WO2013071074A1

WO2013071074A1 - Methods of producing butadiene

Info

Publication number: WO2013071074A1
Application number: PCT/US2012/064407
Authority: WO
Inventors: Adriana L. BOTES; Changlin Chen; Alex van Eck CONRADIE
Original assignee: Invista North America S.A. R.L.
Priority date: 2011-11-11
Filing date: 2012-11-09
Publication date: 2013-05-16

Abstract

Methods and materials for producing butadiene and butadiene precursors in recombinant host cells are described.

Description

METHODS OF PRODUCING BUTADIENE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Application No. 61/558,700, filed November 1 1, 201 1, and U.S. Application No. 61/558,712, filed November 1 1, 2011, the disclosures of which are incorporated by reference in their entirety.

TECHNICAL FIELD

Aspects of the invention relate to methods for the production of butadiene in engineered host cells while maintaining the viability of the host. In particular, aspects of the invention describe the use of metabolic pathways, components of metabolic pathways, enzymes and genes associated with the production of butadiene from carbohydrate and other feedstocks in metabolically engineered host cells such that the host maintains the ability to produce the essential metabolites isopentenyl diphosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Embodiments of the invention describe the use of metabolic pathways in engineered host cells for the production of butadiene via a tetritol 4-phosphate utilizing enzymes in the 2-C-methyl-D-erythritol- 4-phosphate/l-deoxy-D-xylulose-5-phosphate pathway (MEP/DOXP pathway or non- mevalonate pathway) while maintaining the ability to produce IPP and DMAPP via the mevalonate pathway. Examples of tetritol-4-phosphates include but are not limited to erythritol-4-phosphate.

BACKGROUND

1,3 -Butadiene (hereinafter butadiene) is an important monomer for synthetic rubbers including styrene-butadiene rubber (SBR), plastics including polybutadiene (PB), acrylonitrile butadiene styrene (ABS), acrylonitrile butadiene (NBR), and as a raw material for adiponitrile for Nylon-66 and other chemicals. Butadiene is typically produced as a byproduct in the steam cracking process and isolated from the cracker streams via extraction. On-purpose butadiene has been prepared among other methods by dehydrogenation of n-butane, oxydehydrogenation of 1-butene or 2- butene, and chemical dehydrolysis of 1,4-, 1,3- and 2,3-butanediol. Industrially, butadiene has been synthesized using petrochemical-based feedstocks. The current commercial practices for producing on-purpose butadiene have several drawbacks including high cost of production and low yield processes. Currently, methods for the production of on-purpose butadiene rely on petro-chemical feedstocks and on energy intensive catalytic steps. Accordingly, it is clear that there is a need for sustainable methods for producing intermediates, in particular butadiene. In this regard, biotechnology offers an alternative approach through metabolic pathway engineering of host organisms to utilize non-petrochemically derived feedstocks and milder process conditions to produce chemicals. In the case of butadiene, however, there are no known naturally occurring metabolic pathways in which butadiene is formed either as an intermediate or product. Surprisingly, the inventors have now discovered for the first time a method to exploit naturally occurring pathways to construct metabolically engineered host strains to produce butadiene.

SUMMARY

Aspects of the invention relate to methods for the production of butadiene and non-2-C-methylated butadiene precursors in engineered host cells while maintaining the viability of the host. In particular, aspects of the invention describe the use of metabolic pathways, components of metabolic pathways, enzymes and genes associated with the production of butadiene and butadiene precursors from

carbohydrate and other feedstocks in metabolically engineered host cells such that the host maintains the ability to produce the essential metabolites isopentenyl diphosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). More specifically, aspects of the invention describe the use of metabolic pathways in engineered host cells for the production of butadiene via a tetritol-4-phosphate such as erythritol-4-phosphate utilizing enzymes in the 2-C-methyl-D-erythritol-4-phosphate/l-deoxy-D-xylulose-5- phosphate pathway (MEP/DOXP pathway or non-mevalonate pathway) while maintaining the ability to produce IPP and DMAPP via the mevalonate pathway

In one aspect, this document features a method of producing a butadiene or a butadiene precursor in a recombinant host cell having a 2-C-methyl-D-erythritol-4- phosphate/ l-deoxy-D-xylulose-5 -phosphate (MEP/DOXP) pathway and a mevalonate pathway, where the recombinant host cell includes an exogenous nucleic acid encoding an isoprene synthase. The method includes incubating the recombinant host cell with a tetritol or a fermentable carbon source under conditions that the recombinant host cell i) produces a tetritol-4-phosphate from the tetritol or the fermentable carbon source and ii) converts at least some of the tetritol- 4-phosphate into butadiene or a non-2-C-methylated butadiene precursor. The non-2-C- methylated butadiene precursor can be selected from the group consisting of 4- diphospocytidyl-tetritol, 2-phospho-4-(cytidine 5' diphospho)-tetritol, 2-tetritol-2,4- cyclopyrophosphate, l-hydroxy-2-butenyl-4-pyrophosphate, methylallyl diphosphate, and butenylpyrophosphate (e.g., 4-diphosphocytidyl-erythritol, 2-phospho-4-(cytidine 5' diphospho)-erythritol, or 2-erythritol-2,4-cyclopyrophosphate). The tetritol can be erythritol, and the recombinant host cell can produce erythritol 4-phosphate from erythritol.

In any of the methods described herein, the MEP/DOXP pathway can be endogenous to the recombinant host cell.

In any of the methods described herein, the MEP/DOXP pathway can be heterologous to the recombinant host cell, and can include, for example, at least one exogenous nucleic acid encoding a 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD), a 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), a 2-C- methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF), a 4-hydroxy-3-methylbut- 2-en-l-yl (HMB) diphosphate synthase (IspG), a HMB-PP reductase (IspH), and an isopentenyl diphospate isomerase.

In any of the methods described herein, the mevalonate pathway can be endogenous to the recombinant host cell.

In any of the methods described herein, the mevalonate pathway can be heterologous to the recombinant host cell, and can include, for example, at least one exogenous nucleic acid encoding a mevalonate kinase (MVK), a phosphomevalonate kinase (PMK), and a mevalonate-5 -pyrophosphate decarboxylase (PMD). The recombinant host also can include at least one exogenous nucleic acid encoding a thiolase, a HMG-CoA synthase, and a HMG-CoA reductase.

In any of the methods described herein, the recombinant host cell can be deficient in l-deoxy-D-xylulose-5 -phosphate (DXP) reductoisomerase activity and/or l-deoxy-D-xylulose-5-phosphate (DOXP) synthase activity.

In any of the methods described herein, the recombinant host cell can be incubated with the tetritol or the fermentable carbon source in the presence of an inhibitor. For example, the inhibitor can be FR-900098, fosmidomycin or a fosmidomycin analog, or an aryl phosphonate.

This document also features a recombinant host cell having a MEP/DOXP pathway and a mevalonate pathway, wherein the cell is deficient in 1-deoxy-D- xylulose-5 -phosphate (DXP) reductoisomerase activity and DOXP synthase activity, and includes an exogenous nucleic acid encoding an isoprene synthase. The recombinant host cell can produce a butadiene or a non-2 -C-methylated butadiene precursor from a tetritol. The MEP/DOXP pathway can be endogenous to the recombinant host cell. The MEP/DOXP pathway can be heterologous to the recombinant host cell, and can include, for example, at least one exogenous nucleic acid encoding a 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD), a 4- diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), a 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF), a 4-hydroxy-3-methylbut-2-en-l-yl (HMB) diphosphate synthase (IspG), a HMB-PP reductase (IspH), and an isopentenyl diphospate isomerase. The mevalonate pathway can be endogenous to the recombinant host cell. The mevalonate pathway can be heterologous to the recombinant host cell, and can include, for example, at least one exogenous nucleic acid encoding a mevalonate kinase (MVK), a phosphomevalonate kinase (PMK), and a mevalonate-5 -pyrophosphate decarboxylase (PMD). The recombinant host also can include at least one exogenous nucleic acid encoding a thiolase, a HMG-CoA synthase, and a HMG-CoA reductase.

This document also features a method of producing a butadiene or a butadiene precursor in a recombinant host cell having an endogenous mevalonate (MEV) pathway, where the recombinant host cell includes at least one exogenous nucleic acid encoding a 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD), a 4- diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), a 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF), a 4-hydroxy-3-methylbut-2-en-l-yl (HMB) diphosphate synthase (IspG), a HMB-PP reductase (IspH), an isopentenyl diphospate isomerase, and an isoprene synthase. The method includes incubating the recombinant host cell with a tetritol or a fermentable carbon source under conditions that the host cell (i) produces a tetritol-4-phosphate from the tetritol or said fermentable carbon source; and (ii) converts at least some of the tetritol- 4-phosphate into butadiene or a non-2-C-methylated butadiene precursor.

In another aspect, the document features a method of producing a butadiene or a butadiene precursor in a recombinant host cell having an endogenous MEP/DOXP pathway, wherein the recombinant host cell includes at least one exogenous nucleic acid encoding a mevalonate kinase (MVK), a phosphomevalonate kinase (PMK), and a mevalonate-5 -pyrophosphate decarboxylase (PMD), a thiolase, a HMG-CoA synthase, a HMG-CoA reductase, and an isoprene synthase. The method includes incubating the recombinant host cell with a tetritol or a fermentable carbon source under conditions that the host cell (i) produces a tetritol-4-phosphate from the tetritol or the fermentable carbon source; and (ii) converts at least some of the tetritol- 4- phosphate into butadiene or a non-2-C-methylated butadiene precursor.

In any of the methods described herein, the fermentable carbon source can be selected from the group comprising: glycerol, a sugar from a foodstuff; and sugar from a non- foodstuff. The sugar from foodstuff can be sucrose or glucose. The sugar from the non- foodstuff can becellulosic or hemicellulosic derived sugars.

This document also features a method of converting erythritol 4-phosphate to 4-diphospocytidyl-D-erythritol. The method includes contacting erythritol 4- phosphate with a cytidylyltransferase or a recombinant host cell expressing the cytidylyltransferase, wherein the incubation converts erythritol 4-phosphate to 4- diphospocytidyl-D-erythritol.

This document also features a method of converting 4-diphospocytidyl-D- erythritol to 2-phospho-4-(cytidine 5 ' diphospho)-D-erythritol. The method includes contacting 4-diphospocytidyl-d-erythritol with a 4-(cytidine 5'-diphospho)-2-C- methyl-D -erythritol kinase or a recombinant host cell expressing the 4-(cytidine 5'- diphospho)-2-C-methyl-D-erythritol kinase, wherein the incubation converts 4- diphospocytidyl-D-erythritol to 2-phospho-4-(cytidine 5' diphospho)-D-erythritol.

In another aspect, this document features a method of converting 2-phospho-4- (cytidine 5' diphospho)-D-erythritol to D-erythritol-2,4-cyclodiphosphate. The method includes contacting 2-phospho-4-(cytidine 5' diphospho)-D-erythritol with a 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase or a recombinant host cell expressing the 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, wherein the incubation converts 2-phospho-4-(cytidine 5' diphospho)-D-erythritol to erythritol- 2,4-cyclodiphosphate.

This document also features a method of converting erythritol-2,4- cyclodiphosphate to 1 -hydroxy -2 -butenyl-4-diphosphate. The method includes contacting erythritol-2,4-cyclodiphosphate with a l-hydroxy-2-methyl-2-butenyl 4- diphosphate synthase or a recombinant host cell expressing the 1 -hydroxy-2-methyl- 2-butenyl 4-diphosphate synthase, wherein the incubation converts erythritol-2,4- cyclodiphosphate to 1 -hydroxy -2 -butenyl-4-diphosphate.

In any of the methods described herein, the method can include recovering butadiene or the non-2-C-methylated butadiene precursor.

In another aspect, this document features a method of producing butadiene or a butadiene precursor using a recombinant host cell. The method includes incubating the recombinant host with a feedstock, wherein the host (i) converts at least some of the feedstock to a tetritol or a tetritol 4-phosphate; and (ii) converts the tetritol or the tetritol 4-phosphate to butadiene. The recombinant host can be deficient in DOXP synthase activity and/or DXP reductoisomerase activity. The recombinant host can include one or more of an exogenous nucleic acid encoding an enzyme classified under EC 2.7.7.60, an exogenous nucleic acid encoding an enzyme classified under EC 2.7.1.14, an exogenous nucleic acid encoding an enzyme classified under EC 4.6.1.12, an exogenous nucleic acid encoding an enzyme classified under EC 1.17.7.1, or an exogenous nucleic acid encoding an enzyme classified under EC 1.17.1.2.

In one aspect, this document features a method of producing a butadiene or a butadiene precursor in an organism having a partial 2-C-methyl-D-erythritol 4- phosphate/l-deoxy-D-xylulose 5-phosphate pathway (MEP/DOXP pathway) and a mevalonate pathway to produce IPP and DMAPP, comprising the steps of : a. a supplying an organism with a tetritol such as erythritol or a fermentable carbon source that can be utilized by that organism to produce a tetritol-4-phosphate such as erythritol-4-phosphate, b. utilizing enzymes in the MEP/DOXP pathway and isoprene synthase (IspS) to convert at least some of the tetritol- or erythritol-4-phosphate into butadiene or a non-2-C-methylated analog of a pathway intermediate and c. utilizing the mevalonate pathway to maintain viability by producing IPP and DMAPP . The butadiene precursor can be selected from a group comprising 4-diphospocytidyl- tetritol, 4-diphospocytidyl-tetritol-2-phosphate, 2-tetritol-2,4 cyclopyrophosphate, 1- hydroxy-2-butenyl-4-pyrophosphate, methylallyl diphosphate,or

butenylpyrophosphate.

This document also features an organism having a partial MEP/DOXP pathway which produces a butadiene or a butadiene precursor from tetritol- or erythritol-4-phosphate, but is unable to produce l-deoxy-D-xylulose-5-phosphate and/or 2-C-methyl-D-erythritol -4-phosphate. The host organism further has a mevalonate pathway which produces isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) in the biosynthesis of isoprenoids. The host organism can also produce tertritol 4-phosphate such as erythritol-4-phosphate.

In another aspect, this document features a non-natural organism which produces butadiene from a feedstock via a tetritol- or erythritol-4-phosphate intermediate. The host cell organism can be an erythritol producing organism which contains a mevalonate isoprenoid pathway. The host cell organism can be a eurkaryote organism which contains a mevalonate isoprenoid pathway but does not contain a complete non-mevalonate isoprenoid pathway (MEP/DOXP pathway). The host cell organism can be a prokaryote organism which contains a mevalonate isoprenoid pathway but does not contain a complete non-mevalonate isoprenoid pathway (MEP/DOXP pathway). The host cell organism can be an organism which contains a non-mevalonate isoprenoid pathway (MEP/DOXP pathway) but does not contain a complete mevalonate isoprenoid pathway. The host cell organism can contain both a complete non-mevalonate isoprenoid pathway (MEP/DOXP pathway) and a complete mevalonate isoprenoid pathway. Depending on the host cell background, i.e. native pathways to produce IPP and DMAPP, the host is then engineered to be able to produce butadiene via a partial MEP/DOXP pathway (i.e. not able to produce the precursors l-deoxy-D-xylulose-5-phosphate and/or 2-C-methyl- D-erythritol -4-phosphate) and IspS from erythritol-4-phosphate while producing IPP and DMAPP via a complete mevalonate pathway (See, Figure 3).

This document also features a method of producing butadiene utilizing a non- naturally occurring organism comprising the steps of a) converting feedstocks to tetritol- or erythritol 4-phosphate; and b) converting tetritol- or erythritol-4-phosphate into butadiene. The feedstock can be any fermentable carbon source such as glycerol, sugars from foodstuffs, such as sucrose or glucose; or sugars from non- foodstuffs such as cellulosic or hemicellulosic derived sugars or sugars produced by the Calvin cycle in plants or autotrophic bacteria, a CI carbon source such as syngas (comprised of CO/CO₂/H₂), methane, or C2 fermentable carbon sources such as acetate or ethanol. The feedstock can also be carbon rich waste streams derived from petrochemical based processes or from the paper and pulp industry.

In another aspect, this document features a method of producing a butadiene intermediate utilizing a non-naturally occurring organism comprising the steps of: a) converting feedstocks to erythritol-4-phosphate; and b) converting erythritol-4- phosphate into butadiene intermediate. The feedstock can be any fermentable carbon source such as glycerol, sugars from foodstuffs, such as sucrose or glucose; or sugars from non-foodstuffs such as cellulosic or hemicellulosic derived sugars or sugars produced by the Calvin cycle in plants or autotrophic bacteria, a CI carbon source such as syngas (comprised of CO/CO₂/H₂), methane, or C2 fermentable carbon sources such as acetate or ethanol. The feedstock can also be carbon rich waste streams derived from petrochemical based processes or from the paper and pulp industry. This document also features a method of converting erythritol-4-phosphate to 4-diphospocytidyl-d-erythritol via a cytidylyltransferase enzyme.

This document also features a method of converting erythritol-4-phosphate to 4-diphospocytidyl-d-erythritol via a cytidylyltransferase enzyme.

This document also features a method of converting 4-diphospocytidyl-d- erythritol to 2-phospho-4-(cytidine 5' diphospho)-D-erythritol via a cdp-me kinase enzyme.

This document also features a method of converting 2-phospho-4-(cytidine 5' diphospho)-D-erythritol to 2-erythritol-2,4- cyclopyrophosphate via a mecdp synthase enzyme.

This document also features a method of converting 2-erythritol-2,4- cyclopyrophosphate to 1 -hydroxy -2 -butenyl-4-pyrophosphate via ISPH enzyme.

In another aspect, this document features a genetically modified host containing an enzyme for producing a butadiene intermediate utilizing a non-naturally occurring organism comprising the steps of: a) converting feedstocks to tetritol 4- phosphate such as erythritol 4-phosphate; and b) converting tetritol 4-phosphate such as erythritol 4-phosphate into butadiene intermediate.

This document also features a genetically modified host containing an enzyme for producing a butadiene intermediate utilizing a non-naturally occurring organism comprising the steps of converting tetritol-4-phosphate such as erythritol-4-phosphate into butadiene intermediate.

In another aspect, this document features a genetically modified host containing an enzyme from the enzyme class 2,7,7.-. such as 2-C-methyl-D-erythritol- 4-phosphate cytidylyltransferase enzyme (EC 2.7.7.60) which converts erythritol-4- phosphate to 4-diphospocytidyl-d-erythritol.

This document also features a genetically modified host containing an enzyme from the enzyme class 2,7,1.-. such as 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (EC-2.7.1.14) enzyme which converts 4-diphospocytidyl-d-erythritol to 2- phospho-4-(cytidine 5' diphospho)-D-erythritol.

This document also features a genetically modified host containing an enzyme from the enzyme class 4.6.1.-. such as 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase enzyme (EC-4.6.1.12) which converts 2-phospho-4-(cytidine 5' diphospho)- D-erythritol to 2-erythritol-2,4- cyclopyrophosphate.

In yet another aspect, this document features a genetically modified host containing an enzyme from the enzyme class 1.17.7.-. such as (E)-4-hydroxy-3- methylbut-2-enyl-diphosphate synthase enzyme (EC-1.17.7.1) which converts 2- erythritol-2,4-cyclopyrophosphate to 1 -hydroxy-2-butenyl-4-pyrophosphate..

A genetically modified host also is described containing an enzyme from the enzyme class 1.17.1.-. such as 4-hydroxy-3-methylbut-2-enyl-diphosphate reductase enzyme (EC- 1.17.1.2) which converts 1 -hydroxy -2 -butenyl-4-pyrophosphate to methylallyl diphosphate, or butenylpyrophosphate.

Any of the genetically modified hosts described herein can include one or more of the enzymes described above (e.g., any two of the enzymes).

This document also features a method to increase the uptake of a tetritol phosphate by a non-mevalonate pathway in a host organism with a native

MEP/DOXP pathway via deletion of one or more enzymatic steps in terpenoid backbone synthesis pathway such that the organism has an impaired ability to produce 2-C-methyl-D-erythritol 4-phosphate. The deletion of the at least one enzymatic step can be due to the deletion of the dxs gene (EC 2.2.1.7, l-deoxy-D-xylulose-5- phosphate synthase) or the dxr gene (EC 1.1.1.267, l-deoxy-D-xylulose-5-phosphate reductoisomerase).

Unless otherwise defined, 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 pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. The word "comprising" in the claims may be replaced by

"consisting essentially of or with "consisting of," according to standard practice in patent law.

DESCRIPTION OF DRAWINGS

FIG. 1A is a schematic of reactions 1 and 2 in the MEP/DOXP pathway. Reaction 1 converts 2-C-methyl-D-erythritol-4-phosphate to 4-(cytidine 5'- diphospho)-2-C-methyl-D-erythritol using 2-C-methyl-D-erythritol-4-phsophate cytidylyltransferase (also known as 4-diphosphocytidyl-2C-methyl-D-erythritol synthase), classified under EC 2.7.7.60 (IspD). Reaction 2 converts 4-(cytidine 5'- diphospho)-2-C-methyl-D-erythritol to 2-phospho-4-(cytidine 5'-diphospho)-2-C- methyl-D-erythritol using 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, classified under EC 2.7.1.148 (IspE).

FIG. IB is a schematic of reactions 3 and 4 in the MEP/DOXP pathway.

Reaction 3 converts 2-phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol to

2-C-methyl-D-erythritol-2,4-cyclodiphosphate using 2-C-methyl-D-erythritol 2,4- cyclodiphosphate synthase, classified under EC 4.6.1.12 (IspF). Reaction 4 converts 2-C-methyl-D-erythritol-2,4-cyclodiphosphate to 1 -hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate using l-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, classified under EC 1.17.7.1 (IspG).

FIG. 1C is a schematic of reactions 5 and 6 in the MEP/DOXP pathway, and an additional reaction, reaction 7, that can be performed. Reaction 5 converts 1- hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate to dimethylallyl diphosphate

(DMAPP) and isopentenyl pyrophosphate (IPP) using 1 -hydroxy -2-methyl-2-(E)- butenyl 4-diphosphate reductase, classified under EC 1.17.1.2 (IspH). Reaction 6 converts IPP to DMAPP using isopenteny diphosphate isomerase, classified under EC 5.3.3.2. Reaction 7 converts DMAPP to isoprene using isoprene synthase, classified under EC 4.2.3.27 (IspS).

FIG. 2A is a schematic of reactions 1 and 2 in the production of 1 ,3 butadiene and butadiene precursors. In reaction 1, 2-C-methyl-D-erythritol-4-phsophate cytidylyltransferase (also known as 4-diphosphocytidyl-2C-methyl-D-erythritol synthase), classified under EC 2.7.7.60 (IspD), converts D-erythritol-4-phosphate to 4-(cytidine 5'-diphospho)-D-erythritol. In reaction 2, 4-(cytidine 5'-diphospho)-D- erythritol is converted to 2-phospho-4-(cytidine 5'-diphospho)-D-erythritol using 4- diphosphocytidyl-2-C-methyl-D-erythritol kinase, classified under EC 2.7.1.148 (IspE).

FIG. 2B is a schematic of reactions 3 and 4 in the production of 1 ,3 butadiene and butadiene precursors. In reaction 3, 2-phospho-4-(cytidine 5'-diphospho)-D- erythritol is converted to D-erythritol-2,4-cyclodiphosphate using 2-C-methyl-D- erythritol 2,4-cyclodiphosphate synthase, classified under EC 4.6.1.12 (IspF). In reaction 4, D-erythritol-2,4-cyclodiphosphate is converted to l-hydroxy-2-(E)-butenyl 4-diphosphate using l-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, classified under EC 1.17.7.1 (IspG).

FIG. 2C is a schematic of reactions 5, 6, and 7 in the production of 1,3 butadiene and butadiene precursors. In reaction 5, 1 -hydroxy -2 -(E)-butenyl 4- diphosphate is converted to methylallyl diphosphate and butenyl diphosphate using 1- hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase, classified under EC 1.17.1.2 (IspH). In reaction 6, methylallyl diphosphate is converted to butenyl diphosphate using isopentenyl diphosphate isomerase, classified under EC 5.3.3.2. In reaction 7, methyallyl diphosphate is converted to 1,3 -butadiene using isoprene synthase, classified under EC 4.2.3.27 (IspS).

FIG. 3 is a schematic of isoprene synthesis using the MEP/DOXP pathway and mevalonate (MEV) pathway. In the MEP/DOXP pathway, the reactions catalyzed by the endogenous dxr gene (EC 1.1.1.267, l-deoxy-D-xylulose-5-phosphate reductoisomerase) and/or dxs gene (l-deoxy-D-xylulose-5-phosphate synthase, EC 2.2.1.7) can be disrupted.

DETAILED DESCRIPTION

The present invention includes the use of enzymes in the MEP/DOXP pathway (e.g., IspD, IspE, IspF, IspG, IspH, and isopentenyl diphosphate isomerase) and isoprene synthase (IspS) as shown in FIGs. 1A-1C to act on substrate analogs in which the 2-C-methyl group is absent to produce butadiene and non-2-C-methylated butadiene precursors from a tetritol-4-phosphate such as erythritol-4-phosphate instead of producing isoprene from 2-C-methyl-D-erythritol 4-phosphate. The term non 2-C-methylated butadiene precursors refers to isoprene precursors formed in the MEP pathway by IspD, IspE, IspF, IspG, IspH & isopentenyl diphosphate isomerase. Non-2-C-methylated butadiene precursors include 4-diphospocytidyl-tetritol, 4- diphospocytidyl-tetritol-2-phosphate, 2-tetritol-2,4- cyclopyrophosphate, 1 -hydroxy - 2-butenyl-4-pyrophosphate, methylallyl diphosphate or butenylpyrophosphate. In some embodiments, the non-2-C methylated butadiene precursor can be one or more of 4-diphospocytidyl-d-erythritol, 2-phospho-4-(cytidine 5' diphospho)-D-erythritol, 2-erythritol-2,4-cyclopyrophosphate, l-hydroxy-2-butenyl-4-pyrophosphate, methylallyl diphosphate, or butenylpyrophosphate.

In some embodiments, the tetritol-4-phosphate is erythritol-4-phosphate, and the host organism produces erythritol-4-phosphate through either an endogenous or an engineered pathway as described herein. As used herein, an organism containing an endogenous pathway indicates that the organism naturally expresses all of the enzymes catalyzing the reactions within the pathway (e.g., the MEP/DOX pathway or mevalonate pathway). An organism containing an engineered pathway does not naturally express all of the enzymes catalyzing the reactions within the pathway but has been engineered such that all of the enzymes within the pathway are expressed in the organism. These engineered organisms can naturally express none or some (e.g., one or more, two or more, three or more, four or more, five or more, or six or more) of the enzymes of the pathway. Endogenous genes of the engineered organisms can also be disrupted to prevent the formation of undesirable metabolites such as substrate analogs of erythritol-4-phosphate such as 2-C methyl-D-erythritol-4-phosphate or to prevent the loss of intermediates in the pathway through other enzymes acting on such intermediates. Such engineered organisms also can be referred to as recombinant host cells.

Another aspect of the invention provides methods to ensure that the host cell retains the ability to produce the isoprenoid precursors isopentenylpyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are essential for the biosynthesis of molecules used in diverse cellular processes and are thus required for host organism viability.

In one embodiment, a host organism with an endogenous functional mevalonate pathway for the production of IPP and DMAPP, is engineered to express the heterologous enzymes IspD, IspE, IspF, IspG, IspH ,and isopentenyl diphosphate isomerase of the MEP/DOXP pathway & IspS as a route to produce butadiene or non- 2-C methylated butadiene precursor such as 4-diphospocytidyl-D-erythritol, 2- phospho-4-(cytidine 5' diphospho)-D-erythritol, 2-erythritol-2,4-cyclopyrophosphate, l-hydroxy-2-butenyl-4-pyrophosphate, methylallyl diphosphate, or

butenylpyrophosphate. If the host organism has an endogenous dxr gene (EC 1.1.1.267, l-deoxy-D-xylulose-5-phosphate reductoisomerase) or dxs gene (1-deoxy- D-xylulose-5-phosphate synthase, EC 2.2.1.7) present (see FIG. 3), one or both of the genes can be disrupted (e.g., knocked out) to produce a deficiency in the activity of the enzyme and decreased formation of the competing substrate, 2-C-methyl-D- erythritol 4-phosphate. Alternatively, an enzyme inhibitor such as FR-900098, fosmidomycin or a fosmidomycin-related analog, or a non-fosmidomycin-like inhibitor such as an aryl phosphonate can be used to reduce the formation of 2-C- methyl-D-erythritol 4-phosphate, that could enter the engineered MEP pathway. Instead, a tetritol-4-phosphate such as D-erythritol-4-phosphate can serve as a substrate analog of 2-C-methyl-D-erythritol 4-phosphate for IspD. The enzymes in the MEP pathway thus convert erythritol-4-phosphate to the non-2-C-methylated analog of IPP & DMAPP, ultimately producing butadiene via the enzyme product of IspS.

In some embodiments, the host is also able to produce erythritol-4-phosphate from erythrose-4-phosphate or erythritol through endogenous or engineered pathways. Examples of organisms capable of producing erythritol-4-phosphate that are useful as host organisms or as a source of genes to construct a metabolically engineered host capable of producing erythritol-4-phosphate include eukaryotic organisms such as Arthroderma benhamiae, Ashbya gossypii (eremothecium gossypii), Aspergillus flavus, Aspergillus fumigates, Aspergillus niger, Aspergillus spp., Asperigillus clavatus, Asperigillus nidulans, Asperigillus orza, Aureobasidium sp, Botryotinia fuckeliana, Candida albicans, Candida bordini, Candida dubliniensis, Candida glabrata, Candida magnoliae, Candida maltosa, Candida sonorensis, Saccharomyces cerevisiae, Candida tropicalis, Clavispora lusitaniae, Coccidioides immitis,

Coccidioides immitis, Coccidioides pasadasii, Coccidioides posadasii, Coprinopsis cinerea, Cryptococcus neoformans, Debaryomyces hansenii, Encephalitozoon cuniculi, Fusarium gramiearum, Hansenula polymorpha (Pichia angusta),

Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces waltii, Laccaria bicolour, Lachancea thermotolerans, Lodderomyces elongisporus, Magnaporthe orzae, Malassezia globosa, Meyerozyma guiliermondii, Moniliella pollinis,

Moniliophthora perniciosa, Nectria haematococca, Neosartorya fischeri, Neurospora crassa, Nosema ceranae, Penicillium chrysogenum, Phaeosphaeria nodorum, Phanerochaete chrysosporium, Pichia methanolica, Pichia pastoris, Podospora anserina, Postia placenta, Pseudozyma tsukubaensis, Pyrenophora teres,

Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces bayanus, Schefferosmyces stipitis, Schizophyllum commune, Schizosaccharomyces pombe, Sclerotinia sclerotoirum, Sordaria macrospora, Torula corallina, Trichophyton verrucosum, Tuber melanosporum, Uncinocarpus reesii, Ustilago maydis,

Vanderwaltozyma polyspora, Yarrowia lipolytica, Zygosaccharomyces rouxii;

bacteria such as Acholeplasma laidlawii, Aerococcus urinae, Borrelia burgdorferi B31, Borrelia burgdorferi ZS7, Borrelia spp (e.g., Borrelia garinii), Carnobacterium sp, Enterococcus faecalis, Erysipelothrix rhusiopathiae, Gardnerella vaginalis, Haliscomenobacter hydrossis, Lactibacukkys crispatu , Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus brevis, Lactobacillus buchneri,

Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus fermentum ,

Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus keflranofaciens, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus,

Lactobacillus sake, Lactobacillus salivarius, Lactoccus garvieae, Lactococcus lactis sub lactis, Lactococcus lactis subs. Cremoris, Leuconostoc gasicomitatum,

Leuconostoc kimchii, Leuconostoc oenos (Oenococcus oeni), Leuconostoc sp C2 , Melissococcus plutonium, Pediococcus pentosaceus, Staphyloccus aureus,

Staphylococcus lugdunensis, Staphylococcus saprophyticus, Staphylococcus simulans, Staphylococcus spp, Steptococcus pneumonia, Steptocococcus mutans, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus equi subs., Streptococcus gallolyticusm Streptococcus mitis, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus parauberis, Streptococcus pseudopneumoniae, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguis, Streptococcus suis, Streptococcus uberis, Streptocococcus thermophilus, Weissella koreensis protists such as Dictyostelium discoideum, Giardia lamblia, Leishmania braziliensis, Leishmania infantum, Leishmania major, Monosiga brevicollis, Naegleria gruberi, Paramecium tetraurelia, Phytophthora infestans, Tetrahymena thermophila, Trichomonas vaginalis, Trypanosoma brucei, Trypanosoma cruzi and other organisms such as mammals, birds, reptiles, lancelets, fish, ascidians, echinoderms, arthropods, molluscs nematodes, flatworms, cnidarians, sponges, plants, and placozoans.

In another embodiment of the invention, a host organism with an endogenous functional MEP/DOXP pathway for the production of IPP and DMAPP, is engineered to express genes in the mevalonate pathway to allow it to produce IPP and DMAPP via the mevalonate pathway while the MEP/DOXP pathway is exploited to produce butadiene or a non-2-C methylated butadiene precursor such as 4-diphospocytidyl-D- erythritol, 2-phospho-4-(cytidine 5' diphospho)-D-erythritol, 2-erythritol-2,4- cyclopyrophosphate, l-hydroxy-2-butenyl-4-pyrophosphate, methylallyl diphosphate, or butenylpyrophosphate. If the host organism produces mevalonic acid

endogenously, it may only be required to express some of the enzymes in the mevalonate pathway to allow the host organism to produce IPP and DMAPP, e.g., mevalonate kinase (MVK, EC 2.7.1.36), phosphomevalonate kinase (PMK, EC 2.7.4.2) and mevalonate-5-pyrophosphate decarboxylase (PMD, EC 4.1.1.33). If the host organism does not produce mevalonic acid endogenously, additional enzymes that may be required include a thiolase (EC:2.3.1.9), a HMG-CoA synthase (EC 2.3.3.10), and a HMG-CoA reductase (EC 1.1.1.88 or EC 1.1.1.34).

The early steps in the endogenous MEP/DOXP pathway catalyzed by dxs (1- deoxy-D-xylulose-5 -phosphate synthase, EC 2.2.1.7) or IspC (dxr or 1-deoxy-D- xylulose-5-phosphate reductoisomerase, EC 1.1.1.267) can be disrupted to produce a deficiency in the activity of the enzymes such that the formation of 2-C-methyl-D- erythritol 4-phosphate can be reduced or eliminated. For example, deletion of the endogenous dxr gene (EC 1.1.1.267, l-deoxy-D-xylulose-5-phosphate

reductoisomerase) in this pathway, or inhibition of the enzyme using an inhibitor such as FR-900098, fosmidomycin or a fosmidomycin-related analog, or a non- fosmidomycin-like inhibitor such as an aryl phosphonate can reduce or eliminate the formation of the competing substrate for IspD, 2-C-methyl-D-erythritol 4-phosphate. Instead, erythritol-4-phosphate then serves as a substrate analog of 2-C-methyl-D- erythritol 4-phosphate for IspD. The enzymes in the disrupted MEP pathway thus convert a tetritol-4-phosphate such as erythritol-4-phosphate to the non-2-C- methylated analogs of IPP and DMAPP. The host can be further engineered to express IspS to convert the non-2-C-methylated analogs of IPP and DMAPP, i.e., methylallyl-diphosphate and butenyl-diphsophate, to butadiene.

Non-limiting examples of such organisms with an endogenous functional MEP/DOXP pathway for the production of IPP and DMAPP that are useful as host organisms or as a source of genes to construct a metabolically engineered host capable of producing IPP and DMAPP via the MEP/DOXP pathway enzymes include bacteria such as Accumulibacter phosphatis, Acetobacter pasteurianus,

Acetohalobium arabaticum, Achromobacter spp. (e.g., Archromobacter

xylosoxidans), Acidaminococcus fermentans, Acidimicrobium ferrooxidans,

Acidiphilium spp. (e.g., Acidiphilium cryptum), Acidithiobacillus spp (e.g.,

Acidithiobacillus ferrooxidans), Acidobacterium spp (e.g., Acidobacterium capsulatum), Acidothermus cellulolyticus, Acidovorax spp (e.g., Acidovorax avenae), Acinetobacter spp (e.g., Acinetobacter baumannii), Actinobacillus spp (e.g., Actinobacillus pleuropneumoniae), Actinosynnema mirum, Aeromonas hydrophila, Aeromonas spp (e.g., Aeromonas salmonicida) , Aggregatibacter spp (e.g.,

Aggregatibacter aphrophilus), Agrobacterium spp (e.g., Agrobacterium radiobacter), Agrobacterium tumefaciens, Aicanivorax borkumensis, Alicycliphilus denitriflcans, Alicyclobacillus acidocaldarius, Aliivibrio salmonicida, Alkalilimnicola ehrlichei, Alkalilimnicola ehrlichei, Alkaliphilus spp (e.g., Alkaliphilus metalliredigens), Allochromatium vinosum, Alteromonas spp (Alteromonas macleodii), Ammonifex degensii, Amycolatopsis mediterranei, Amycolicicoccus subflavus, Anaerococcus prevotii, Anaeromyxobacter spp. (e.g., Anaeromyxobacter dehalogenans), Anaplasma spp (e.g., Anaplasma marginale), Anoxybacillus spp (e.g., Anoxybacillus

flavithermus), Arcanobacterium haemolyticum, Arcobacter spp. (e.g., Arcobacter butzleri), Aromatoleum aromaticum, Arthrobacter spp (e.g., Arthrobacter aurescens), Asticcacaulis excentricus, Atopobium parvulum, Azoarcus sp, Azorhizobium caulinodans, Azospirillum sp, Bacillulus cellulosilyticus, Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus spp (e.g., Bacillus anthracis), Bacillus subtilis, Bacillus tusciae, Bacteroides spp. (e.g., Bacteroides fragilis), Bartonella spp. (e.g., Bartonella henselae), Baumannia cicadellinicola, Beijerinckia indica,

Beutenbergia cavernae, Bifidobacterium adolescentis, Bordetella spp (e.g., Bordetella pertussis), Brachybacterium faecium, Brachyspira spp (e.g., Brachyspira

hyodysenteriae), Bradyrhizobium spp (e.g., Bradyrhizobium japonicum),

Brevibacillus brevis, Brevundimonas subvibrioides, Brucella spp (e.g., Brucella melitensis), Buchnera spp (e.g., Buchnera aphidicola), Burkholderia spp (e.g., Burkholderia mallei), Butyrivibrio proteoclasticus, Caldicellulosiruptor spp (e.g., Caldicellulosiruptor saccharolyticus), Campylobacter spp (e.g., Campylobacter jejuni), Candidatus azobacteroides pseudotrichonymphae, Candidatus desulfococcus oleovorans, Candidatus desulforudis audaxviator, Candidatus koribacter versatilis, Candidatus protochlamydia amoebophila, Candidatus puniceispirillum marinum, Candidatus solibacter usitatus, Candidatus spp (e.g., Candidatus blochmannia pennsylvanicus), Candidatus spp (e.g., Cadidatus ruthia magnifica), Candidatus spp (e.g., Candidatus pelagibacter ubique), Candidatus arthromitus, Carboxydothermus hydrogenoformans, Catenulispora acidiphila, Caulobacter spp (e.g., Caulobactaer crescenthus), Cellulomonas spp (e.g., Cellulomonas flavigena), Cellvibrio (e.g., Cellvibrio japonicus), Chitinophaga pinensis, Chlamydia muridarum, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila spp (e.g., Chlamydophila caviae), Chromobacteriumvioiaceum, Chromohaibacter saiexigens, Citrobacter spp (e.g., Citrobacter Koseri), Clavibacter michiganesis subs michiganesis, Clostridiales genomosp, Clostridium acetobutylicum, Clostridium autoethanogenum, Clostridium beijerinckii, Clostridium botulinum A, Clostridium cellulolyticum, Clostridium cellulovorans, Clostridium difficile, Clostridium kluyveri, Clostridium lentocellum, Clostridium ljungdahlii, Clostridium novyi, Clostridium perfringens, Clostridium phytofermentans, Clostridium saccharolyticum, Clostridium saccharolyticum, Clostridium sp, Clostridium tetani, Clostridium thermocellum, Clostridium sticklandii, Colwellia psychrerythraea, Comamonas testosterone, Conexibacter woesei, Coprotheromobacter proteolyticus, Coriobacterium glomerans,

Corynebacterium efficiens, Corynebacterium glutamicum, Corynebacterium spp (e.g., Corynebacterium diphtheria), Cryptobacterium curium, Cupriavidus spp (e.g., Cupriavidus metallidurans), , Dechloromonas aromatic, Delftia spp (e.g., Delftia acidovorans), Delsulfobacca acetoxidans, Desulfarculus baarsii, Desulfatibacillum alkenivorans, Desulfltobacterium hafniense, Desulfobacterium autotrophicum, Desulfobulbus propionicus, Desulfomicrobium spp. (e.g., Desulfomicrobium baculatum), Desulfotalea psychrophila, Desulfotomaculum spp (e.g.,

Desulfotomaculum reducens), Desulfovibrio spp (e.g., Desulfovibrio vulgaris Hildenborough), Desulfurispirillum indicum, Desulfurivibrio alkaliphilus, Dickeya spp (e.g., Dickeya dadantii, or Dickeya zeae), Dinoroseobacter shibae, Edwardsiella spp (e.g., Edwardiella ictaluri), Eggerthella spp (e.g., Eggerthella lenta), Ehrlichia spp (e.g., Ehrlichia ruminantium Welgevonden), Enterobacter cloacae, Enterobacter spp (e.g., Enterobacter cloacae), Erwinia spp (e.g., Erwinia tasmaniensis),

Erythrobacter litoralis, Escherichia coli, Ethanoligenens harbinense, Eubacterium spp (e.g., Eubacterium eligens), Exiguobacterium spp. (e.g., Exiguobacterium sibiricum), Ferrimonas balearica, Finegoldia magna, Francisella spp (e.g.,

Francisella tularensis), Frankia spp (e.g., Frankia symbiont), Gallibacterium anatis, Gallionella capsiferriformans, Gemma proteobacterium, Geobacillus spp (e.g., Geobacillus kaustophilus), Geobacter spp. (e.g., Geobacter sulfurreducens),

Geodermatophilus obscurus, Glaciecola sp, Gluconacetobacter diazotrophicus, Gluconobacter oxydans, Granulibacter bethesdensis, Haemophilus spp (e.g., Haemophilus influenzae), Hahella chejuensis, Halanaerobium hydrogeniformans, Halomonas elongata, Halorhodospira haiophiia, Halothermothrix orenii,

Halothiobacillus neapolitanus, Helicobacter spp (e.g., Helicobacter felis),

Helicobacter spp (e.g., Helicobacter pylori), Heliobacterium modesticaldum, Herbaspirillum seropedicae, Herminiimonas arsenicoxydans, Hippea maritima, Hirschia baltica, Hyphomicrobium spp (e.g., Hyphomicrobium denitriflcans), Hyphomonas neptunium, Idiomarina loihiensis, Intrasporangium calvum, Isoptericola variabilis, Jannaschia sp, Jonesia denitriflcans, Kangiella koreensis,

Ketogulonicigenium vulgare, Kineococcus radiotolerans, Klebsiella spp (e.g., Klebsiella pneumonia), Kocuria rhizophila, Kribbella flavida, Kytococcus sedentarius, Lactobacillus sakei, Lactobacillus sp, Laribacter hongkongensis, Lawsonia intracellularis, Leifsonia xyli xyli, Leptospira interrrogans serovar lai, Leptospira spp (e.g., Leptospira borgpetersenii), Leptothrix cholodnii, Lysinibacillus sphaericus, Macrococcus caseolyticus, Magnetococcus sp, Magnetospirillum magneticum, Mahella australiensis, Maricaulis maris, Marinobacter aquaeolei, Marinomonas spp (e.g., Marinomonas mediterranea), Mesorhizobium spp (e.g., Mesorhizobium loti), Methylibium petroleiphilum, Methylobacillus flagellates, Methylobacterium spp (e.g., Methylobacterium extorquens), Methylocella silvestris, Methylococcus capsulatus, Methylomonas methanica, Methylotenera spp (e.g., Methylotenera mobilis), Methylovorus spp (e.g., Methylovorus glucosetrophus), Microbacterium testaceum, Micrococcus luteus, Microlunatus phosphovorus, Micromonospora spp (e.g., Micromonospora aurantiaca), Minibacterium

massiliensis, Mobiluncus curtisii, Moorella thermoacetica, Moraxella catarrhalis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium smegamatis, Mycobacterium spp (e.g., Mycobacterium leprae), Mycobacterium tuberculosis, Mycoplasma gallisepticum, Mycoplasma spp (e.g, Mycoplasma penetrans),

Nakamurella multipartita, Natranaerobius thermophilus, Nautilia profundicola, Neisseria spp (e.g., Neisseria meningitidis), Neorickettsia sennetsu, Nitratifractor salsuginis, Nitratiruptor sp, Nitrobacter spp.(e.g., Nitrobactaer winogradskyi), Nitrosococcus spp (e.g., Nitrosococcus oceani), Nitrosomonas spp. (e.g.,

Nitrosomonas europaea), Nocardioides sp, Novosphingobium spp (e.g.,

Novosphigobium aromaticivorans), Ochrobacterum anthropi, Odoribacter splanchnicus, Oligotropha spp (e.g., Oligotropha carboxidovorans), Olsenella uli, Ostreococcus lucimarinus, Ostreococcus tauri, Paenibacillus sp, Paenibacillus spp (e.g., Paenibacillus polymyxa), Paludibacter propionicigenes, Pantoea spp (e.g., Pantoea ananatis), Parabacteroides distasonis, Parachlamydia acanthamoebae, Paracoccus denitrificans, Parvibaculum lavamentivorans, Parvularcula bermudensis, Pasteurella multocida, Pectobacterium spp (e.g., Pectobacterium atrosepticum), Pedobacter spp (e.g., Pedobacter heparinus), Pelobacter spp (e.g., Pelobacter carbinolicus), Pelotomaculum thermopropionicum, Phenylobacterium zucineum, Photorhabdus spp (e.g., Photorhabdus luminescens), Polaromonas spp (e.g., Polaromonas naphthalenivorans), Polymorphum gilvum, Polynucleobacter spp (e.g., Polynucleobacter necessaries), Porphyromonas spp (e.g., Porphyromonas gingivalis), Prevotella spp (e.g., Prevotella ruminicola), Propionibacterium acnes, Proteus mirabilis, Pseudoalteromonas spp (e.g., Pseudoalteromonas haloplanktis),

Pseudomonas (e.g., Pseudomonas aeruginosa), Pseudomonas fluorescens,

Pseudomonas putida, Psychrobacter spp (e.g., Psychrobacter arcticum),

Psychromonas ingrahamii, Pusillimonas sp, Ralstonia spp (e.g. Ralstonia

solanaerarum), Ramlibacter tataouinensis, Renibacterium salmoninarum, Rhizobium spp (e.g., Rhizobium etli), Rhodobacter spp (e.g., Rhodobacter sphaeroides),

Rhodococcus, Rhodococcus spp (e.g., Rhodococcus equi), Rhodoferax spp (e.g., Rhodoferax ferrieducens), Rhodomicrobium vannielii, Rhodopseudomonas spp (e.g., Rhodopseudomonas palustris), Rhodospirillum spp (e.g., Rhodospirillum rubrum), Rhodothermus spp (e.g., Rhodothermus marinus), Rosebacter spp (e.g., Rosebacter denitrificans), Rothia spp (e.g., Rothia mucillaginosa), Rubrobacter xylanophilus, Ruegeria sp, Ruminococcus albus, Saccharophagus degradans (Microbulbifer degradans), Saccharopolyspora spp (e.g., Saccharopolyspora erythraea),

Salinibacter spp (e.g., Salinibacter ruber), Salinispora spp (e.g., Salinispora tropica), Salmonella sp, Sanguibacter keddieii, Segniliparus rotundus, Selenomonas sputigena, Serratia spp (e.g., Serrtia proteamaculans), Shewanella spp (e.g., Shewanella denitrificano), Shigella spp (e.g., Shigella flexneri), Sideroxydans lithotrophicus, Silicibacter pomeroyi, Simkania negevensis, Sinorhizobium spp (e.g., Sinorhizobium meliloti), Slackia heliotrinireducens, Sodalis glossinidius, Sphingobacterium sp, Sphingopyxis spp (e.g., Sphingopyxis alaskensis), Spirochaeta smaragdinae, Spirochaeta spp (e.g., Stackebrandtia nassauensis, Starkeya novella,

Stenotrophomonas spp (e.g., Stenotrophomonas maltophilia), Streptococcus sp, Streptomyces spp (e.g., Streptomyces coelicolor), Streptosporangium roseum, Sulfobacillus acidophilus, Sulfurimonas spp (e.g., Sulfurimonas denitriflcans), Sulfurospirillum deleyianum, Sulfurovum sp, Symbiobacterium thermophilum, Syntrophobacter fumaroxidans, Syntrophobotulus glycolicus, Syntrophomonas wolfei, Syntrophothermus lipocalidus, Syntrophus aciditrophicus, Taylorella equigenitalis, Tepidanaerobacter sp, Terriglobus saanensis, Thauera sp, Thermaerobacter marianensis, Thermincolapotens, Thermoanaerobacter spp (e.g.,

Thermoanaerobacter tengcongensis), Thermoanaerobacterium

thermosaccharolyticum, Thermoanaerobacterium xylanolyticum, Thermobispora bispora, Thermodesulfobium narugense, Thermobiflda fusca, Thermosediminibacter oceani, Thioalkalivibrio sp, Thiobacillus denitriflcans, Thiomonas intermedia, Tolumonas auensis, Treponema spp (e.g., Treponema pallidum), Treponema succinifaciens, Tropheryma whipplei, Tsukamurella paurometabola, Variovorax spp (e.g., Variovorax paradoxus), Veillonella parvula, Verminephrobacter eiseniae, Verrucosispora maris, Vibrio spp (e.g., Vibrio cholerae), Wigglesworthia glossinidia, Wolbachia spp (e.g., Wolbachia pipientis), Wolinella succinogenes, Xanthomonas spp (Xanthomonas campestris), Xanthobacter autotrophicus, Xenorhabdus (e.g.,

Eexnohabdus bovienii), Xylanimonas cellulosilytica, Xylella spp (e.g., Xylella fastidiosa), Yersinia spp (e.g., Yersinia pestis), Zymomonas mobilis, or Zymomonas sp, algae such as Chlamydormonas reinhardtii, Volvox carter f. Nagariensis and Cyanidioschyzon merolae, and protozoans such as Plasmodium berghei, Plasmodium chabaudi, Plasmodium falciparum, Plasmodium knowlesi, Plasmodium vivax and Plasmodium yoelii

In some embodiments, the host is also able to produce erythritol-4-phosphate via endogenous or engineered pathways.

In another embodiment, the host organism has both an endogenous functional mevalonate pathway and a functional endogenous MEP/DOXP pathway for the production of IPP and DMAPP. In these host organisms, the mevalonate pathway can be used to produce IPP and DMAPP, while the MEP/DOXP pathway can be exploited for the production of butadiene and non-2-C methylated butadiene precursors such as 4-diphospocytidyl-D-erythritol, 2-phospho-4-(cytidine 5' diphospho)-D-erythritol, 2- erythritol-2,4-cyclopyrophosphate, l-hydroxy-2-butenyl-4-pyrophosphate, methylallyl diphosphate, or butenylpyrophosphate. The early steps in the endogenous MEP/DOXP pathway that are catalyzed by dxs (l-deoxy-D-xylulose-5 -phosphate synthase, EC 2.2.1.7) or IspC (dxr or l-deoxy-D-xylulose-5-phosphate

reductoisomerase, EC 1.1.1.267) can be disrupted to produce an enzyme deficiency, and reduce or eliminate the formation of 2-C-methyl-D-erythritol 4-phosphate. For example, deletion of the endogenous dxr gene (EC 1.1.1.267, l-deoxy-D-xylulose-5- phosphate reductoisomerase) in this pathway, or inhibition of the enzyme by the addition of inhibitors such as fosmidomycin, would reduce or eliminate the formation of the competing substrate for IspD, 2-C-methyl-D-erythritol 4-phosphate. Instead, erythritol-4-phosphate can serve as a substrate analog of 2-C-methyl-D-erythritol 4- phosphate for IspD. The enzymes in the disrupted MEP pathway thus convert a tetritol-4-phosphate such as erythritol-4-phosphate to the non-2-C-methylated analogs of IPP and DMAPP. The host can be further engineered to express IspS to convert these non-2-C-methylated analogs of IPP and DMAPP, i.e., methylallyl-diphosphate and butenyl-diphosphate, to butadiene or precursors of butadiene. The interruption of the early steps of the MEP pathway may advantageously also attenuate the pathogenicity of the organism.

In some embodiments, the host is also able to produce erythritol-4-phosphate via endogenous or engineered pathways. Non-limiting examples of organisms with both an endogenous functional mevalonate pathway and an endogenous functional MEP/DOXP pathway for the production of IPP and DMAPP that are useful as host organisms or as a source of genes to construct a metabolically engineered host capable of producing IPP and DMAPP via the mevalonate pathway and butadiene via enzymes in the MEP/DOXP pathway enzymes include bacteria such as Listeria monocytogenes, Listeria grayi, Listeria innocua, Listeria ivanovii, Listeria murrayi, Listeria seeiigeri, Listeria weishimeri, Nocardia farcinica; plants such as Arabidopsis lyrata, Arabidopsis thaiiana, Brachypodium distachyon, Lotus japonicas, Medicago truncatula, Orzya sativa japonica, Populous trichocarpa, Ricinus communis, Seiagineiia moellendorffli, Sorghum bicolour, Vitis vinifera, Zea mays, mosses such as Fontinalis antipyretica, Physcomitrella patens, Polytrichum commune, and diatoms such as Phaeodactylum tricornutum, Thalassiosira pseudonana.

In another embodiment, the host organism has an endogenous functional mevalonate pathway and an endogenous MEP/DOXP pathway, which may lack one or more genes encoding one or more of the enzymes IspD, IspE, IspF, IspG, or IspH. In these host organisms, the mevalonate pathway can be used to produce IPP and DMAPP, while the MEP pathway can be exploited for the production of butadiene after engineering the MEP pathway to express the enzymes in the pathway that are absent in the native host. For example, Listeria innocua lacks IspG & IspH, and the MEP pathway in this organism can be augmented to be fully functional by expression of the genes gcpE and lytB, encoding IspG and IspH respectively. In addition, interruption of the early steps in the MEP pathway catalyzed by dxs (1-deoxy-D- xylulose-5 -phosphate synthase, EC 2.2.1.7) or IspC (dxr or l-deoxy-D-xylulose-5- phosphate reductoisomerase, EC 1.1.1.267) can reduce or eliminate the formation of 2-C-methyl-D-erythritol 4-phosphate. Instead, erythritol-4-phosphate then serves as a substrate analog of 2-C-methyl-D-erythritol 4-phosphate for IspD. The enzymes in the disrupted MEP pathway thus convert a tetritol-4-phosphate such as erythritol-4- phosphate to the non-2-C-methylated analogs of IPP and DMAPP. The host is further engineered to express IspS to convert these non-2-C-methylated analogs of IPP and DMAPP, i.e., methylallyl-diphosphate and butenyl-diphosphate, to butadiene.

In another embodiment, butadiene or a non-2-C methylated butadiene precursor such as 4-diphospocytidyl-D-erythritol, 2-phospho-4-(cytidine 5' diphospho)-D-erythritol, 2-erythritol-2,4-cyclopyrophosphate, 1 -hydroxy-2-butenyl- 4-pyrophosphate, methylallyl diphosphate, or butenylpyrophosphate can be produced in an organism having an MEP/DOXP pathway by supplying an organism with an excess of a tetritol under conditions that the organism can produce tetritol 4-phosphate from the tetritol and convert at least some of the tetritol 4-phosphate into butadiene or a butadiene precursor such as a non-methylated analog of the MEP/DOXP pathway precursor. For example, the organism can convert at least 20%, 21%, 22%, 23%, 24%, or 25% of the tetritol 4-phosphate to butadiene.

It is understood by those skilled in the art that the pathway for the production of butadiene from a tetritol 4-phosphate (e.g., erythritol 4-phosphate) using the MEP pathway consisting of the IspD, IspE, IspF, IspG, IspH enzymes, and isopentenyl diphosphate isomerase, in combination with isoprene synthase (IspS) can be engineered to produce butadiene more efficiently. Flux through the pathway can be improved by increasing the level of expression of each of the individual enzymes or by improving the catalytic efficiency of each of the enzymes in the pathway by techniques known to persons skilled in the art.

For example, a host organism expressing the enzymes in the MEP pathway and IspS can be adapted via classical selection techniques or mutagenesis techniques to catalyze the respective reactions of the analog substrates lacking a 2-C methyl group more efficiently and thus to produce each intermediate in the pathway and finally butadiene from a tetritol-4-phosphate such as erythritol-4-phospahte.

Enzyme levels in the host cells can also be increased by genetic modification of the host to express more copies of the genes encoding the enzymes IspD, IspE, IspF, IspG, IspH, IspS, and isopentenyl diphosphate isomerase or combinations thereof, under strong promoters or by inserting the heterologous genes in loci with high transcriptional efficiency in the genome of the host.

Enzymes which convert non-methylated analogs of the MEP/DOXP pathway precursors to other butadienes or non-2-C methylated butadiene precursor such as 4- diphospocytidyl-D-erythritol, 2-phospho-4-(cytidine 5' diphospho)-D-erythritol, 2- erythritol-2,4-cyclopyrophosphate, l-hydroxy-2-butenyl-4-pyrophosphate, methylallyl diphosphate, or butenylpyrophosphate can be useful in the production of butadiene through the above described pathways, as shown in FIGs. 2A-2C. In some embodiments, the document provides an enzyme from the enzyme class 2,7,7.-. such as 2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase enzyme (EC 2.7.7.60), which converts erythritol-4-phosphate to 4-diphospocytidyl-d-erythritol. In some embodiments, the document provides an enzyme from the enzyme class EC 2,7, 1.-., such as 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (EC-2.7.1.14) enzyme, which converts 4-diphospocytidyl-d-erythritol to 2-phospho-4-(cytidine 5' diphospho)-D-erythritol. In some embodiments, the document provides an enzyme from the enzyme class EC 4.6.1.-., such as 2-C-methyl-D-erythritol 2,4- cyclodiphosphate synthase enzyme (EC-4.6.1.12), which converts 2-phospho-4-

(cytidine 5' diphospho)-D-erythritol to 2-erythritol-2,4-cyclopyrophosphate. In some embodiments, the document provides an enzyme from the enzyme class EC 1.17.7.-, such as (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase enzyme (EC- 1.17.7.1), which converts 2-erythritol-2,4-cyclopyrophosphate to l-hydroxy-2- butenyl-4-pyrophosphate. In some embodiments, the document provides an enzyme from the enzyme class EC 1.17.1.-., such as 4-hydroxy-3-methylbut-2-enyl diphosphate reductase enzyme (EC-1.17.1.2), which converts l-hydroxy-2-butenyl-4- pyrophosphate to methylallyl diphosphate, or butenylpyrophosphate. In some embodiments, the document provides an enzyme from the enzyme class EC 4.2.3.-., such as Isoprene synthase (EC-4.2.3.27), which converts methylallyl diphosphate or butenylpyrophosphate to butadiene. In further embodiments, the document provides an enzyme capable of carrying two or more enzymatic conversions of butadiene precursors derived from erythritol-4-phosphate. In any of the embodiments described herein, the recombinant host can include a nucleic acid encoding one or more enzymes from such classes. Nucleic acids encoding isoprene synthase or enzymes in the MEP/DOXP pathway have been identified from various organisms and are readily available in publicly available databases such as GenBank or EMBL.

Protein engineering techniques known to those skilled in the art can be applied to improve the substrate selectivity, substrate affinity and kinetic constants K_m and max Or other properties such as stability of the enzymes. Where structural information is available for the enzymes, rational design approaches can be used to predict advantageous amino acid substitutions. Directed evolution of the enzymes can be applied to improve the enzymes, using techniques such as random mutagenesis, DNA shuffling and the like.

It is understood by those skilled in the art, that for instance, IspD can be engineered to have a lower K_m or higher V_max for a tetritol-4-phosphate such as erythritol-4-phosphate than for the natural substrate 2-C-methyl-D-erythritol-4- phosphate. In such a case, it may not be necessary to disrupt the early MEP pathway by deletion of for example the endogenous dxr gene (EC 1.1.1.267, 1-deoxy-D- xylulose-5 -phosphate reductoisomerase) in this pathway, or inhibition of the enzyme by the addition of inhibitors for example fosmidomycin, so as to avoid the formation of the natural substrates 2-C-methyl-D-erythritol- 4-phosphate that competes with a tetritol-4-phosphate such as erythritol-4-phosphate for binding in the active site of IspD. Similarly, the IspS enzyme that is expressed in the host organism can be engineered to have a lower K_m or higher V_max for a methylallyl diphosphate than for the natural substrate dimethylallyl diphosphate (DMAPP), thereby reducing or eliminating the formation of isoprene from IPP and DMAPP formed in the mevalonate pathway of the host by the engineered IspS.

In one embodiment, erythritol-4-phosphate can be produced in the host organism from erythrose-4-phosphate, which in turn may be derived from any fermentable carbon source such as hexoses, pentoses, or glycerol. Tetritol-4- phosphates include the isomers erythritol-4-phosphate or threitol-4-phosphate. The sugar alcohol erythritol-4-phosphate is formed from the sugar aldehyde erythrose-4- phosphate by the enzyme EPDH (erythritol-4-phosphate dehydrogenase). Erythrose- 4-phosphate is formed in the Bifidobacterium shunt present in organisms such as Bifidobacterium animalis, B. longum, and Leuconostoc msenteroides from D- fructose-6-phosphate via a fructose-6-phosphate phosphoketolase (EC4.1.2.22), during 5,6-dimethylbenzimidazole biosynthesis in organisms such as Bacillus megaterium, Prauserella rugosa, Propionibacterium freudenreichii, Salmonella enterica enterica serovar Typhimurium, and Sinorhizobium meliloti.

Erythrose-4-phosphate also is an intermediate in the non-oxidative branch of the pentose phosphate pathway and is formed from glyceraldehydes-3 -phosphate and D-sedoheptulose-7-phosphate by a transaldolase B (EC 2.2.1.2), in the 3- dehydroquinate biosynthesis I and chorisrmate biosynthesis I pathways where it is formed from 3-deoxy-D-arabino-heptulosonate-7-phosphate by an aldolase (EC 4.1.2.15), the Calvin-Benson-Brasham cycle, formaldehyde assimilation II (RuMP Cycle), formaldehyde assimilation II (dihydroxyacetone cycle) and Rubsco shunt where it is formed from glyceraldehyde-3 -phosphate and fructose-6-phosphate via a transketolase (EC 2.2.1.1), or from D-sedoheptulose-l,7-bisphosphate by an aldolase (f aA, EC 4.1.2.-), or in the chorismate biosynthesis 1 pathway from 3-deoxy-D- arabino-heptulosonate-7-phosphate by the action of 3-deoxy-7-phosphoheptulonate synthase (EC 2.5.1.54).

It is understood by persons skilled in the art that, since erythrose-4-phosphate is a precursor to erythritol-4-phosphate, it is advantageous to engineer the various pathways present in the host organism so as to maximize the formation of erythrose- 4-phosphate. This can be achieved by classical selection techniques or for example by overexpression of the genes involved in its formation, or through protein engineering of the enzymes involved in its formation to shift the equilibrium of the reversible reactions towards erythrose-4-phosphate synthesis or through alleviating feedback inhibition of allosteric enzymes. It is also understood by those skilled in the art of metabolic engineering that it may be advantageous to delete or inhibit enzymes involved in the consumption of erythrose-4-phosphate, such as erythrose-4-phosphate dehydrogenase (EC 1.2.1.72) in the pyridoxal-5 'phosphate biosynthesis pathway, and to prevent the dephosphorylation of erythrose-4-phosphate and erythritol-4-phosphate by sugar phosphatases involved in the hydrolysis of the phospho-ester bond of sugar phosphates, such as phosphatises and kinases, such as erythrose-4-phosphate-kinase or erythritol-4-phosphate phosphatase. Nucleic acids encoding such enzymes have been identified from various organisms and are readily available in publicly available databases such as GenBank or EMBL.

In another embodiment, erythritol-4-phosphate is produced in the host organism from erythritol. For example, erythrose-4-phosphate can be

dephosphorylated to erythrose, and the erythrose thus formed is reduced to erythritol by erythrose reductase in a host such as Candida magnoliae. In another example, erythritol may be added to the fermentation broth, or produced in situ through a co- fermentation by any host organism able to secrete erythritol. Erythritol is then taken up into the cells by facilitated diffusion or active transport by sugar transporters and phosphorylated by a kinase to erythritol-4-phosphate. It is understood by people skilled in the art that the uptake of erythritol can be increased by protein engineering of sugar transporters to improve the rate of uptake and affinity for erythritol. It also is advantageous to prevent the consumption or degradation of erythritol-4-phosphate by phosphatases in order to maximize the flux of erythritol-4-phosphate into the MEP pathway for conversion to butadiene. Host organisms useful for the production of erythritol include for example yeast strains belonging to the genera Yarrowia, Moniliella and Trichosporonoides, such as Yarrowia Upolytica, Moniliella poiiinis, M. acetobuten, Trichosporonoides nigrescens, T. oedocephaiis, T. megachiiiienses as well as other microorganisms such as Toruia corallina, Aureobasidium sp, Pseudozyma tsukubaensis, Candida magnoliae, Leuconostoc oenos (Oenococcus oeni).

The present document provides methods and means to convert a range of molecules detailed herein into butadiene. As used herein, the term "non-naturally occurring" when used in reference to a recombinant host cell is intended to mean that the host cell has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding polypeptides (e.g., enzymes or metabolic polypeptides), other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial genetic material. It is understand that a recombinant host can express a plurality of polypeptides (e.g., one, two, three, four, five, or six polypeptides) from one or more exogenous nucleic acids. In some embodiments, an exogenous nucleic acid encodes multiple polypeptides of interest (e.g., multiple enzymes). In some embodiments, an exogenous nucleic acid encodes a single polypeptide of interest (e.g., a single enzyme). In some embodiments, a recombinant host includes a plurality of different exogenous nucleic acids, where each exogenous nucleic acid encodes a single polypeptide of interest (e.g., one enzyme). Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon, as explained in more detail below.

Provided is a method of using the genetic engineering of a host to increase tetritol-4-phosphate conversion in the non-mevalonate (MEP/DOXP) pathway. For instance, a method to increase the uptake of tetritol-4-phosphate, such as erythritol-4- phosphate, into a non-mevalonate pathway comprising the deletion of one or more of the genes that encode one or more enzymes catalyzing one or more steps in the terpenoid backbone synthesis pathway (see FIG. 3). For example, deletion of either the dxs gene (EC 2.2.1.7, 1 -deoxy-D-xylulose-5 -phosphate synthase) or the dxr gene (EC 1.1.1.267, l-deoxy-D-xylulose-5-phosphate reductoisomerase) would disable the complete MEP/DOXP pathway, and force the engineered host to use the alternative substrate, erythritol-4-Phosphate (EP), as the 2-C-methyl-D-erythritol-4-phosphate would not be produced. OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A method of producing a butadiene or a butadiene precursor in a recombinant host cell having a 2-C-methyl-D-erythritol-4-phosphate/l-deoxy-D-xylulose-5- phosphate (MEP/DOXP) pathway and a mevalonate pathway, wherein said recombinant host cell comprises an exogenous nucleic acid encoding an isoprene synthase, said method comprising incubating said recombinant host cell with a tetritol or a fermentable carbon source under conditions that said recombinant host cell i) produces a tetritol-4-phosphate from said tetritol or said fermentable carbon source and ii) converts at least some of the tetritol- 4-phosphate into butadiene or a non-2-C- methylated butadiene precursor.

2. The method of claim 1, wherein said MEP/DOXP pathway is endogenous to said recombinant host cell.

3. The method of claim 1, wherein said MEP/DOXP pathway is heterologous to said recombinant host cell.

4. The method of claim 3, wherein said recombinant host comprises at least one exogenous nucleic acid encoding a 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD), a 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), a 2-C- methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF), a 4-hydroxy-3-methylbut- 2-en-l-yl (HMB) diphosphate synthase (IspG), a HMB-PP reductase (IspH), and an isopentenyl diphospate isomerase.

5. The method of any one of claims 1-4, wherein said mevalonate pathway is endogenous to said recombinant host cell.

6. The method of any one of claims 1-4, wherein said mevalonate pathway is heterologous to said recombinant host cell.

7. The method of claim 6, wherein said recombinant host cell comprises at least one exogenous nucleic acid encoding a mevalonate kinase (MVK), a

phosphomevalonate kinase (PMK), and a mevalonate-5 -pyrophosphate decarboxylase (PMD).

8. The method of claim 6 or claim 7, said recombinant host cell comprising at least one exogenous nucleic acid encoding a thiolase, a HMG-CoA synthase, and a HMG-CoA reductase.

9. The method of anyone of claims 1-8, wherein said tetritol is erythritol, and said recombinant host cell produces erythritol 4-phosphate from erythritol.

10. The method of anyone of claims 1-9, wherein said recombinant host cell is deficient in l-deoxy-D-xylulose-5-phosphate (DXP) reductoisomerase activity.

1 1. The method of anyone of claims 1-10, wherein said recombinant host cell is incubated with said tetritol or said fermentable carbon source in the presence of an inhibitor.

12. The method of claim 11, wherein said inhibitor is FR-900098, fosmidomycin or a fosmidomycin analog, or an aryl phosphonate.

13. The method of any one of claims 1-11, wherein said recombinant host cell is deficient in l-deoxy-D-xylulose-5-phosphate (DOXP) synthase activity.

14. The method of any one of claims 1-13, wherein said non-2-C-methylated butadiene precursor is selected from the group consisting of 4-diphospocytidyl- tetritol, 2-phospho-4-(cytidine 5' diphospho)-tetritol, 2-tetritol-2,4- cyclopyrophosphate, l-hydroxy-2-butenyl-4-pyrophosphate, methylallyl diphosphate, and butenylpyrophosphate.

15. The method of claim 14 wherein said non-2-C-methylated butadiene precursor is 4-diphosphocytidyl-erythritol, 2-phospho-4-(cytidine 5' diphospho)-erythritol, or 2- erythritol-2,4-cyclopyrophosphate.

16. A recombinant host cell having a MEP/DOXP pathway and a mevalonate pathway, wherein said cell is deficient in l-deoxy-D-xylulose-5-phosphate (DXP) reductoisomerase activity and DOXP synthase activity, and comprises an exogenous nucleic acid encoding an isoprene synthase, wherein said cell produces a butadiene or a non-2-C-methylated butadiene precursor from a tetritol.

17. The recombinant host cell of claim 16, wherein said MEP/DOXP pathway is endogenous to said host cell.

18. The recombinant host cell of claim 16, wherein said MEP/DOXP pathway is heterologous to said host cell.

19. The recombinant host cell of claim 18, wherein said recombinant host comprises at least one exogenous nucleic acid encoding a 4-diphosphocytidyl-2-C- methyl-D-erythritol synthase (IspD), a 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), a 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF), a 4- hydroxy-3-methylbut-2-en-l-yl (HMB) diphosphate synthase (IspG), a HMB-PP reductase (IspH), and an isopentenyl diphospate isomerase.

20. The recombinant host cell of any one of claims 16-19, wherein said mevalonate pathway is endogenous to said recombinant host cell.

21. The recombinant host cell of anyone of claims 16-19, wherein said mevalonate pathway is heterologous to said recombinant host cell.

22. The recombinant host cell of claim 21, said host cell comprising an exogenous nucleic acid encoding a MVK, a phosphomevalonate kinase (PMK), and a mevalonate-5 -pyrophosphate decarboxylase (PMD).

23. The recombinant host cell of claim 22, further comprising an exogenous nucleic acid encoding a thiolase, a HMG-CoA synthase, and a HMG-CoA reductase.

24. A method of producing a butadiene or a butadiene precursor in a recombinant host cell having an endogenous mevalonate (MEV) pathway,

said recombinant host cell comprising at least one exogenous nucleic acid encoding a 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD), a 4- diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), a 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF), a 4-hydroxy-3-methylbut-2-en-l-yl (HMB) diphosphate synthase (IspG), a HMB-PP reductase (IspH), an isopentenyl diphospate isomerase, and an isoprene synthase,

said method comprising incubating said recombinant host cell with a tetritol or a fermentable carbon source under conditions that said host cell (i) produces a tetritol- 4-phosphate from said tetritol or said fermentable carbon source; and (ii) converts at least some of the tetritol- 4-phosphate into butadiene or a non-2-C-methylated butadiene precursor.

90 25. A method of producing a butadiene or a butadiene precursor in a recombinant

91 host cell having an endogenous MEP/DOXP pathway,

92 said recombinant host cell comprising at least one exogenous nucleic acid

93 encoding a mevalonate kinase (MVK), a phosphomevalonate kinase (PMK), and a

94 mevalonate-5 -pyrophosphate decarboxylase (PMD), a thiolase, a HMG-CoA

95 synthase, a HMG-CoA reductase, and an isoprene synthase,

96 said method comprising incubating said recombinant host cell with a tetritol or

97 a fermentable carbon source under conditions that said host cell (i) produces a tetritol-

98 4-phosphate from said tetritol or said fermentable carbon source; and (ii) converts at

99 least some of the tetritol- 4-phosphate into butadiene or a non-2-C-methylated

100 butadiene precursor.

101

102 26. The method of anyone of claims 1-15, 24, or 25, wherein said fermentable

103 carbon source is selected from the group comprising: glycerol, a sugar from a

104 foodstuff; and sugar from a non- foodstuff.

105 27. The method of claim 26, wherein said sugar from foodstuff is sucrose or

106 glucose.

107 28. The method of claim 26, wherein said sugar from said non- foodstuff is

108 cellulosic or hemicellulosic derived sugars.

109 29. A method of converting erythritol 4-phosphate to 4-diphospocytidyl-D-

I I o erythritol, said method comprising contacting erythritol 4-phosphate with a

I I I cytidylyltransferase or a recombinant host cell expressing said cytidylyltransferase,

1 12 wherein the incubation converts erythritol 4-phosphate to 4-diphospocytidyl-D-

1 1 3 erythritol.

1 14 30. A method of converting 4-diphospocytidyl-D-erythritol to 2-phospho-4-

1 15 (cytidine 5' diphospho)-D-erythritol, said method comprising contacting 4-

1 16 diphospocytidyl-d-erythritol with a 4-(cytidine 5 ' -diphospho)-2-C-methyl-D-

1 1 7 erythritol kinase or a recombinant host cell expressing said 4-(cytidine 5'-diphospho)-

1 1 8 2-C-methyl-D-erythritol kinase, wherein the incubation converts 4-diphospocytidyl-

1 1 9 D-erythritol to 2-phospho-4-(cytidine 5' diphospho)-D-erythritol.

120 31. A method of converting 2-phospho-4-(cytidine 5' diphospho)-D-erythritol to

121 D-erythritol-2,4-cyclodiphosphate, said method comprising contacting 2-phospho-4-

122 (cytidine 5' diphospho)-D-erythritol with a 2-C-methyl-D-erythritol 2,4-

123 cyclodiphosphate synthase or a recombinant host cell expressing said 2-C-methyl-D-

124 erythritol 2,4-cyclodiphosphate synthase, wherein the incubation converts 2-phospho-

125 4-(cytidine 5' diphospho)-D-erythritol to erythritol-2,4-cyclodiphosphate.

126 32. A method of converting erythritol-2,4-cyclodiphosphate to l-hydroxy-2-

127 butenyl-4-diphosphate, said method comprising contacting erythritol-2,4-

128 cyclodiphosphate with a l-hydroxy-2-methyl-2-butenyl 4-diphosphate synthase or a

129 recombinant host cell expressing said l-hydroxy-2-methyl-2-butenyl 4-diphosphate

130 synthase, wherein the incubation converts erythritol-2,4-cyclodiphosphate to 1-

131 hydroxy-2-butenyl-4-diphosphate.

132 33. The method of any of the preceding claims, comprising recovering butadiene or

133 said non-2-C-methylated butadiene precursor.

134 34. A method of producing butadiene or a butadiene precursor using a recombinant

135 host cell, said method comprising incubating said recombinant host with a feedstock,

136 wherein said host (i) converts at least some of the feedstock to a tetritol or a tetritol 4-

137 phosphate; and (ii) converts said tetritol or said tetritol 4-phosphate to butadiene.

138 35. The method of claim 34, wherein said recombinant host is deficient in DOXP

139 synthase activity.

140 36. The method of claim 34 or claim 35, wherein said recombinant host is

141 deficient in DXP reductoisomerase activity.

142 37. The method of anyone of claims 34-36, wherein said recombinant host

143 comprises an exogenous nucleic acid encoding an enzyme classified under EC

144 2.7.7.60.

145 38. The method of anyone of claims 34-37, wherein said recombinant host

146 comprises an exogenous nucleic acid encoding an enzyme classified under EC

147 2.7.1.14.

148 39. The method of anyone of claims 34-38, wherein said recombinant host

149 comprises an exogenous nucleic acid encoding an enzyme classified under EC

150 4.6.1.12.

151 40. The method of anyone of claims 34-39, wherein said recombinant host

152 comprises an exogenous nucleic acid encoding an enzyme classified under EC

153 1.17.7.1.

154 41. The method of anyone of claims 34-40, wherein said recombinant host

155 comprises an exogenous nucleic acid encoding an enzyme classified under EC

156 1.17.1.2.

157