WO2009085068A1

WO2009085068A1 - Compositions comprising artemisinic acid and methods of their preparation

Info

Publication number: WO2009085068A1
Application number: PCT/US2008/011734
Authority: WO
Inventors: Glenn Dorin; Ronald Henry
Original assignee: Amyris Biotechnologies, Inc.
Priority date: 2007-12-27
Filing date: 2008-10-14
Publication date: 2009-07-09

Abstract

Provided herein are compositions comprising purified artemisinic acid and methods of their preparation. The artemisinic acid can be used, for example, for the preparation of artemisinin, an anti-malaria compound.

Description

COMPOSITIONS COMPRISING ARTEMISINIC ACID AND METHODS OF THEIR PREPARATION

[0001] This application claims the benefit of U.S. Provisional Application Nos. 61/017,097, filed December 27, 2007, and 61/029,315, filed February 16, 2008, each of which is incorporated by reference in its entirety.

1. FIELD OF THE INVENTION

[0002] Provided herein are methods for producing artemisinic acid and compositions produced by such methods. Artemisinic acid is useful, for example, for the production of artemisinin, a compound useful for the treatment of malaria.

2. BACKGROUND OF THE INVENTION

[0003] Approximately 270 million people are infected with malaria, making it one of the world's major infectious diseases. Developing new anti-malarial drugs, and alternative methods of producing anti-malarial drugs, is therefore an important world health objective.

[0004] One of these anti-malarial drugs is artemisinin. Artemisinin is a component of the traditional Chinese medicinal herb Artemisia annua, which has been utilized for controlling symptoms of fever in China for over 1000 years. In the scientific literature, artemisinin is also sometimes referred to by its Chinese name, Qinghaosu. Recent strides have been made in understanding the properties and structure of this molecule. The compound was first isolated in 1972. Its anti-malarial activity was discovered in 1979 {Chinese Med. J, 92: 811 (1979)). The total synthesis of the molecule was accomplished in 1983 (Schmid, G., Hofheinz, W., J. Am. Chem. Soc, 105: 624 (1983)).

[0005] Production of artemisinin can be accomplished through several routes. One method involves extracting artemisinin from Artemisia annua. A drawback of this method is the low and inconsistent yields (0.01-0.8%) of artemisinin from the plant (Wallart, et al., Planta Med 66: 57-62 (2000); Abdin, et al, Planta Med 69: 289-299 (2003)). An alternate production procedure involves extracting an artemisinin precursor, artemisinic acid, from Artemisia annua and then synthetically converting this molecule into artemisinin. Because artemisinic acid can be present in Artemisia annua at levels approximately 10 times higher than artemisinin, the conversion of the former to the latter has received a great deal of attention. However, the yields of artemisinic acid from Artemisia annua are variable and despite the quick growth of Artemisia annua, it is currently estimated that the world's supply of the plant would meet less than 10% of the world's demand for artemisinic acid and artemisinin. Therefore, artemisinic acid is generally considered to be inaccessible (Haynes et al., Chem. Bio. Chem., 6: 659-667 (2005)) and, a need for an economical and scalable method of producing artemisinin remains.

[0006] A synthetic route for the conversion of artemisinic acid to artemisinin has been described in U.S. Pat. No. 4,992,561 to Roth et al. Therefore, a reliable and cost-effective source of artemisinic acid would provide an important step towards a sustainable method of producing the anti-malaria compound artemisinin.

3. SUMMARY OF THE INVENTION

[0007] Provided herein are methods useful for the purification of artemisinic acid from compositions comprising artemisinic acid and one or more contaminants. In certain embodiments, the methods are capable of providing compositions comprising artemisinic acid in high yield, high purity or both. In certain embodiments, the methods can be carried out in as few as three steps. The resulting artemisinic acid can be used, for example, for the preparation of artemisinin, a compound useful for the treatment of malaria.

[0008] In one aspect, provided herein are methods for the purification of artemisinic acid from compositions comprising artemisinic acid. In certain embodiments, the compositions comprising artemisinic acid are aqueous compositions and the methods comprise 1) solvent extraction of the aqueous composition, yielding a first solvent phase, followed by 2) aqueous extraction of the first solvent phase, yielding a second aqueous phase, followed by 3) precipitation of artemisinic acid from the second aqueous phase. In certain embodiments, the methods are capable of providing solid, crystalline artemisinic acid in pure form and high yield. As shown in the examples below, the methods can provide 80-90% yield at each step and can provide artemisinic acid at 90% or greater purity following the precipitation step.

[0009] The starting aqueous composition comprising artemisinic acid (the "first aqueous composition" in the sections below) can be any composition comprising artemisinic acid known to those of skill in the art. In certain embodiments, the first aqueous composition is derived from a cell extract comprising artemisinic acid. In certain embodiments, the first aqueous composition is derived from a plant extract comprising artemisinic acid. In certain embodiments, the first aqueous composition is derived from a cell culture broth, e.g. a fermentation broth, comprising artemisinic acid. Advantageously, in certain embodiments, the starting material, e.g. cell extract, plant extract or cell culture broth, can be used directly in the methods provided herein. In further embodiments, the starting material can be treated according to any technique deemed suitable to those of skill in the art prior to the initial step of the methods provided herein. For instance, in certain embodiments, an extract or broth can be separated from cells and cellular debris by, for example, centrifugation or filtration to yield a first aqueous composition. For convenience, the resulting composition can be described as "derived" from the extract or broth.

[0010] The solvent extraction step can be carried out be any solvent extraction technique apparent to those of skill in the art. Exemplary solvent extraction techniques are described in the sections below. Typically, the aqueous solution ("first aqueous solution") is contacted with a first solvent under conditions suitable for artemisinic acid to partition into a resulting solvent phase ("the first solvent phase"). The resulting aqueous phase ("first aqueous phase") can be discarded. In certain embodiments, the pH of the first aqueous suspension is adjusted to acidic conditions. In certain other embodiments, the solvent is immiscible with the first acqueous solution. Illustrative examples of such solvents include as butyl acetate, ethyl acetate, isopropyl myristate, methyl isobutyl ketone, methyl oleate, and toluene.

[0011] The aqueous extraction step can be according to any aqueous extraction technique deemed suitable by one of skill in the art. Exemplary techniques are described below. Typically, the first solvent phase is contacted with a second aqueous solution under conditions suitable for artemisinic acid to partition into the resulting aqueous phase ("second aqueous phase"). This can be accomplished with an aqueous solution at high pH. The resulting solvent phase ("second solvent phase") can be discarded. In certain embodiments, the pH is between about 10 and about 11.5. The pH can be maintained by any buffer known to those of skill in the art to be capable of buffering at the chosen pH. Useful buffers include phosphate, carbonate, borate and citrate buffers.

[0012] The precipitation step can be carried out by any technique deemed suitable by one of skill in the art. Exemplary precipitation techniques are described in the sections below. In certain embodiments, artemisinic acid can be precipitated by lowering the pH of the second aqueous phase with acid. In other embodiments, a surfactant is added prior to acidification. In still other embodiments, the surfactant is sodium dodecyl sulfate. In certain embodiments, lowering the pH to about 5 or lower is sufficient to precipitate artemisinic acid. The precipitated artemisinic acid can be collected from the second aqueous phase by standard techniques. In certain embodiments, the resulting artemisinic acid is in crystalline form. [0013] In another aspect, the present invention provides compositions prepared by the methods of the invention. In certain embodiments, the compositions comprise artemisinic acid in high yield. In certain embodiments, the compositions comprise artemisinic acid in high purity. In certain embodiments, the compositions comprise artemisinic acid in solid form. In certain embodiments, the compositions comprise artemisinic acid in crystalline form.

[0014] The artemisinic acid provided herein can be used for any purpose one of skill in the art deems suitable for artemisinic acid. In certain embodiments, the artemisinic acid is used for the preparation of artemisinin by any technique known to those of skill. The artemisinin can be used for the treatment of malaria in a subject in need thereof by methods known to those of skill in the art.

[0015] In another aspect, provided herein are processes for purification of artemisinic acid from a first aqueous composition comprising artemisinic acid and one or more contaminants, said method comprising the steps of:

(a) extracting artemisinic acid from the first aqueous composition with a solvent immiscible with said aqueous composition to yield a first solvent phase comprising artemisinic acid;

(b) extracting artemisinic acid from said first solvent phase with a second aqueous composition at a pH of from about 10 to about 11.5 to yield a second aqueous phase comprising artemisinic acid; and

(c) precipitating artemisinic acid from the second aqueous phase.

[0016] In some embodiments, the first aqueous composition comprises fermentation broth in which a microbial strain engineered to make artemisinic acid occurs. When such fermentation occurs in the presence of an immiscible solvent, this type of fermentation is called extractive fermentation. The solvent is generally a nonpolar solvent such as isopropyl myristate, or methyl oleate. Under these circumstances, the artemisinic acid is extracted from the fermentation broth into the immiscible solvent. In other words, a solvent extraction step effectively occurs during fermentation.

[0017] In still other embodiments, precipitation occurs from the second aqueous phase by adding an acid and lowering the pH into the acidic range (below 7). In certain embodiments, a surfactant is added prior to the acidification. In certain other embodiments, the surfactant is sodium dodecyl sulfate. [0018] In another aspect, provided herein are processes for purification of artemisinic acid from a first aqueous composition comprising artemisinic acid and one or more contaminants, said method comprising the steps of:

(a) contacting the first aqueous composition having a pH about 7 or less with an organic solvent under conditions suitable for artemisinic acid to partition into the resulting first solvent phase;

(b) recovering said first solvent phase;

(c) contacting said first solvent phase with an aqueous sodium phosphate solution at a pH of about 10 or greater, said aqueous solution being immiscible with said solvent phase, under conditions suitable for the artemisinic acid to partition into the resulting second aqueous phase;

(d) recovering said second aqueous phase;

(e) acidifying said second aqueous phase to a pH of about 5 or less under conditions suitable for precipitation of said artemisinic acid; and

(f) recovering said artemisinic acid.

[0019] In some embodiments, the first aqueous composition is derived from a fermentation broth. In other embodiments, the first aqueous composition is derived from a cell extract. In further embodiments, the first aqueous composition is derived from a plant extract.

[0020] In some embodiments, the pH of the first aqueous composition is from about 2 to about 7. In other embodiments, the pH of the first aqueous composition from about 3 to about 6. In further embodiments, the pH of the first aqueous composition is about 3, about 4, about 5 or about 6.

[0021] In some embodiments, the extraction of the aqueous solution is in the presence of a surfactant. The presence of a surfactant in the aqueous solution in the presence of a solvent phase expedites the extraction of artemisinic acid into the solvent phase. In other embodiments, the surfactant is sodium dodecyl sulfate. In certain embodiments, the surfactant is sodium dodecyl sulfate at a concentration from about 0.2% to about 5%, from about 0.2% to about 3%, from about 0.2% to about 2%, or at a concentration of about 0.2%, about 0.5%, about 1%, about 1.25% or about 2%. In further embodiments, the surfactant is sodium dodecyl sulfate at a concentration of about 0.2%, about 0.5%, about 1%, about 1.25% or about 2%.

[0022] In some embodiments, steps (a) and (b) occur during extractive fermentation. In other words, fermentation of host cells capable of making artemisinic acid occurs in the presence of a solvent that is immiscible with the fermentation medium. Under these circumstances, the fermentation medium is the first aqueous composition and the immiscible solvent comprises the first solvent phase. During the course of fermentation, the artemisinic acid made by the host cells is extracted into the immiscible solvent (in effect an in situ solvent extraction).

[0023] In some embodiments, the solvent is a nonpolar solvent. In other embodiments, the solvent is selected from the group consisting of butyl acetate, ethyl acetate, isopropyl myristate, methyl isobutyl kethone, methyl oleate, and toluene. In certain embodiments, the solvent is a polar solvent. In further embodiments, the solvent is selected from the group consisting of butanol, methyl ethyl ketone and 2-butanol.

[0024] In some embodiments, the second aqueous composition is buffered with a buffer wherein the buffer comprises phosphate, carbonate, borate, citrate or a combination thereof. In other embodiments, the second aqueous composition is buffered with potassium phosphate or potassium borate. In certain embodiments, the second aqueous composition is buffered with sodium phosphate, sodium carbonate, sodium borate or sodium citrate. In other embodiments, the second aqueous composition is buffered with about 1 -5 % w/v potassium phosphate or about 1-5 % w/v sodium phosphate. In further embodiments, the second aqueous composition is buffered with about 1% w/v sodium phosphate. The potassium phosphate and the sodium phosphate can be present in the mixture at a concentration of, for example, about 0.1 to about 6% (w/v), about 0.2 to about 5% (w/v), about 0.3 to about 4% (w/v), about 0.5 to about 3% (w/v), about 0.6 to about 2% (w/v), about 0.7 to about 1% (w/v) or about 1% (w/v), where the concentration is measured by the weight (in gram) of the salt over the volume (in ml) of the buffer.

[0025] In some embodiments, the artemisinic acid is precipitated by acidifying the second aqueous phase. In other embodiments, the second aqueous phase is acidified to a pH less than about 6, less than about 5, from about 2 to about 6, or from about 2.5 to about 5.

[0026] In some embodiments, a surfactant is added to the second aqueous phase prior to acidification. In this instance where the surfactant is added to the aqueous phase in the absence of a solvent phase, the presence of the surfactant in the aqueous phase appears to keep contaminants from co-precipitating with the artemisinic acid by increasing the solubility of the contaminants in the second aqueous phase. In other embodiments, the surfactant is sodium dodecyl sulfate. In certain embodiments, the surfactant is sodium dodecyl sulfate at a concentration from about 0.01% to about 5%, from about 0.01% to about 3%, from about 0.01% to about 1%, from about 0.01% to about 0.5%, from about 0.01% to about 0.1%, or from about 0.1% to about 0.05%.

[0027] In another aspect, provided herein are processes for purification of artemisinic acid from an extractive fermentation, said method comprising the steps of:

(a) growing host cells capable of making artemisinic acid in an aqueous fermentation medium and in the presence of a solvent phase;

(b) recovering the solvent said solvent phase;

(c) contacting said solvent phase with an aqueous sodium phosphate solution at a pH of about 10 or greater, said aqueous solution being immiscible with said solvent phase, under conditions suitable for the artemisinic acid to partition into the resulting an aqueous phase;

(d) recovering said aqueous phase;

(e) acidifying said aqueous phase to a pH of about 5 or less under conditions suitable for precipitation of said artemisinic acid; and

(f) recovering said artemisinic acid.

[0028] In some embodiments, the aqueous composition is buffered with a buffer wherein the buffer comprises phosphate, carbonate, borate, citrate or a combination thereof. In other embodiments, the second aqueous composition is buffered with potassium phosphate or potassium borate. In certain embodiments, the aqueous composition is buffered with sodium phosphate, sodium carbonate, sodium borate or sodium citrate. In other embodiments, the aqueous composition is buffered with about 1-5 % w/v potassium phosphate or about 1-5 % w/v sodium phosphate. In further embodiments, the aqueous composition is buffered with about 1% w/v sodium phosphate. The potassium phosphate and the sodium phosphate can be present in the mixture at a concentration of, for example, about 0.1 to about 6% (w/v), about 0.2 to about 5% (w/v), about 0.3 to about 4% (w/v), about 0.5 to about 3% (w/v), about 0.6 to about 2% (w/v), about 0.7 to about 1% (w/v) or about 1% (w/v), where the concentration is measured by the weight (in gram) of the salt over the volume (in ml) of the buffer. [0029] In some embodiments, the artemisinic acid is precipitated by acidifying the aqueous phase. In other embodiments, the second aqueous phase is acidified to a pH less than about 6, less than about 5, from about 2 to about 6, or from about 2.5 to about 5.

[0030] In some embodiments, a surfactant is added to the aqueous phase prior to acidification. In other embodiments, the surfactant is sodium dodecyl sulfate. In certain embodiments, the surfactant is sodium dodecyl sulfate at a concentration from about 0.01% to about 5%, from about 0.01% to about 3%, from about 0.01% to about 1%, from about 0.01% to about 0.5%, from about 0.01% to about 0.1%, or from about 0.1% to about 0.05%.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 provides extraction of artemisinic acid from a first aqueous composition as a function of time; and

[0033] FIG. 2 provides extraction yield of artemisinic acid by butyl acetate as a function of sodium dodecyl sulfate (SDS) concentration.

[0034] FIG. 3 provides extraction yield of artemisinic acid by ethyl acetate as a function of sodium dodecyl sulfate (SDS) concentration.

4. DETAILED DESCRIPTION OF THE INVENTION 4.1 Definitions

[0035] A composition that is a "substantially pure" compound refers to a composition that is substantially free of one or more other compounds, i.e., the composition contains greater than 80%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, greater than 99.6%, greater than 99.7%, greater than 99.8%, or greater than 99.9% of the compound; or less than 20%, less than 10%, less than 5%, less than 3%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the one or more other compounds, based on the total volume or weight of the composition.

[0036] In the following description, all numbers disclosed herein are approximate values, regardless whether the word "about" or "approximate" is used in connection therewith. They may vary by 1 percent, 2 percent, 5 percent, or, sometimes, 10 to 20 percent. Whenever a numerical range with a lower limit, R^L, and an upper limit, R^u, is disclosed, any number falling within the range is specifically disclosed. In particular, the following numbers within the range are specifically disclosed: R=R^L+k*(R^u-R^L), wherein k is a variable ranging from 1 percent to 100 percent with a 1 percent increment, i.e., k is 1 percent, 2 percent, 3 percent, 4 percent, 5 percent,..., 50 percent, 51 percent, 52 percent,..., 95 percent, 96 percent, 97 percent, 98 percent, 99 percent, or 100 percent. Moreover, any numerical range defined by two R numbers as defined in the above is also specifically disclosed.

4.2 Embodiments of the Invention

[0037] Provided herein are methods for the preparation of artemisinic acid from compositions such as cell extracts, plant extracts and fermentation broths. Also provided herein are compositions comprising artemisinic acid prepared according to a method herein. In certain embodiments, the compositions can display advantageous yield or purity. In certain embodiments, the compositions provide pure solid forms of artemisinic acid.

4.3 Compounds

[0038] Provided herein are compositions comprising artemisinic acid and methods for their preparation. "Artemisinic acid" is the compound according to the following:

A systematic name of artemisinic acid is 2-((lR,4R,4aS,8aR)-4,7-dimethyl-l,2,3,4,4a,5,6,8a- octahydronaphthalen-l-yl)acrylic acid. Unless otherwise specified, the term "artemisinic acid" includes salts of the above compound, pharmaceutically acceptable salts of the above compound, isotopic variants of the above compound, solid forms of the above compound and forms of the above compound in solution.

[0039] In certain embodiments, provided herein are compositions comprising substantially pure artemisinic acid. In certain embodiments, provided herein are compositions comprising artemisinic acid in a solid form. In certain embodiments, provided herein are compositions comprising artemisinic acid in crystalline form.

4.4 Preparation of the Compounds

[0040] In certain embodiments, artemisinic acid is prepared from a starting mixture comprising artemisinic acid and one or more contaminants. The contaminant can be any substance to be separated from the artemisinic acid, for instance, a peptide, a polypeptide, a fatty acid, another small molecule, etc. In advantageous embodiments, the starting mixture is derived from a biological system capable of producing crude artemisinic acid. For instance, in certain embodiments, the starting mixture can be derived from a plant capable of producing crude artemisinic acid. In certain embodiments, the starting mixture can be derived from extract from such a plant. In further embodiments, the starting mixture can be derived from a cell capable of producing artemisinic acid. In certain embodiments, the starting mixture can be derived from an extract of such a cell. In certain embodiments, the starting mixture can be derived from a fermentation medium or a cell culture medium in contact with such a cell. In certain embodiments, the starting mixture can be derived from a fermentation broth in contact with such a cell.

[0041] In particular embodiments, the starting mixture can be derived from a cell that natively produces artemisinic acid. Useful cells include cells of Artemisia anuua L. In further particular embodiments, the starting mixture can be derived from a cell that is engineered to produce artemisinic acid. Exemplary host cells are described in a section below.

[0042] Starting mixtures can be derived from the above source materials by any technique apparent to those of skill in the art. In certain embodiments, the source material {e.g. cell extract, plant extract, fermentation broth, cell culture medium) can be used in the methods provided herein directly. In other words, the source material is the starting mixture in these embodiments. For instance, in certain embodiments, the methods provided herein are applied to a fermentation broth from a host cell capable of producing artemisinic acid. In further embodiments, the source material can be treated with any technique apparent to those of skill in the art to facilitate purification of artemisinic acid. For instance, a crude cell extract can be treated to remove cellular debris by, for example, centrifugation, filtration or other techniques deemed appropriate to those of skill in the art. Such starting mixtures are describe as "derived from" the source material to indicate that the source material may be altered prior to the steps of the methods provided herein.

[0043] In typical embodiments, the starting material is aqueous and monophasic, i.e. it is a first aqueous composition. In certain embodiments, insoluble material can be removed from the starting material to yield the first aqueous composition. Techniques to remove insoluble material should be readily apparent to one of skill in the art such as centrifugation and filtration. In certain embodiments, the starting material can comprise more than one phase. If so, in certain embodiments, the method herein can be applied to the aqueous portion of the starting material, i.e. that phase is the first aqueous composition. In certain embodiments, the aqueous portion is removed from any other phase prior to the methods provided herein. In other embodiments, the methods provided herein are applied to the entire starting material including its aqueous portion, i.e. the aqueous portion is the first aqueous composition.

4.4.1. Solvent extraction

[0044] In a first step, artemisinic acid is extracted from the first aqueous composition with a solvent. The solvent can be any solvent apparent to those of skill in the art to be capable of extracting artemisinic acid from the first aqueous solution. The solvent extraction can be carried out by any extraction technique apparent to those of skill in the art.

[0045] In useful embodiments, the solvent is selected to be immiscible with the first aqueous composition. In other words, mixture of the solvent with the first aqueous composition should be capable of providing a two phase mixture after equilibration.

[0046] In certain embodiments, the solvent is a polar solvent immiscible with the first aqueous composition. In certain embodiments the solvent is selected from butanol and methyl ethyl ketone. In certain embodiments, the solvent is butanol, e.g. sec-butanol. In certain embodiments, the solvent is methyl ethyl ketone.

[0047] In further embodiments, the solvent is a nonpolar solvent immiscible with the first aqueous composition. In certain embodiments, the solvent is selected from butyl acetate, ethyl acetate, isopropyl myristate, methyl isobutyl ketone, methyl oleate, and toluene. In certain embodiments, the solvent is butyl acetate. In certain embodiments, the solvent is ethyl acetate. In certain embodiments, the solvent is toluene.

[0048] The solvent can be added at any volume deemed suitable by one of skill in the art. In certain embodiments, the solvent is added at a volume of from about 0.5 to about 2.0 volumes of the first aqueous composition, from about 0.2 to about 1.0 volumes of the first aqueous composition, from about 0.3 to about 1.0 volumes of the first aqueous composition, from about 0.4 to about 1.0 volumes of the first aqueous composition, or from about 0.5 to about 1.0 volumes of the first aqueous composition. In certain embodiments, the solvent is added at a volume of about 0.5 volumes of the first aqueous composition.

[0049] In certain embodiments, the first aqueous composition is at a low pH for the solvent extraction. It is believed that more artemisinic acid can be extracted from the first aqueous composition at a lower pH. In certain embodiments, the pH less than about 7, less than about 6, less than about 5, less than about 4, less than about 3, or less than about 2. In certain embodiments, the pH is from about 2 to about 6, from about 2 to about 5, from about 2 to about 4. hi certain embodiments, the pH is about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, or about 6.5.

[0050] The pH can be adjusted with any acid or base deemed suitable to those of skill in the art. In certain embodiments, the pH is adjusted with an acid to the desired pH. In particular embodiments, the acid is sulfuric acid.

[0051] In certain embodiments, the solvent extraction is carried out with a surfactant. The presence of the surfactant in the aqueous phase in the presence of a solvent phase increases the kinetics of the extraction of artemisinic acid into the solvent phase from the aqueous phase, The surfactant can be any surfactant known to those of skill in the art. The surfactant can be added to the first aqueous composition, to the solvent, or to the mixture of the first aqueous composition and the solvent. Useful surfactants include anionic surfactants such as sodium dodecyl sulfate and ammonium laurel sulfate. In certain embodiments, the surfactant is sodium dodecyl sulfate. The surfactant can be present in the mixture at a concentration of, for example, about 0.1 to about 4% (w/v), about 0.1 to about 3% (w/v), about 0.1 to about 2% (w/v), where the concentration is measured by the weight (in gram) of the surfactant over the volume (in ml) of the first aqueous composition. In certain embodiments, the surfactant is present in the mixture at a concentration of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 1%, about 1.25%, about 1.5%, about 2%, about 2.5%, about 3% or about 4%.

[0052] The solvent extraction can be carried out at any temperature deemed suitable by one of skill in the art. In certain embodiments, the extraction is carried out at a temperature between about 20 °C and about 40 ⁰C or between about 20 °C and about 30 °C. In certain embodiments, the extraction is carried out at ambient temperature.

[0053] The solvent and the first aqueous composition can be mixed by standard techniques such as inversion, shaking, stirring etc.

[0054] Mixing the first aqueous composition with the solvent can be carried out for any length of time deemed suitable by one of skill in the art to extract a sufficient amount of artemisinic acid from the first aqueous composition, hi certain embodiments, the extraction is carried out for at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 45 minutes, or at least about 60 minutes.

[0055] After mixing, the mixture can be allowed to equilibrate to two phases, an aqueous phase (the "first aqueous phase" of the methods) and a solvent phase (the "first solvent phase" of the methods). The first solvent phase can be removed from the first aqueous phase by any technique deemed suitable by one of skill in the art. The first aqueous phase is not needed in the methods below and can be discarded, hi certain embodiments, the first aqueous phase can be extracted further with solvent one or more times to improve yields.

[0056] When artemisinic acid is produced from non-extractive fermentation of host cells, the fermentation broth itself can comprises the first aqueous composition and solvent extraction can occur as described above.

[0057] However, when artemisinic acid is produced from extractive fermentation, solvent extraction effectively occurs during the fermentation process. The fermentation medium is the first aqueous composition and the immiscible solvent comprises the first solvent phase. During the course of fermentation, the artemisinic acid made by the host cells is extracted into the immiscible solvent (in effect an in situ solvent extraction). If desired, solvent extraction of the fermentation broth can also occur to increase the total amount of artemisinic acid that is recovered.

[0058]

4.4.2. Aqueous extraction

[0059] In the second step of the methods provided herein, artemisinic acid is extracted from the first solvent phase, prepared in the step above, with an aqueous composition ("the second aqueous composition").

[0060] The second aqueous composition can be any aqueous composition deemed capable of extracting artemisinic acid from the first solvent phase. In particular embodiments, the second aqueous composition is at a pH sufficient to extract the artemisinic acid.

[0061] In certain embodiments, the pH of the second aqueous composition is from about 10 to about 11. 5. In certain embodiments, the pH of the second aqueous composition is about 10, about 10.1, about 10.2, about 10.3, about 10.4, about 10.5, about 10.6, about 10.7, about 10.8, about 10.9, about 11, about 11.1, about 11.2, about 11.3, about 11.4, or about 11.5. In certain embodiments, the pH is about 10.7.

[0062] The pH of the second aqueous composition can be maintained by any technique apparent to those of skill in the art. In certain embodiments, the second aqueous composition is buffered at the desired pH. Useful buffers include potassium, carbonate, borate and citrate buffers. In certain embodiments, the buffer is selected from potassium phosphate and potassium carbonate. In certain embodiments, the buffer is selected from sodium phosphate, sodium carbonate, sodium borate and sodium citrate. In particular embodiments, the buffer is sodium phosphate. The buffer can be at any concentration deemed useful by one of skill in the art. In certain embodiments, the buffer is at a concentration of about 50 mM to about 350 mM, about 50 mM to about 300 mM, about 50 mM to about 200 mM, or about 50 mM to about 10OmM. In certain embodiments, the concentration of the buffer is about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, or about 350 mM.

[0063] The aqueous extraction can be carried out at any temperature deemed suitable by one of skill in the art. In certain embodiments, the extraction is carried out at a temperature between about 20 °C and about 40 ⁰C or between about 20 °C and about 30 °C. In certain embodiments, the extraction is carried out at ambient temperature.

[0064] The second aqueous composition can be added at any volume deemed suitable by one of skill in the art. In certain embodiments, the second aqueous composition is added at a volume of from about 0.5 to about 2.0 volumes of the first solvent phase, or from about 0.5 to about 1.0 volumes of the first solvent phase. In certain embodiments, the second aqueous composition is added at a volume of about 0.5 volumes of the first solvent phase. In certain embodiments, the second aqueous composition is added at a volume of about 1.0 volumes of the first solvent phase.

[0065] The first solvent phase and the second aqueous composition can be mixed by standard techniques such as inversion, shaking, stirring etc.

[0066] Mixing the second aqueous composition with the first solvent phase can be carried out for any length of time deemed suitable by one of skill in the art to extract a sufficient amount of artemisinic acid from the first aqueous composition. In certain embodiments, the extraction is carried out for at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 45 minutes, or at least about 60 minutes.

[0067] After mixing, the mixture can be allowed to equilibrate to two phases, an aqueous phase (the "second aqueous phase" of the methods) and a solvent phase (the "second solvent phase" of the methods). The second aqueous phase can be removed from the second solvent phase by any technique deemed suitable by one of skill in the art. The second solvent phase is not needed in the methods below and can be discarded. In certain embodiments, the second solvent phase can be extracted further with solvent one or more times to improve yields.

4.4.3. Precipitation of artemisinic acid

[0068] In a third step, purified artemisinic acid can be precipitated from the second aqueous phase prepared in the method steps above. Precipitation can be carried out by any technique apparent to those of skill in the art.

[0069] In certain embodiments, artemisinic acid is precipitated by acidifying the second aqueous phase. The acidification can be to any pH deemed suitable for precipitation of the artemisinic acid. In certain embodiments, the pH is less than about 6, less than about 5, less than about 4, less than about 3, or less than about 2. In certain embodiments, the pH is from about 2 to about 6, from about 2 to about 5, from about 2 to about 4. In certain embodiments, the pH is about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, or about 6.5. In certain embodiments, the pH is about 5. The second aqueous phase can be acidified with any acid deemed useful by one of skill in the art. In certain embodiments, the second aqueous phase is acidified by sulfuric acid.

[0070] In certain embodiments, a surfactant is added to the second aqueous phase prior to acidification. In this instance, the presence of the surfactant in the second aqueous phase in the absence of a solvent phase increases the solubility of contaminants in the second aqueous phase thereby minimizing the likelihood that they will co-precipitate with the artemisinic acid. Useful surfactants include anionic surfactants such as sodium dodecyl sulfate and ammonium laurel sulfate. In certain embodiments, the surfactant is sodium dodecyl sulfate. In certain embodiments, the surfactant is sodium dodecyl sulfate at a concentration from about 0.01% to about 5%, from about 0.01% to about 3%, from about 0.01% to about 1%, from about 0.01% to about 0.5%, from about 0.01% to about 0.1%, or from about 0.1% to about 0.05%. [0071] In certain embodiments, the acidified second aqueous phase is homogenized to facilitate precipitation. Homogenization can be by any homogenization technique known to those of skill in the art including, for example, a homogenizer, a high-pressure homogenizer and a sonicator. In certain embodiments, the second aqueous phase is homogenized with a high pressure homogenizer.

[0072] The precipitation can be carried out at any temperature deemed suitable by one of skill in the art. In certain embodiments, the precipitation is carried out at a temperature between about 20 °C and about 40 °C or between about 20 ⁰C and about 30 °C. In certain embodiments, the precipitation is carried out at ambient temperature.

[0073] The precipitation can be carried out for any length of time deemed suitable for recovery of artemisinic acid.

[0074] Precipitated artemisinic acid can be captured by any technique apparent to those of skill including, for example, centrifugation and filtration. The resulting solid artemisinic acid can be washed and dried by standard techniques.

4.4.4. Purification methods

[0075] In certain embodiments, the above steps are carried out in the order described. In particular, the first aqueous composition is extracted with solvent at low pH, the resulting first solvent phase then is extracted with a second aqueous composition at high pH, and the resulting second aqueous phase is acidified to precipitate purified artemisinic acid.

[0076] Those of skill in the art will recognize that the above steps can also be carried out in other sequences. As described above, artemisinic acid will precipitate or partition into a solvent phase from a low pH aqueous composition, and it will dissolve or partition into a high pH aqueous composition.

[0077] Accordingly, provided herein are further methods of purification. In one embodiment, a first aqueous composition is acidified to a low pH to precipitate artemisinic acid. The resulting solids are resuspended in aqueous solution, and the pH of the solution can be brought to about 12 to dissolve artemisinic acid. The solids can be further washed with aqueous solution to dissolve further artemisinic acid. The resulting pooled aqueous solutions can be acidified to a pH of about 3 to precipitate artemisinic which can be captured by centrifugation. The resulting artemisinic acid can then be extracted with solvent in high purity and high yield. 4.4.5. Host cells

[0078] Useful host cells for the production of crude artemisinic acid include those described in Ro et al, 2006, Nature 440:940-943. Such cells can comprise, for example, polynucleotides that encode enzymes of a farnesyl pyrophosphate biosynthetic pathway, a polynucleotide encoding armophadiene synthase from A. annua, and a polynucleotide encoding CYPl IAVl from A. annua. See Ro et al, the contents of which are hereby incorporated by reference in their entirety. A farnesyl pyrophosphate pathway is capable of catalyzing the production of farnesyl pyrophosphate from simple sugars. Amorphadiene synthase is capable of catalyzing the production of amorpha-4, 11-diene from farnesyl pyrophosphate. CYP71AV1 is capable of facilitating the production of artemisinic acid from amorpha-4, 11-diene.

[0079] The host cell can be grown according to any technique known to those of skill in the art. In particular, the host cell can be grown in culture medium appropriate for the host cell. In certain embodiments, the host cell is grown or cultured by contact with a simple sugar under conditions suitable for their growth and production of farnesyl pyrophosphate. In certain embodiments, the host cell can be grown or cultured by contact with glucose, galactose, mannose, fructose, ribose or a combination thereof.

[0080] Any suitable host cell may be used in the practice of the present invention. In one embodiment, the host cell is a genetically modified host microorganism in which nucleic acid molecules have been inserted, deleted or modified {i.e., mutated; e.g., by insertion, deletion, substitution, and/or inversion of nucleotides), to either produce farnesyl pyrophosphate or, or increased yields of farnesyl pyrophosphate or farnesyl pyrophosphate. In another embodiment, the host cell is capable of being grown in liquid growth medium.

[0081] Illustrative examples of suitable host cells include archae cells, bacterial cells, eukaryotic cells and microorganisms. Some non-limiting examples of archae cells include those belonging to the genera: Aeropyrum, Archaeglobus, Halobacterium, Methanococcus, Methanobacterium, Pyrococcus, Sulfolobus, and Thermoplasma. Some non-limiting examples of archae strains include Aeropyrum pernix, Archaeoglobus fulgidus, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Pyrococcus abyssi, Pyrococcus horikoshii, Thermoplasma acidophilum, Thermoplasma volcanium.

[0082] Some non-limiting examples of bacterial cells include those belonging to the genera: Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Arthrobacter, Azobacter, Bacillus, Brevibacterium, Chromatium, Clostridium, Corynebacterium, Enterobacter, Erwinia, Escherichia, Lactobacillus, Lactococcus, Mesorhizobium, Methylobacterium, Microbacterium, Phormidium, Pseudomonas, Rhodobacter, Rhodopseudomonas, Rhodospirillum, Rhodococcus, Salmonella, Scenedesmun, Serratia, Shigella, Staphlococcus, Strepromyces, Synnecoccus, and Zymomonas.

[0083] Some non-limiting examples of bacterial strains include Bacillus subtilis, Bacillus amyloliquefacines, Brevibacterium ammoniagenes, Brevibacterium immariophilum, Clostridium beigerinckii, Enterobacter sakazakii, Escherichia coli, Lactococcus lactis, Mesorhizobium loti, Pseudomonas aeruginosa, Pseudomonas mevalonii, Pseudomonas pudica, Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodospirillum rubrum, Salmonella enterica, Salmonella typhi, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, and the like.

[0084] In general, if a bacterial host cell is used, a non-pathogenic strain is preferred. Some non-limiting examples of non-pathogenic strains include Bacillus subtilis, Escherichia coli, Lactibacillus acidophilus, Lactobacillus helveticus, Pseudomonas aeruginosa, Pseudomonas mevalonii, Pseudomonas pudita, Rhodobacter sphaeroides, Rodobacter capsulatus, Rhodospirillum rubrum, and the like.

[0085] Some non-limiting examples of eukaryotic cells include fungal cells. Some non- limiting examples of fungal cells include those belonging to the genera: Aspergillus, Candida, Chrysosporium, Cryotococcus, Fusarium, Kluyveromyces, Neotyphodium, Neurospora, Penicillium, Pichia, Saccharomyces, and Trichoderma.

[0086] Some non-limiting examples of eukaryotic strains include Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Candida albicans, Chrysosporium lucknowense, Fusarium graminearum, Fusarium venenatum, Kluyveromyces lactis, Neurospora crassa, Pichia angusta, Pichia finlandica, Pichia kodamae, Pichia membranaefaciens, Pichia methanolica, Pichia opuntiae, Pichia pastor is, Pichia pijperi, Pichia quercuum, Pichia salictaria, Pichia thermotolerans, Pichia trehalophila, Pichia stipitis, Streptomyces ambofaciens, Streptomyces aureofaciens, Streptomyces aureus, Saccaromyces bayanus, Saccaromyces boulardi, Saccharomyces cerevisiae, Streptomyces fungicidicus, Streptomyces griseochromogenes, Streptomyces griseus, Streptomyces lividans, Streptomyces olivogriseus, Streptomyces rameus, Streptomyces tanashiensis, Streptomyces vinaceus, and Trichoderma reesei. [0087] In general, if a eukaryotic cell is used, a non-pathogenic strain is preferred. Some non-limiting examples of non-pathogenic strains include Fusarium graminearum, Fusarium venenatum, Pichia pastoris, Saccaromyces boulardi, and Saccaromyces cerevisiae.

[0088] In addition, certain strains have been designated by the Food and Drug Administration as GRAS or Generally Regarded As Safe. Some non-limiting examples of these strains include Bacillus subtilis, Lactibacillus acidophilus, Lactobacillus helveticus, and Saccharomyces cerevisiae.

[0089] There are two known biosynthetic pathways that synthesize IPP and its isomer, dimethylallyl pyrophosphate ("DMAPP"). Eukaryotes other than plants use the mevalonate- dependent ("MEV") isoprenoid pathway exclusively to convert acetyl-coenzyme A ("acetyl- CoA") to IPP, which is subsequently isomerized to DMAPP. Prokaryotes, with some exceptions, use the mevalonate-independent or deoxyxylulose 5-phosphate ("DXP") pathway to produce IPP and DMAPP separately through a branch point. In general, plants use both the MEV and DXP pathways for IPP synthesis.

[0090] In general, the MEV pathway comprises six steps.

[0091] In the first step, two molecules of acetyl-coenzyme A are enzymatically combined to form acetoacetyl-CoA. An enzyme known to catalyze this step is, for example, acetyl- CoA thiolase. Some non-limiting examples of nucleotide sequences include the following GenBank accession numbers and the organism from which the sequences derived: (NC_000913 REGION: 2324131..2325315; Escherichia colϊ), (D49362; Paracoccus denitrificans), and (L20428; Saccharomyces cerevisiae).

[0092] 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 synthase. Some non-limiting examples of nucleotide sequences include (NC_001145. complement 19061..20536; Saccharomyces cerevisiae), (X96617; Saccharomyces cerevisiae), (X83882; Arabidopsis thaliana), (AB037907; Kitasatospora griseola), (BT007302; Homo sapiens), and (NC_002758, Locus tag SAV2546, GeneID 1 12257 '1; Staphylococcus aureus).

[0093] In the third step, HMG-CoA is enzymatically converted to mevalonate. An enzyme known to catalyze this step is, for example, HMG-CoA reductase Some non-limiting examples of nucleotide sequences include (NM_206548; Drosophila melanogaster), (NC_002758, Locus tag SAV2545, GeneID 1122570; Staphylococcus aureus), (NM_204485; Gallus gallus), (AB015627; Streptomyces sp. KO 3988), (AF542543; Nicotiana attenuatά), (AB037907; Kitasatospora griseola), (AX128213, providing the sequence encoding a truncated HMGR; Saccharomyces cerevisiae), and (NC_001145: complement (115734..118898; Saccharomyces cerevisiae).

[0094] In the fourth step, mevalonate is enzymatically phosphorylated to form mevalonate 5-phosphate. An enzyme known to catalyze this step is, for example, mevalonate kinase. Some non-limiting examples of nucleotide sequences include (L77688; Arabidopsis thaliana), and (X55875; Saccharomyces cerevisiae).

[0095] 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. Some non-limiting examples of nucleotide sequences include (AF429385; Hevea brasiliensis), (NM 006556; Homo sapiens), and (NC_001145. complement 712315..713670; Saccharomyces cerevisiae).

[0096] In the sixth step, mevalonate 5-pyrophosphate is enzymatically converted into IPP. An enzyme known to catalyze this step is, for example, mevalonate pyrophosphate decarboxylase. Some non-limiting examples of nucleotide sequences include (X97557; Saccharomyces cerevisiae), (AF290095; Enterococcus faecium), and (U49260; Homo sapiens);

[0097] If IPP is to be converted to DMAPP using the mevalonate pathway, then a seventh step is required. An enzyme known to catalyze this step is, for example, IPP isomerase. Some non-limiting examples of nucleotide sequences include (NC_000913, 3031087..3031635; Escherichia coli), and (AF082326; Haematococcus pluvialis).

[0098] In general, the DXP pathway comprises seven steps. In the first step, pyruvate is condensed with D-glyceraldehyde 3-phosphate to make l-deoxy-D-xylulose-5-phosphate. An enzyme known to catalyze this step is, for example, l-deoxy-D-xylulose-5-phosphate synthase. Some non-limiting examples of nucleotide sequences include (AF035440; Escherichia coli), (NC_002947, locus tag PP0527; Pseudomonas putida KT2440), (CP000026, locus tag SPA2301 ; Salmonella enterica Paratyphi, see ATCC 9150), (NC_007493, locus tag RSP_0254; Rhodobacter sphaeroides 2.4.1), (NC_005296, locus tag RPA0952; Rhodopseudomonas palustris CGA009), (NC_004556, locus tag VOX292,; Xylella fastidiosa Temeculal), and (NC_003076, locus tag AT5G11380; Arabidopsis thaliana). [0099] 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. Some non-limiting examples of nucleotide sequences include (ABOl 3300; Escherichia colϊ), (AF148852; Arabidopsis thaliana), (NC_002947, locus tag PP1597; Pseudomonas putida KT2440), (AL939124, locus tag SCO5694; Streptomyces coelicolor A3(2)), (NC_007493, locus tag RSP_2709; Rhodobacter sphaeroides 2.4.1), and (NC_007492, locus tag Pfl_l 107; Pseudomonas fluoresce™ PfO-I).

[00100] In the third step, 2C-methyl-D-erythritol-4-phosphate is converted to 4- diphosphocytidyl-2C-methyl-D-erythritol. An enzyme known to catalyze this step is, for example, 4-diphosphocytidyl-2C-methyl-D-erythritol synthase. Some non-limiting examples of nucleotide sequences include (AF230736; Escherichia coli), (NC_007493, locus_tag RSP_2835; Rhodobacter sphaeroides 2.4.1), (NC_003071, locus tag AT2G02500; Arabidopsis thaliana), and (NC_002947, locusjag PP1614; Pseudomonas putida KT2440).

[00101] In the fourth step, 4-diphosphocytidyl-2C-methyl-D-erythritol is converted to 4- diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate. An enzyme known to catalyze this step is, for example, 4-diphosphocytidyl-2C-methyl-D-erythritol kinase. Some non-limiting examples of nucleotide sequences include (AF216300; Escherichia coli) and (NC_007493, locusjag RSP_1779; Rhodobacter sphaeroides 2.4.1).

[00102] In the fifth step, 4-diphosphocytidyl-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-cyclodiphosphate synthase. Some non- limiting examples of nucleotide sequences include (AF230738; Escherichia coli), (NC_007493, locusjag RSP 6071; Rhodobacter sphaeroides 2.4.1), and (NC_002947, locusjag PP1618; Pseudomonas putida KT2440).

[00103] In the sixth step, 2C-methyl-D-erythritol 2,4-cyclodiphosphate is converted to 1- hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate. An enzyme known to catalyze this step is, for example, l-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase. Some non-limiting examples of nucleotide sequences include (AY033515; Escherichia coli), (NC_002947, locusjag PP0853; Pseudomonas putida KT2440), and (NC_007493, locusjag RSP_2982; Rhodobacter sphaeroides 2.4.1).

[00104] In the seventh step, l-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate is converted into either IPP or its isomer, DMAPP. An enzyme known to catalyze this step is, for example, isopentyl/dimethylallyl diphosphate synthase. Some non-limiting examples of nucleotide sequences include (AY062212; Escherichia colϊ) and (NC 002947, locus tag PP0606; Pseudomonas putida KT2440).

[00105] In some embodiments, "cross talk" (or interference) between the host cell's own metabolic processes and those processes involved with the production of IPP as provided by the present invention are minimized or eliminated entirely. For example, cross talk is minimized or eliminated entirely when the host microorganism relies exclusively on the DXP pathway for synthesizing IPP, and a MEV pathway is introduced to provide additional IPP. Such a host organisms would not be equipped to alter the expression of the MEV pathway enzymes or process the intermediates associated with the MEV pathway. Organisms that rely exclusively or predominately on the DXP pathway include, for example, Escherichia coli.

[00106] In some embodiments, the host cell produces IPP via the MEV pathway, either exclusively or in combination with the DXP pathway. In other embodiments, a host's DXP pathway is functionally disabled so that the host cell produces IPP exclusively through a heterologously introduced MEV pathway. The DXP pathway can be functionally disabled by disabling gene expression or inactivating the function of one or more of the DXP pathway enzymes.

[00107] Like IPP, farnesyl pyrophosphate ("FPP") also can be made biologically. In some embodiments, two molecules of IPP and one molecule of DMAPP are condensed to form FPP. In some embodiments, the reaction can be catalyzed by an enzyme known to catalyze this step, for example, farnesyl pyrophosphate synthase.

[00108] Some non-limiting examples of nucleotide sequences for farnesyl pyrophosphate synthase include (ATU80605; Arabidopsis thalianά), (ATHFPS2R; Arabidopsis thaliana), (AAU36376; Artemisia annua), (AF461050; Bos taurus), (D00694; Escherichia coli K-12), (AE009951, Locus AAL95523; Fusobacterium nucleatum subsp. nucleatum ATCC 25586), (GFFPPSGEN; Gibber ellafujikuroi), (CP000009, Locus AAW60034; Gluconobacter oxydans 621H), (AF019892; Helianthus annuus), (HUMFAPS; Homo sapiens), (KLPFPSQCR; Kluyveromyces lactis), (LAUl 5777; Lupinus albus), (LAU20771; Lupinus albus), (AF309508; Mus musculus), (NCFPPSGEN; Neurospora crassa), (PAFPSl; Parthenium argentatum), (PAFPS2; Parthenium argentatum), (RATFAPS; Rattus norvegicus), (YSCFPP; Saccharomyces cerevisiae), (D89104; Schizosaccharomyces pombe), (CP000003, Locus AAT87386; Streptococcus pyogenes), (CP000017, Locus AAZ51849; Streptococcus pyogenes), (NC_008022, Locus YP 598856; Streptococcus pyogenes MGAS10270), (NC_008023, Locus YP_600845; Streptococcus pyogenes MGAS2096), (NC_008024, Locus YP_602832; Streptococcus pyogenes MGAS 10750), and (MZEFPS; Zea mays).

[00109] Methods for the biological production of both IPP and FPP have been previously described by references including WO 2007/140339, WO 2006/014837 and U.S. Publication Nos. 2003/0148479; 2004/0005678; and, 2006/0079476.

[00110] Such host cells can be further modified to express a polynucleotide encoding an enzyme capable of producing amorpha-4,11-diene from farnesyly pyrophosphate. Exemplary enzymes include amrophadiene synhtase, as described in Ro et al. , supra. Such host cells can be further modified to express a polynucleotide encoding the cytochrome P450 CYP71AV1 as described in Ro et al, supra. This cytochrome P450 is capable of facilitating the conversion of artemisinic acid from amorpha-4,11-diene.

4.5 Methods of Use

[00111] Artemisinic acid prepared as described herein can be used for the preparation of artemisinin according to any technique apparent to one of skill in the art. Exemplary techniques for the preparation of artemisinin from artemisinic acid are provided in Acton & Roth, 1992, J Org. Chem. 57:3610-3614, U.S. Patent No. 5,310,946 and U.S. Patent Application Publication No. 20060270863 Al, the contents of which are hereby incorporated by reference in their entireties.

[00112] The resulting artemisinin can be used for any purpose deemed suitable by those of skill in the art. hi certain embodiments, the artemisinin can be used for the treatment of malaria in a subject having malaria. In certain embodiments, the artemisinin can be used for the treatment of cancer in a subject having cancer. The artemisinin can be administered to such subjects in pharmaceutical compositions and by methods of administration deemed suitable by those of skill in the art.

5. EXAMPLES

5.1 Example 1: A first solvent extraction step for extracting artemisinic acid from fermentation broth

[00113] A fermentor broth comprising artemisinic acid produced by a host cell was adjusted to pH 3.0 with H₂SO₄. Sodium dodecyl sulfate (SDS) added to a final concentration of 2% w/v. 0.5 volumes of butyl acetate were added to the one volume of fermentation broth. Extraction was carried out at ambient temperature for 30 minutes with mixing. After extraction, the phases (solids, solvent, aqueous) were separated by centrifugation. The artemisinic acid partitioned into the solvent phase, which was isolated and subsequently processed as described in Example, 2.

[00114] FIG. 1 provides artemisinic acid yields in the solvent phase over time. After 30 minutes, a yield of about 90% was obtained. Purity was evaluated at 20-30% artemisinic acid by weight.

[00115] The above method was repeated with sodium dodecyl sulfate concentrations of 0, 0.2%, 0.5%, 1%, 1.25% & 2%. FIG. 2 provides yield of artemisinic acid in the solvent phase as a function of sodium dodecyl sulfate concentration.

5.2 Example 2: A second aqueous extraction step for extracting artemisinic acid from the solvent phase

[00116] One volume of solvent phase was extracted with 0.5 volumes of 50 mM sodium phosphate buffer at pH 10.7. The two phases were mixed for 10 minutes, and then separated by centrifugation. The solvent phase was subsequently extracted with phosphate buffer a second time under the same conditions. The extractions were run at ambient temperature

[00117] After extraction, the phases (solvent, aqueous) were separated by centrifugation. The artemisinic acid partitioned into the aqueous phase, which was isolated and subsequently processed in Example 3.

[00118] After 5 minutes, a yield of about 80-85% was obtained. Purity was evaluated at 40% artemisinic acid by weight.

5.3 Example 3: A third precipitation step for precipitating artemisinic acid from the aqueous phase

[00119] The aqueous phase from Example 2 was adjusted to pH 5.0 with H₂SO₄. The acidified solution was passed through a high-pressure homogenizer, one pass at 1000 bar pressure. After precipitation, the artemisinic acid solid was capture by filtration, washed and dried.

[00120] A yield of about 95% was obtained. Purity was evaluated at 90% artemisinic acid by weight. 5.4 Example 4: First solvent extraction steps for extracting artemisinic acid from the fermentation broth

[00121] The first solvent extraction step for extracting artemisinic acid from fermentation broth was repeated for several times to evaluate suitable solvents. Five solvents used for this evaluation include 2-butanol, butyl acetate, ethyl acetate, methyl ethyl ketone, and toluene.

[00122] The artemisinic extract used for this example was first precipitated from a base extraction of fermentation broth, with a purity of about 30% (w/w) at 10 mg/ml artemisinic acid concentration. Fractions of the precipitate were sonicated to homogeneity and adjusted to pH 3.0 in 25 mM sodium citrate, pH 6.0 in 25 mM sodium citrate, and pH 9.0 in 25 mM sodium borate for use in the solvent extraction. The solvent extraction was carried as follows: to a 2-ml microfuge tube was added 0.9 ml of artemisinic acid precipitate suspension and 0.9 ml of the test solvent. The tube was then vortexed for 10 seconds, held at ambient temperature for 30 minutes, and then centrifuged to separate the phases. Samples of the solvent phase was analyzed for artemisinic acid content by reverse phase high performance liquid chromatography by reverse phase high performance liquid chromatography (RP- HPLC) and dry weight. Results of the extractions at pH 3.0, pH 6.0, and pH 9.0 are shown in Tables 1-3 respectively.

Table 1 - pH 3 Extraction Results

Table 2 - pH 6 Extraction Results

Table 3 - pH 9 Extraction Results

[00123] Emulsions were observed in toluene samples at pH 6 and pH 9. The results show that high yields of artemisinic acid extraction can be obtained at pH 3. Of the solvents evaluated, both butyl acetate and ethyl acetate can extract artemisinic acid at high yields and with good selectivity.

[00124] Extraction of whole cell broth with ethyl acetate and butyl acetate showed little difference in yield and selectivity (data not shown). Extractions with butyl acetate tend to generate fewer interface solids than with ethyl acetate.

5.5 Example 5: Effect of sodium dodecyl sulfate (SDS) on the efficiency of the first solvent extraction step

[00125] Experiments were conducted to evaluate the effect of varying amounts of sodium dodecyl sulfate (SDS) on solvent extraction of artemisinic acid from the whole cell broth. Whole cell broth from an artemisinic acid fermentation adjusted to pH 3 was extracted with an equal volume of ethyl acetate in the presence of varying concentrations of sodium dodecyl sulfate (SDS) ranging from 0 to 2% w/v. The solution was vortex mixed, held at ambient temperature, and subsequently centrifuged for phase separation. Samples from the ethyl acetate phase were analyzed for artemisinic acid content by reverse phase high performance liquid chromatography (RP-HPLC) to determine yield. Results are shown in Figure 3, which shows that increasing the concentration of sodium dodecyl sulfate (SDS) tend to result in increased artemisinic acid yield from the solvent extraction process. Another experiment was conducted to evaluate the effect of SDS concentration on solvent extraction efficiency of artemisinic acid while using butyl acetate as the extraction solvent, which generated similar results with Figure 3 (data not shown). 5.6 Example 6: Effect of Solvent to Aqueous Ratio on the efficiency of the first solvent extraction step

[00126] The whole cell broth of artemisinic acid adjusted to pH 3 containing 2% w/v of SDS was extracted with varying amounts of butyl acetate, from 20% v/v to 50% v/v, based on the total volume of the whole cell broth. The solution was vortex mixed, held at ambient and subsequently centrifuged to separate the phases. Samples of the butyl acetate phase were analyzed for artemisinic acid content by reverse phase high performance liquid chromatography (RP-HPLC) to determine yield. Results are shown in Table 4 below.

Table 4.

[00127] The results suggest that butyl acetate volumes down to 30% v/v based on the total volume of the whole cell broth can be used in a solvent extraction step with no loss of yield.

5.7 Example 7: Extraction Kinetics of the first solvent extraction step

[00128] Experiments were conducted to determine the extraction kinetics of artemisinic acid from the whole cell broth. The whole cell broth of artemisinic acid was adjusted to pH 3 with 2% w/v of SDS and was extracted with 0.5 volumes of butyl acetate at ambient temperature. The solution was mixed with a high-shear mixer (ultra turrax). Samples were taken at 5, 10, 15, 20, 25 and 30 minutes. The samples taken were subsequently centrifuged for phase separation. Samples of the butyl acetate phase were assayed for artemisinic acid content by reverse phase high performance liquid chromatography (RP-HPLC) to determine yield. Results are shown in Figure 4, which show that artemisinic acid yields of about 95- 100% could be obtained after extraction for 30 minutes.

5.8 Example 8: Second aqueous extraction steps for extracting artemisinic acid from the solvent phase

[00129] Second aqueous extraction steps were conducted as follows to evaluate the effectiveness of three different buffers: sodium phosphate buffer, the potassium phosphate buffer and the potassium carbonate buffer.

[00130] The 5% w/v potassium carbonate buffer (~ 360 mM) was adjusted to pH 11.7. Buffer solutions of sodium and potassium phosphate were made to 350 mM and adjusted to pH 11.7 with the corresponding base. Butyl acetate phase (4 ml) from a whole cell broth extraction similar to Example 1 was dispensed into tubes and extracted with 3ml of each of the aqueous buffers. The tubes were mixed for 3 minutes, and the phases subsequently separated by centrifugation. Samples from the aqueous phase were analyzed for artemisinic acid content by reverse phase high performance liquid chromatography (RP-HPLC), the results of which are shown in Table 5 below. The results in Table 5 show that high artemisinic acid yields could be obtained by phosphate buffer. Samples from the aqueous phase were also acidified to pH 3.0 to precipitate artemisinic acid, and the precipitate was measured for weight purity, the results of which are shown in Table 6 below.

Table 5.

Table 6.

5.9 Example 9: Effect of pH on the second aqueous extraction step

[00131] To determine the optimum pH for the aqueous extraction step, a butyl acetate extract was extracted with an equal volume of 50 mM sodium phosphate buffer from pH 8.1 to pH 11.5. Samples from the aqueous phase were analyzed to determine the artemisinic acid content by reverse phase high performance liquid chromatography (RP-HPLC), the results of which are shown in Table 7 below.

Table 7.

5.10 Example 10: Extraction Kinetics of the second aqueous extraction step

[00132] Experiments were conducted to determine the extraction kinetics of artemisinic acid from the butyl acetate phase. Butyl acetate phase was extracted with an equal volume of 50 mM sodium phosphate buffer, pH 10.7, at ambient temperature. The suspension was mixed with a high-shear mixer (ultra turrax), and samples were taken at 5, 10, 15, 20, 25 and 30 minutes. The samples taken were subsequently centrifuged for phase separation. Samples of the aqueous phase were assayed for the artemisinic acid content by reverse phase high performance liquid chromatography (RP-HPLC) to determine yield, the results of which are shown in Table 8 below. The results in Table 8 show that the extractions of the artemisinic acid into the aqueous phase were relatively rapid.

Table 8.

5.11 Example 11: Third precipitation steps for precipitating artemisinic acid from the aqueous phase

[00133] Several precipitations were conducted to optimize the pH values for precipitation. The aqueous extract obtained in a phosphate buffer extraction step similar to Example 2 was acidified to pH 3.0, pH 4.0, and pH 5.0, respectively followed by ultrasonication to produce precipitation. While little difference was observed in the yield (data not shown), precipitation under pH 5.0 generated less pigmented contaminants than that under pH 3.0 and pH 4.0. Another experiment was conducted to produce precipitation with pH values ranging from pH 4.0 to pH 5.0. The filtration losses at varying pH values were tested, the results of which are shown in Table 9 below. Table 9.

5.12 Example 12: Precipitation Kinetics of the third precipitation step

[00134] Precipitations were conducted the determine kinetics of the artemisinic acid precipitation reaction at pH 5.0.

[00135] An aliquot of phosphate buffer-extracted artemisinic acid was adjusted to pH 5.0 with 5N H₂SO₄ and was ultrasonicated to initiate precipitation. Samples were taken from 0 to 22 hours and filtered. The resulting filtrate was analyzed for artemisinic acid content by reverse phase high performance liquid chromatography (RP-HPLC), the results are shown in Table 10 below. The results in Table 10 show that the precipitation reaction could be completed within 1 hour. Subsequent precipitations using multiple 2-liter process runs showed that product losses in the filtrate for this precipitation step was not higher than 5%.

Table 10.

[00136] Precipitation of artemisinic acid at low pH suggested variable precipitation kinetics, where some samples remained as stable "emulsions" for days without significant precipitate formation. Homogenization of this "emulsion" by ultrasonication or high- pressure homogenization with an APV/Gaulin unit may promote rapid precipitation. High pressure homogenization at 1000 bar pressure might be sufficient to initiate precipitation of artemisinic acid that has been extracted into the phosphate buffer.

5.13 Example 13: Extractive Fermentation of yeast producing artemisinic acid

[00137] An artemisinic acid producing yeast strain was run in a carbon restricted, mixed glucose and ethanol feed, fed-batch fermentation. The starting aqueous medium volume was 690 mL, which consisted of 620 mL batch medium and 70 mL seed culture. The OD600 of the two seed flasks were 6.3. Prior to inoculation, 200 mL of isopropyl myristate (IPM) was added to the reactor. Exponential feed was initiated at 22.5 hours after both the glucose and ethanol in the batch medium was consumed.

[00138] The exponential phase of the feed continued until 43.1 hours when it reached a maximum of 10 g glucose & ethanol/hr/L fermentor volume. The feed was then reduced to a linear feed of 5 g glucose & ethanol/hr/L fermentor volume. The aqueous fermentor volume increased to 1.52 L by the end of the run (approximately 122 hr after inoculation) and no volume was removed from the fermentation except for sampling. Because the aqueous volume in the reactor was increasing throughout the run (and sample volume was being removed), the ratio of IPM to the total volume (IPM + aqueous phase) changed from approximately 0.23 at the start of the fermentation to 0.09 at the end of the fermentation.

[00139] In the reactor, the IPM and aqueous cell broth forms a well mixed mixture or emulsion. At early times in the process (28 hrs or less), samples of the mixture, taken from the fermentor, will begin to phase separate when left standing for several minutes. At later times (40 hrs or more), the mixture becomes a more stable emulsion as the yeast cell density increases.

[00140] At approximately 123.4 hours after inoculation, fermentation was ended and the cell broth and IPM mixture was harvested. The liquid mixture was removed from the reactor vessel by gravity flow (siphoning) and the total volume was measured prior to further processing. The total volume of the cell broth and IPM mixture was approximately 1680 mL. The liquid mixture was transferred to two 1-L centrifuge bottles, and centrifuged for 25 minutes at 24°C and 7459 X G to separate the phases. The organic and aqueous phases appeared to separate with little interface emulsion remaining and the cells pelletted firmly to bottom and side of the centrifuge bottle. After centrifugation, the light phase (IPM) was removed from the centrifuge bottles by serological pipette and collected in a graduated cylinder. The remaining aqueous phase and cells were centrifuged again for 40 minutes at 24°C and 7459 X G to separate any addition IPM that was missed in the first pass. Approximately 154-165 mL total of IPM was collected.

5.14 Example 14: Purification of artemisinic acid from IPM layer

[00141] Isopropyl myristate (IPM) was isolated from an artemisinic acid fermentation described in Example 13. IPM ( 100 mis) was mixed with 400 mis of 1 % NaPO₄* 12H₂O and the pH adjusted to 10.7 by the addition of 5N NaOH. The solution was then stirred at ambient temperature for 60 minutes. After mixing, the solution was allowed to gravity settle in a separatory funnel for 60 minutes at ambient temperature. The bottom aqueous phase was drawn off from the upper IPM phase. The bottom aqueous phase was run through a liquid: liquid annular centrifugal contactor (CINC industries) to ensure complete removal of any residual IPM. The solution was mixed and the pH adjusted to 5.0 with 5N H₂SO₄. The acidification resulted in the formation of a fine white precipitate which was captured on a 0.45 micron PTFE filter, rinsed with purified water and then dried. Analysis of the dried precipitate by GC-FID gave artemisinic acid purities of 87-90% by area and 82-84% by weight.

5.15 Example 15: Purification of artemisinic acid from IPM layer with SDS

[00142] Isopropyl myristate (IPM) was isolated from an artemisinic acid fermentation described in Example 13. IPM ( 100 mis) was mixed with 900 mis of 1 % NaPO₄* 12H₂O and the pH adjusted to 10.7 by the addition of 5N NaOH. The solution was then stirred at ambient temperature for 60 minutes. After mixing, the solution was allowed to gravity settle in a separatory funnel for 60 minutes at ambient temperature. The bottom aqueous phase was drawn off from the upper IPM phase. The bottom aqueous phase was run through a liquid:liquid annular centrifugal contactor (CINC industries) to ensure complete removal of any residual IPM. A 10% w/v sodium dodecyl sulfate (SDS) solution was added to approximately 850 mis of the aqueous phase to bring the final SDS concentration to 0.03%. The solution was mixed and the pH adjusted to 5.0 with 5N H₂SO₄. The acidification resulted in the formation of a fine white precipitate which was captured on a 0.45 micron PTFE filter, rinsed with purified water and then dried. Analysis of the IPM before and after aqueous extraction showed that 5% of the artemisinic acid remained in the IPM after extraction (-95% yield). Analysis of the filtrate after precipitation showed that 2% of the artemisinic acid present in the aqueous phase remained in the filtrate after acidification (~ 98% yield). Analysis of the dried precipitate by GC-FID gave artemisinic acid purities of- 96% by area and ~ 98% by weight.

[00143] All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

What is claimed is;

1. A method for purification of artemisinic acid from a first aqueous composition comprising artemisinic acid and one or more contaminants, said method comprising the steps of: a. extracting artemisinic acid from the first aqueous composition with a solvent immiscible with said aqueous composition to yield a first solvent phase comprising artemisinic acid; b. extracting artemisinic acid from said first solvent phase with a second aqueous composition at a pH of from about 10 to about 11.5 to yield a second aqueous phase comprising artemisinic acid; and c. precipitating artemisinic acid from the second aqueous phase.

2. The method of claim 1, wherein the first aqueous composition is derived from a fermentation broth.

3. The method of claim 1, wherein the first aqueous composition is derived from a cell extract.

4. The method of claim 1, wherein the first aqueous composition is derived from a plant extract.

5. The method of any of claim 1, wherein the pH of the first aqueous composition is from about 2 to about 6.

6. The method of any of claim 1 , wherein the solvent is a nonpolar solvent.

7.. The method of claim 9, wherein the solvent is butyl acetate, ethyl acetate, isopropyl myristate, methyl isobutyl ketone, methyl oleate, toluene or a combination thereof.

8. The method of any of claim 1, wherein the solvent is a polar solvent.

9. The method of claim 8, wherein the solvent is butanol, methyl ethyl ketone, 2- butanol or a combination thereof.

10. The method of any of claims 1 , wherein the second aqueous composition is buffered with a buffer, wherein the buffer comprises phosphate, carbonate, borate, citrate or a combination thereof.

11. The method of any of claims 1 , wherein the artemisinic acid is precipitated by acidifying the second aqueous phase.

12. The method of claim 11, wherein a surfactant is added to the second aqueous phase prior to acidification.

13. A method for purification of artemisinic acid from a first aqueous composition comprising artemisinic acid and one or more contaminants, said method comprising the steps of:

(b) recovering said first solvent phase;

(d) recovering said second aqueous phase;

(f) recovering said artemisinic acid.

14. The method of claim 13, wherein the first aqueous composition is derived from a fermentation broth.

15. The method of claim 13, wherein the first aqueous composition is derived from a cell extract.

16. The method of claim 13, wherein the first aqueous composition is derived from a plant extract.

17. The method of claim 13, wherein the pH of the first aqueous composition is between about 2 and about 6.

18. The method of claim 13, wherein the extraction of the first aqueous composition comprises a surfactant.

19. The method of claim 18, wherein the surfactant is sodium dodecyl sulfate and is present in an amount from about 0.2% to about 3.0%, as measured by the weight of sodium dodecyl sulfate over the volume of the first aqueous composition.

20. The method of claim 13, wherein the solvent is a nonpolar solvent.

21. The method of claim 13, wherein the second aqueous phase is buffered with a buffer, wherein the buffer comprises phosphate, carbonate, borate, citrate, or a combination thereof.

22. The method of claim 21, wherein the second aqueous phase is buffered with a buffer selected from the group consisting of potassium phosphate, potassium borate, sodium phosphate, sodium carbonate, sodium borate, sodium cirtrate, and combinations thereof wherein the amount of buffer is from about 1% and 5% by weight per volume.

23. The method of claim 13, wherein a surfactant is added to the second aqueous phase prior to acidification.

24. The method of claim 23, wherein the surfactant is sodium dodecyl sulfate and is present in an amount from about 0.01% to about 5.0%, as measured by the weight of sodium dodecyl sulfate over the volume of the second aqueous phase.

25. The method of claim 13, wherein steps (a) and (b) occur during an extractive fermentation of host cells capable of making artemisinic acid.

26. A method of purifying artemisinic acid from an extractive fermentation, said method comprising the steps of:

(a) growing host cells capable of making artemisinic acid in an aqueous fermentation medium and in the presence of an immiscible solvent wherein the immisible solvent forms a solvent phase;

(b) recovering said solvent phase;

(d) recovering said aqueous phase;

(f) recovering said artemisinic acid.

27. The method of claim 26, wherein the immiscible solvent comprises isopropyl myristate.

28. The method of claim 26, wherein the sodium phosphate solution is at a concentration of about 1% w/v.

29. The method of claim 26, further comprising adding a surfactant to said aqueous phase prior to acidification.

30. The method of claim 29, wherein the surfactant is sodium dodecyl sulfate.

31. The method of claim 26, wherein the surfactant is sodium dodecyl sulfate at a concentration from about 0.01% to about 5%, where the concentration is measured by the weight of the sodium dodecyl sulfate over the volume of said aqueous phase.

32. The method of claim 26, wherein the surfactant is sodium dodecyl sulfate at a concentration from about 0.01% to about 1%, where the concentration is measured by the weight of the sodium dodecyl sulfate over the volume of said aqueous phase.

33. The method of claim 26, wherein the second aqueous phase is acidified to a pH from about 2. 5 to about 5.