAAACTTACGATCA

AGATCAATTGACCAACGGACACGCAGTTCACGACGCCCAACACCCATGCAGATC

GAACGTTGCGGTCAAGAAAAGAATTACACAGTCCCTTATCCGATAGGAGTTGTACT

CATTTAGAATTTGATATTTCCAATACTGGACTATCGTATGAAACTGGCGACCATG

TCGGTGTATATGTGGAAAACCTGTCTGAAGTTGTAGATGAAGCCGAAAAATTGAT

TGGGCTTCCTCCACATACATACTTTTTCTGTGCATACAGATAATGAAGATGGTACT

CCACTTGGCGGAGCCTCGTTACCACCTCCCTTTCCACCATGTACACTTAGAAAAG

CTCTTGCATCTTATGCAGATGTACTTTTCTCACCCAAAGAAAAAGTGCATTACTAGCT

CTAGCCGCCCATGCTACCGACTCTACTGAAGCTGACCGTTTGAAATTCTTTGCTTC

ACCTGCTGGCAAAGACGAGTACGCACAGTGGATTGTGGCATCTCACAGATCATT

GCTGGAAGTGATGGAAGCCTTCCCATCGGCAAAGCCACCATTAGGCGTGTTTTTC

GCATCTGTTGCCCCACGTTTACAGCCTAGATACTATTCCATATCTTCTAGCCCAAA

ATTTGCCCCCAATCGTATTCATGTGACGTGTGCGCTGGTGTATGAACAAACTCCA

TCAGGAAGGGTACATAAAGGTGTCTGTAGTACATGGATGAAAAACGCGGTGCCA

ATGACTGAATCTCAAGATTGTTCGTGGGCACCAATTTATGTTCGTACTTCTAATTT

TAGACTACCTAGTGACCCTAAAGTACCAGTGATTATGATCGGGCCTGGGACAGG

ACTAGCGCCATTCAGAGGTTTCTACAAGAAAGATTGGCCCAAAAGGAAGCAGG

TACGGAATTAGGAACCGCAATTCTATTCTTTGGTTGTCGTAATAGAAAAGTTGAC

TTTATATACGAAGATGAGTTAAACAACTTCGTTGAAACTGGAGCGTTATCAGAAT

TAGTGACAGCATTCTCTAGGGAAGGTGCAACAAAAGAATACGTCCAACATAAAA

TGACCCAAAAGGCCAGCGATATATGGAATTTGCTGTCCGAGGGTGCCTATTTGTA

CGTTTGTGGTGATGCAAAGGGAATGGCTAAAGATGTTCACAGGACATTGCATAC

AATTGTTCAGGAACAAGGTTCCTTGGATTCCTCTAAGGCAGAACTTTATGTTAAA

AACCTTCAGATGGCTGGTAGATATTTGCGTGATGTTTGGTGAGCTAGCTAAGATC

CGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTAT

TTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTT

TTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGA

AGGTTTTGGGACGCTCGAAGATCCAGCTGCATTAATGAATCGGCCAACGCGCGG

GGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCT

GCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAAT

ACGGTTATTCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAG

GCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATA

GGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCCAAGTCAGAGGTGGC

GAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCCTGGAAGCTCCCTCGT

GCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTT

CGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTA

GGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCCGTTCAGCCCGACCGC

TGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTAT

CGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCG

GTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAG

TATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAG

CTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTTGTTTGCAAG

CAGCAGATTACGCGCAGAAAAAAAGGATCTCAAAGAAGATCCTTTGATCTTTTTCTA

CGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGA

GATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAA

ATCAATTCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATC

AGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACT

CCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCT

GCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAAC

CAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCC

ATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATA

GTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTT

GGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCC

CCATGTTGTGCAAAAAAGCGGTTAGTCCTTCGGTCCTCCGATCGTTGTCAGAAG

TAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTT

ACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGT

CATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACG

GGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACG

TTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATG

TAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTTCACCAGCGTTTC

TGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGA

CACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTAT

CAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAAATAAAC

AAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAACGAAGCATCT

GTGCTTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTCAAACAA

AGAATCTGAGCTGCATTTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTA

CCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCG

CTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTTACAGAACAGAAATGCAACG

CGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTTCTACAAAAT

GCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTTTAGATTACTTTTTTTTCTCCT

TTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAG

GTTAGAAGAAGGCTACTTTGGTGTCTATTTTTCTCTCTAAAAAAAGCCTGACT

CCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGAT

AAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAAC

AGAAAGTGATAGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCT

TCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTC

GATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAG

AGATAAACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAA

AGGTGGATGGGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGA

TACTTTTGAGCAATGTTTGTGGAAGCGGTATTCGCAATATTTTAGTAGCTCGTTAC

AGTCCGGTGCGTTTTTGGTTTTTTGAAAGTGCGTCTTCAGAGCGCTTTTGGTTTTC

AAAAGCGCTCTGAAGTTCCTATAACTTTTCTAGAGAATAGGAACTTCGGAATAGGA

ACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCGC

ACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATATATAT

ATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGC

GTCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATTCCCATTC

CATGCGGGGTATCGTATGCTTCCTTCAGCACTACCCCTTTAGCTGTTCTATATGCTG

CCACTCCTCCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTG

GATCATACTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTAT

CACGAGGCCCTTTCGTC

Claims (48)

1. the production system of bio-organic compounds comprises:

A. the container of at least 100 liters of capacity;

B. form the aqueous culture medium of first phase in this container;

C. a large amount of host cells in the aqueous culture medium, described host cell can be made at least a bio-organic compounds; With,

D. with the first contacted liquid organic second mutually, described second comprises described at least a bio-organic compounds mutually.

2. system as claimed in claim 2, wherein said at least a bio-organic compounds is C ₅Bio-organic compounds.

3. system as claimed in claim 2, wherein said at least a bio-organic compounds is C ₁₀Bio-organic compounds.

4. system as claimed in claim 2, wherein said at least a bio-organic compounds is C ₁₅Bio-organic compounds.

5. system as claimed in claim 2, wherein said at least a bio-organic compounds is C ₂₀Bio-organic compounds.

6. system as claimed in claim 2, wherein said at least a bio-organic compounds is C ₂₀₊Bio-organic compounds.

7. system as claimed in claim 1, wherein said at least a bio-organic compounds is an isoprenoid compounds.

8. system as claimed in claim 1, the capacity of wherein said container is at least 1000 liters.

9. system as claimed in claim 1, the capacity of wherein said container is at least 10,000 liter.

10. system as claimed in claim 1, the capacity of wherein said container is at least 50,000 liter.

11. system as claimed in claim 1, the capacity of wherein said container is at least 100,000 liter

12. system as claimed in claim 1, the capacity of wherein said container is at least 500,000 liter.

13. system as claimed in claim 1, the capacity of wherein said container is at least 1,000,000 liter.

14. system as claimed in claim 1, wherein said organic second comprises described bio-organic compounds mutually.

15. system as claimed in claim 1, wherein said organic second comprises at least 90% bio-organic compounds mutually.

16. make C ₅-C ₂₀The system of isoprenoid compounds comprises:

A. the container of at least 100 liters of capacity;

B. the aqueous culture medium of forming first phase in this container;

C. a large amount of host cells in the aqueous culture medium, described host cell can be made at least a isoprenoid; With,

D. with the first contacted liquid organic second mutually, comprise described at least a isoprenoid.

17. the system in the claim 16, wherein said isoprenoid compounds is a hemiterpene.

18. the system in the claim 16, wherein said isoprenoid compounds is a monoterpene.

19. the system in the claim 16, wherein said isoprenoid compounds is a sesquiterpene.

20. the system in the claim 16, wherein said isoprenoid compounds is a diterpene.

21. produce the method for bio-organic compounds, comprise:

A. cultivate a large amount of host cells in aqueous culture medium, described host cell produces at least a bio-organic compounds, and wherein this aqueous culture medium forms first mutually with host cell;

B. form with the first contacted liquid organic second mutually, described second comprises described at least a bio-organic compounds mutually;

C. from first at least a portion of separating second phase mutually; With,

D. separate described at least a bio-organic compounds from second mutually.

22. the method for claim 21, wherein said organic second induces formation mutually.

23. the method for claim 21, wherein said organic second communicates associates and first shifts out second in mutually and separate mutually.

24. as the system of claim 21, wherein said at least a bio-organic compounds is C ₅Bio-organic compounds.

25. as the system of claim 21, wherein said at least a bio-organic compounds is C ₁₀Bio-organic compounds.

26. as the system of claim 21, wherein said at least a bio-organic compounds is C ₁₅Bio-organic compounds.

27. as the system of claim 21, wherein said at least a bio-organic compounds is C ₂₀Bio-organic compounds.

28. as the system of claim 21, wherein said at least a bio-organic compounds is C ₂₀₊Bio-organic compounds.

29. the method for claim 21, wherein said separating step comprises absorption.

30. the method for claim 21, wherein said separating step comprises distillation.

31. the method for claim 21, wherein said separating step comprise the solution-air extraction.

32. the method for claim 21, wherein said separating step comprises liquid-liquid extraction.

33. the method for claim 21, wherein said separating step comprises ultrafiltration.

34. the method for claim 21, wherein said at least a bio-organic compounds is an isoprenoid compounds.

35. produce C ₅-C ₂₀The method of isoprenoid compounds comprises:

A. cultivate a large amount of host cells in aqueous culture medium, described host cell is produced at least a C at least in the container of 100 liters of capacity ₅-C ₂₀Isoprenoid compounds;

B. form and comprise described at least a C ₅-C ₂₀The organic phase of isoprenoid compounds;

At least a portion of c. from aqueous culture medium, separating described organic phase; With,

D. from described organic phase, separate described at least a C ₅-C ₂₀Isoprenoid compounds.

36. the method for claim 35, wherein said at least a isoprenoid compounds is a hemiterpene.

37. the method for claim 35, wherein said at least a isoprenoid compounds is a monoterpene.

38. the method for claim 35, wherein said at least a isoprenoid compounds is a sesquiterpene.

39. the method for claim 35, wherein said at least a isoprenoid compounds is a diterpene.

40. the method for claim 35, wherein said organic phase is induced formation.

41. the method for claim 35, wherein said organic phase is separated by shifting out organic phase from aqueous phase.

42. the method for claim 35, wherein said separating step comprises absorption.

43. the method for claim 35, wherein said separating step comprises distillation.

44. the method for claim 35, wherein said separating step comprise the solution-air extraction.

45. the method for claim 35, wherein said separating step comprises liquid-liquid extraction.

46. the method for claim 35, wherein said separating step comprises ultrafiltration.

47. the fuel fabrication biological production system comprises:

A. one or more fermentation systems comprise:

I) container of at least 100 liters of at least one capacity;

The aqueous culture medium of ii) in described at least one container, forming first phase;

Iii) a large amount of host cells in the aqueous culture medium, described host cell can be made, produce or synthetic at least a bio-organic compounds; With,

Iv) with the first contacted liquid organic second mutually, it comprises described at least a bio-organic compounds;

B. one or more first phase-separation system, thus separate with one or more components of second organic phase or second organic phase first;

C. selectively one or more second phase-separation system, thus described at least a bio-organic compounds from second organic phase, separated;

D. selectively one or more reactors or container, wherein said at least a bio-organic compounds chemically or biologically is modified;

E. selectively one or more purification systems, thereby purifying or be further purified the bio-organic compounds of described bio-organic compounds or modification;

F. selectively one or more mixing vessels or system are used to mix described at least a bio-organic compounds and one or more additional fuel elements; With

G. selectively one or more systems that are further purified, thereby purifying or be further purified the mixture of described at least a bio-organic compounds and described one or more additional fuel elements.

48. make the method for fuel composition, comprise:

A. in aqueous culture medium, cultivate a large amount of host cells, described host cell production, manufacturing or synthetic at least a bio-organic compounds, wherein said aqueous culture medium is formed first phase;

B. form with the first contacted liquid organic second mutually, it comprises described at least a bio-organic compounds;

C. from first at least a portion of separating second phase mutually;

D. separate described at least a bio-organic compounds from second mutually;

E. selectively chemically or biologically modify described at least a bio-organic compounds;

F. the bio-organic compounds of described bio-organic compounds of purifying or modification selectively;

G. selectively mix described at least a bio-organic compounds and one or more additional fuel elements; With

H. the mixture of described one or more bio-organic compounds of purifying and described one or more additional fuel elements selectively.

Images