CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from International Application No.

PCT/US2012/067463 filed November 30, 2012, which claims priority from U.S. Application No. 61/566,085, filed December 2, 2011, and U.S. Application No. 61/714,883, filed October 17, 2012; and from International Application No.

PCT/US2012/042757, filed June 15, 2012, which claims the benefit of U.S.

Provisional Application No. 61/714,883, filed October 17, 2012; U.S. Provisional Application No. 61/566,085, filed December 2, 2011; and U.S. Provisional

Application No. 61/498,408, filed June 17, 2011, all of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

[0002] This invention relates to methods for biosynthesizing 1,3-butadiene, and more particularly to synthesizing 1,3-butadiene using one or more isolated enzymes such as dehydrogenases, monooxygenases, desaturases, dehydratases, and decarboxylases, or using recombinant host cells expressing one or more of such enzymes.

BACKGROUND

[0003] 1,3 -Butadiene (hereinafter butadiene) is an important monomer for the production of synthetic rubbers including styrene-butadiene-rubber (SBR), polybutadiene (PB), styrene-butadiene latex (SBL), acrylonitrile-butadiene-styrene resins (ABS), nitrile rubber, and adiponitrile, which is used in the manufacture of Nylon-66 (White, Chemico-Biological Interactions, 2007, 166, 10-14).

[0004] Butadiene is typically produced as a co-product from the steam cracking process, distilled to a crude butadiene stream, and purified via extractive distillation (White, Chemico-Biological Interactions, 2007, 166, 10-14).

[0005] On-purpose butadiene has been prepared among other methods by dehydrogenation of n-butane and n-butene (Houdry process); and oxidative dehydrogenation of n-butene (Oxo-D or O-X-D process) (White, Chemico-Biological Interactions, 2007, 166, 10-14).

[0006] 95% of global butadiene production is undertaken via the steam cracking process using petrochemical-based feedstocks such as naphtha. Production of on- purpose butadiene is not significant, given the high cost of production and low process yield (White, Chemico-Biological Interactions, 2007, 166, 10-14). Given a reliance on petrochemical feedstocks and, for on-purpose butadiene, energy intensive catalytic steps; biotechnology offers an alternative approach via biocatalysis.

Biocatalysis is the use of biological catalysts, such as enzymes, to perform biochemical transformations of organic compounds.

[0008] Accordingly, against this background, it is clear that there is a need for sustainable methods for producing intermediates, in particular butadiene, wherein the methods are biocatalyst based (Jang et al., Biotechnology & Bioengineering, 2012, 109(10), 2437 - 2459).

[0009] Both bioderived feedstocks and petrochemical feedstocks are viable starting materials for the biocatalysis processes.

[0010] The generation of two vinyl groups into medium carbon chain length enzyme substrates is a key consideration in synthesizing butadiene via biocatalysis processes.

[0011] There are no known enzyme pathways leading to the synthesis of butadiene in prokaryotes or eukaryotes. Three potential pathways have been suggested for producing 1,3 -butadiene from biomass-sugar: (1) from acetyl-CoA via crotonyl-CoA; (2) from erythrose-4-phosphate; and (3) via a condensation reaction with malonyl- CoA and acetyl-CoA. However, no information using these strategies has been reported (Jang et al, Biotechnology & Bioengineering, 2012, 109(10), 2437 - 2459).

[0012] The closest analogous compound synthesized by prokaryotes or eukaryotes is 2-methyl-l,3-butadiene (isoprene), given the short five carbon chain length and two vinyl groups. Isoprene may be synthesised via two routes leading to the precursor dimethylvinyl-PP, viz. the mevalonate and the non-mevalonate pathway (Kuzuyama, Biosci. Biotechnol. Biochem., 2002, 66(8), 1619-1627). [0013] The mevalonate pathway incorporates a decarboxylase enzyme, mevalonate diphosphate decarboxylase (hereafter MDD), that generates the first vinyl-group in the precursors leading to isoprene (Kuzuyama, Biosci. Biotechnol. Biochem., 2002, 66(8), 1619-1627).

[0014] Mevalonate diphosphate decarboxylase (EC 4.1.1.33) may thus be earmarked as a candidate enzyme in the synthesis of butadiene from non-native substrates.

[0015] In elucidating the role of the 3-methyl group associated with the native substrate, mevalonate diphosphate, it has been demonstrated that the turn-over number, k _cat , for 3-hydroxy-5-diphosphatepentanoic acid as shown in FIG. 12(a) is dramatically lower at 0.23±0.05 [s ¹ ] as opposed to the nominal 8.33±1 [s ¹ ] for the native substrate (Dhe-Paganon et al, Biochemistry, 1994, 33, 13355 - 13362). In addition, the reaction with substrate only progressed as far as phosphorylation of the 3-hydroxyl group, i.e., no decarboxylated product was detectable, implying that the decarboxylation rate is decreased at least 300 fold compared to the native substrate. In conclusion, the 3-methyl group was deemed indispensible in stabilizing the carbo- cation transition state (Dhe-Paganon et ah, Biochemistry, 1994, 33, 13355 - 13362).

[0016] It has been demonstrated that the MDD enzyme from Saccharomyces cerevisiae accepts 3-hydroxy-3-methyl-butyrate (FIG. 12(b)), which includes the 3- methyl group stabilizing the carbocation transition state, as a substrate converting the substrate to isobutene. However, the specific activity is dramatically lower at 4.8· 10 ^"6 ^mol/(min-mg)] as opposed to the native substrate activity of 6.4 ^mol/(min-mg)] (Gogerty & Bobik, Applied & Environmental Microbiology, 2010, 76(24), 8004 - 8010).

[0017] The key substrate binding interactions of serine and arginine residues on the periphery of the catalytic cleft with the pyrophosphate group of the native substrate mevalonate diphosphate have been elucidated. Correct substrate orientation within the catalytic cleft is thus important to enzyme activity, which plausibly accounts for the low activity of MDD when accepting 3-hydroxy-3-methyl-butyrate (Figure 14(b)) as substrate (Barta et ah, Biochemistry, 2012, 51, 5611-5621).

[0018] The importance of the 3-methyl group and the pyrophosphate group associated with the native substrate in underpinning the activity of MDD teaches against using MDD in the synthesis of butadiene from non-native precursors that do not contain these key groups.

[001 ] The enzyme, isoprene synthase (hereinafter ISPS), generates the second vinyl group in the final precursor, dimethylvinyl-PP, of isoprene synthesis.

[0020] Isoprene synthase (EC 4.2.3.27) may thus be earmarked as a candidate enzyme in the synthesis of butadiene from non-native substrates.

[0021] Similar to MDD, the 3-methyl group associated with the native substrate dimethylvinyl-PP plays an important role in stabilizing the carbo-cation that has been postulated as a transient intermediate (Silver & Fall, J. Biol. Chem., 1995, 270(22), 13010 - 13016; Kuzma ei a/., Current Microbiology , 1995, 30, 97 - 103).

[0022] The importance of the 3-methyl group in underpinning the activity of ISPS teaches against using ISPS for the synthesis of butadiene from non-native precursors that do not contain the 3-methyl group.

[0023] In addition to MDD and ISPS, microorganisms can generate vinyl groups in metabolites typically via dehydratase, ammonia lyase, desaturase, or decarboxylase activity. However, these enzyme activities rarely catalyse the formation of terminal vinyl groups. Dehydratases and ammonia lyases typically accept fatty acid analogues that have activated hydrogen atoms or aromatic compounds, where the aromatic ring serves as an electron withdrawing group. Desaturases predominate in fatty acid synthesis, generating unsaturated bonds at fixed non-terminal positions along long chain fatty acids. In turn, decarboxylases acting on the terminal carboxyl group typically leave the associated alpha functional group at the terminal position after catalysis. Therefore, the associated enzymatic activity of these enzymes teaches against their use for the generation of terminal vinyl groups in short or medium chain carbon metabolites leading to the synthesis of butadiene.

SUMMARY

[0024] This document is based at least in part on the discovery that it is possible to construct biochemical pathways for producing medium chain carbon metabolites, in which two vinyl groups can be formed, leading to the synthesis of butadiene. These pathways described herein rely on enzymes such as MDD, ISPS and dehydratases for the final enzymatic step.

[0025] Prior to the inventors' surprising discovery, it was not known that enzymes capable of forming two terminal vinyl groups in a medium chain carbon metabolite existed or could be produced for the synthesis of butadiene.

[0026] Thus, in one aspect, this document provides enzymes that can convert butadiene synthesis substrates into butadiene. As used herein, the term "butadiene synthesis substrate" refers to a substrate for which an enzyme can catalyze a reaction that results directly in 1,3 -butadiene or in a product that, after one or more enzyme- catalyzed reactions, is converted to 1,3-butadiene.

[0027] In some embodiments, the first vinyl group leading to the synthesis of butadiene is enzymatically formed in 4-oxalocrotonate, 2-hydroxymuconate semialdehyde, or 2-hydroxy-6-oxonona-2,4-diene-l,9-dioate to produce 2-oxopent-4- enoate. See, FIG. 2.

[0028] In some embodiments, the first vinyl group leading to the synthesis of butadiene is enzymatically formed in propanoyl-CoA, lactoyl-CoA, or 3- hydroxypropionyl-CoA to produce propenoyl-CoA. See, FIG. 3.

[0029] In some embodiments, the first vinyl group leading to the synthesis of butadiene is enzymatically formed in (R) 3 -hydroxy -pentanoate to produce 3- hydroxypent-4-enoate. See, FIG. 4.

[0030] In some embodiments, the first vinyl group leading to the synthesis of butadiene is enzymatically formed in pent-2-enoyl [acp] to produce 2, 4— pentadienoyl -[acp]. See FIG. 5.

[0031 ] In some embodiments, the first vinyl group leading to the synthesis of butadiene is enzymatically formed in 5-hydroxypentanoyl-CoA (via 5-hydroxy-pent- 2-enoyl-CoA as intermediate) or pent-3-enoyl-CoA to produce 2,4-pentadienoyl-CoA. See, FIG. 6.

[0032] In some embodiments, the first vinyl group leading to the synthesis of butadiene is enzymatically formed in 4-hydroxybutyryl-CoA, (R) 3-hydroxybutanoyl- CoA or glutaconyl-CoA to produce crotonyl-CoA. See, FIG. 7. [0033] In some embodiments, the first vinyl group leading to the synthesis of butadiene is enzymatically formed in 2-butanol to produce 3-buten-2-ol. See, FIG. 8.

[0034] In some embodiments, the second vinyl group leading to the synthesis of butadiene is formed by mevalonate diphosphate decarboxylase (MDD), an enzyme classified under EC 4.1.1.33 (FIG. 9). For example, 2-hydroxypent-4-enoate is converted consecutively by two or more enzymes; producing butadiene in the last enzymatic conversion by decarboxylation directly (FIG. 1, reaction X).

[0035] In some embodiments, the second vinyl group leading to the synthesis of butadiene is formed by isoprene synthase (ISPS), an enzyme classified under EC 4.2.3.27 (FIG. 10). For example, activated butenols (diphosphoesters) may be generated by one or more enzymes from butenols (FIG. 1 , reaction II); producing butadiene in the last enzymatic conversion by dephosphorylation directly (FIG. 1, reaction III).

[0036] In some embodiments, the second vinyl group leading to the synthesis of butadiene is formed by a dehydratase enzyme classified in EC 4.2.1.-, such as linalool dehydratase (EC 4.2.1.127), kievitone hydratase (EC 4.2.1.95), oleate hydratase (EC 4.2.1.53) and carotenoid 1,2-hydratase (EC 4.2.1.131) (FIG. 11). Such dehydratases accept hydroxylated substrates such as butenols. For example, butenols may be generated in one or more enzymatic steps from butanediols, butanols, butenes, butenals or C5 alkenols (FIG. 1, reactions IV, V, VI, VII, IX) by dehydratase, hydratase, desaturase, dehydrogenase or decarboxylase activity; producing butadiene in the last enzymatic conversion by dehydration directly (FIG. 1, reaction I). Butenols include, for example, 1-buten-l-ol, 2-buten-l-ol and 3-buten-2-ol (see FIG. 1).

[0037] For example, this document provides enzymes that convert butenols into butadiene. This conversion can be performed by a single enzyme, or may be performed by two or more enzymes, acting sequentially (that is to say, for example, a first enzyme acts on a four carbon molecule to produce a first butenol, and that first butenol then is acted upon by a second enzyme to produce butadiene) (see, e.g., FIG. 1, reaction I).

[0038] This document also provides methods of producing butadiene from a unsaturated hydroxylated four carbon molecule, comprising at least one biocatalytic step. For example, the butenol can be activated to the corresponding butenol diphosphoester before conversion to butadiene (see, e.g., FIG. 1, reactions II & III). In some embodiments, the butenol is selected from the group consisting of 1 buten 2 ol, 1 buten 3 ol, 1 buten 4 ol, 2 buten 1 ol, 2 buten 2 ol, 2 buten 3 ol or 2 buten 4 ol. For butenol such as 1-buten-l-ol, l-buten-2-ol, 2-buten-2-ol, and 2-buten-3-ol the butenol can be generated in situ as the enolate of the corresponding ketone or aldehyde such as 1-butanal or 2-butanone.

[0039] In some embodiments, a butenol is produced from four carbon molecules selected from the group consisting of a butanediol (1,4-butanediol, l,3-butanediol,2,3- butanediol) (FIG. 1, reaction IV) or a butanol (1-butanol, or 2-butanol) (FIG. 1, reaction V) or a butene (1-butene or 2-butene) (FIG. 1, Reaction VI) or a butenal such as 1-butenal or 2-butenal, or a 2-keto-but-l-ene (FIG. 1, reaction VII) by the action of an enzyme.

[0040] The reactions performed by the enzymes can be net dehydration (i.e., the removal of H ₂ O from the molecule by an enzyme having dehydratase activity, reaction IV), dehydrogenation (i.e., the removal of hydrogen from the molecule, which in the reactions catalysed by the enzymes results in a desaturation of the carbon backbone of the molecule) by an enzyme or enzyme complex having desaturase activity, reaction V), hydroxylation (i.e., the replacement of a hydrogen with a hydroxy 1 group) by an enzyme with hydroxylase activity, such as an alkene monooxygenase or Cytochrome P450 or co-hydroxylase (reaction VI), or reduction by an oxidoreductase/ketone reductase to convert butenals or C4 unstaurated ketones to butenols. For the dehydration step, the enzyme may be the same enzyme class as the enzyme class used for the dehydration of the butenol to butadiene or may be of another enzyme class. Migration of the double bond in the butenols may be catalysed by isomerases.

[0041] This document also provides an enzyme from the enzyme class 4.2.1.-. which converts butanediols to butenol (FIG. 1 , reaction VIII).

[0042] In some embodiments, a butenol such as l-buten-4-ol is produced from a five carbon molecule such as 2-hydroxypent-4-enoate by the action of a decarboxylase (such as a decarboxylase from EC 4.1.1.-) (FIG. 1 , reaction IX). 2-hydroxypent-4- enoate may also be converted directly into butadiene by a decarboxylase or GHMP kinase without formation of the intermediate butenol (FIG. 1, Reaction X).

[0043] In some embodiments, the butenol is selected from the group consisting of 1 buten 2 ol, 1 buten 3 ol, 1 buten 4 ol, 2 buten 1 ol, 2 buten 2 ol, 2 buten 3 ol or 2 buten 4 ol. For butenol such as l-buten-2-ol, 2-buten-2-ol, and 2-buten-3-ol the butenol can be generated in situ as the enolate of the corresponding ketone or aldehyde such as 1- butanal or 2-butanone.

[0044] In one aspect, this document features a method for the biosynthesis of butadiene. The method includes forming two terminal vinyl groups in a butadiene synthesis substrate. A first vinyl group can be enzymatically formed in the butadiene synthesis substrate to produce a compound selected from the group consisting of 2- oxopent-4-enoate, propenyl-CoA, (R) 3-hydroxypent-4-enoate, 2,4-pentadienoyl- [acp], 2,4-pentadienoyl-CoA, crotonyl-CoA, and 3-buten-2-ol.

[0045] In one aspect, 2-oxopent-4-enoate can be produced by forming a first vinyl group in (i) 4-oxalocrotonate using an 4-oxalocrotonate decarboxylase classified in EC 4.1.1.77, (ii) 2-hydroxymuconate semialdehyde using a 2 -hydroxy muconate- semialdehyde hydrolase classified in EC 3.7.1.9, or (iii) 2-hydroxy-6-oxonona-2,4- diene-l,9-dioate using a 2-hydroxy-6-oxonona-2,4-dienedioate hydrolase classified in EC 3.7.1.14. 2-oxopent-4-enoate can be produced by converting 2-hydroxymuconate semialdehyde to 2-hydroxymuconate using a 2 aminomuconate semialdehyde dehydrogenase classified under EC 1.2.1.32, converting 2-hydroxymuconate to 4- oxalocrotonate using a 2-hydroxymuconate tautomerase classified under EC 5.3.2.6, and converting 4-oxalocrotonate to 2-oxopent-4-enoate using a 4-oxalocrotonate decarboxylase classified under EC 4.1.1.77. 2-hydroxymuconate semialdehyde can be produced by converting catechol to 2-hydroxymuconate semilaldehyde using a catechol 2,3-dioxygenase classified under EC 1.13.11.2. Catechol is produced by converting anthranilate using an anthranilate 1 ,2-dioxygenase classified under EC 1.14.12.1 or by converting protocatechuate using protocatechuate decarboxylase classified under EC 4.1.1.63. Anthranilate can be produced by converting chorismate using an anthranilate synthase classified under EC 4.1.3.27. Protocatechuate can be produced by converting 3-dehydroshikimate using a 3-dehydroshikimate dehydratase classified under EC 4.2.1.118. [0046] In one aspect, 2-hydroxymuconate semialdehyde can be produced by converting 5-carboxy-2-hydroxymuconate-6-semiladehyde using a 5-carboxy-2- hydroxymuconate-6-semialdehyde decarboxylase such as a 5-carboxy-2- hydroxymuconate-6-semialdehyde decarboxylase is encoded by praH. The 5- carboxy-2-hydroxymuconate-6-semiladehyde can be produced by converting protocatechuate using a protocatechuate 2,3-dioxygenase such as protocatechuate 2,3-dioxygenase is encoded by praA. 2-hydroxy-6-oxonona-2,4-diene-l,9-dioate can be produced by converting 2,3-dihydroxy phenylpropionoate using a 3- carboxyethylcatechol 2, 3-dioxygenase classified under EC 1.13.11.16. 2, 3- dihydroxyphenylpropionate can be produced by converting cis-3-(carboxy-ethyl)-3,5- cyclo-hexadiene-l,2-diol using a 3-(cis-5, 6-dihydroxycyclohexa-l,3-dien-l-yl) propanoate dehydrogenase classified under EC 1.3.1.87. Cis-3-(carboxy-ethyl)-3,5- cyclo-hexadiene-l,2-diol can be produced by converting 3 -phenyl-propionate using a 3-phenylpropanoate dioxygenase classified under EC 1.14.12.19. The 3-phenyl- propionate can be produced by converting E-cinnamate using a 2-enoate reductase classified under EC 1.3.1.31. E-cinnamate can be produced by converting L- phenylalanine using a phenylalanine ammonia-lyase classified under EC 4.3.1.24.

[0047] In one aspect, the butadiene synthesis substrate can be propanoyl-CoA.

Propenoyl-CoA can be produced by forming a first vinyl group in (i) propanoyl-CoA using a butyryl-CoA dehydrogenase classified under EC 1.3.8.1 or a medium-chain acyl-CoA dehydrogenase classified under EC 1.3.8.7, (ii) lactoyl-CoA using a lactoyl- CoA dehydratase classified under EC 4.2.1.54, or (iii) 3-hydroxypropionyl-CoA using a 3-hydroxypropionyl-CoA dehydratase classified under EC 4.2.1.116. The propanoyl-CoA can be produced by converting (2S)-methylmalonyl-CoA using a methylmalonyl-CoA carboxy transferase classified under EC 2.1.3.1 or a

methylmalonyl-CoA decarboxylase classified under EC 4.1.1.41. The (2S)- methylmalonyl-CoA can be produced by converting (2R)-methylmalonyl-CoA using a methylmalonyl-CoA epimerase classified under EC 5.1.9 .1. The (2R)- methylmalonyl-CoA can be produced by converting succinyl-CoA using a methylmalonyl-CoA mutase classified under EC 5.4.99.2.

[0048] The propanoyl-CoA can be produced by converting 2-oxo-butyrate using a 2- ketobutyrate formate-lyase classified under EC 2.3.1.- such as the 2-ketobutyrate formate-lyase encoded by tdcE. The 2-oxo-butryate can beproduced by converting L-threonine using a threonine ammonia lyase classified under EC 4.3.1.19.

[0049] The propanoyl-CoA can be produced by converting propanol using a propionaldehyde dehydrogenase such as a propionaldehyde dehydrogenase is encoded by pduP Propanol can be produced by converting 1,2-propanediol using a propanediol dehydratase classified under EC 4.2.1.28.

[0050] The propanoyl-CoA can be produced from levulinic acid by converting levulinyl-CoA using a transferase classified under EC 2.3.1.-. The levulinyl-CoA can be produced by converting levulinyl acid using an acyl-CoA synthetase or ligase classified under EC 6.2. 1.-.

[0051 ] The lactoyl-CoA can be produced by converting L-lactate using a proprionate CoA-transferase classified under EC 2.8.3.1. L-lactate can be produced by converting pyruvate using an L-lactate dehydrogenase classified under EC 1.1.1.27.

[0052] The 3-hydroxypropionyl-CoA can be produced by converting 3- hydroxypropionate using a 3-hydroxyisobutyryl-CoA hydrolase classified under EC 3.1.2.4 or by converting malonate semialdehyde using a 3-hydroxypropionate dehydrogenase classified under EC 1.1.1.59. The malonate semiladehyde is produced by converting malonyl-CoA using a ma onyl-CoA reductase classified under EC 1.2.1.75.

[0053] The propanoyl-CoA can be produced by converting propenoyl-CoA using a butyryl-CoA dehydrogenase classified under EC 1.3.8.1 or a medium-chain acyl-CoA dehydrogenase classified under EC 1.3.8.7.

[0054] The (R) 3-hydroxypent-4-enoate propenoyl-CoA can be produced by forming a first vinyl in (R) 3-hydroxypentanoate using a desaturase/monooxygenase or cytochrome P450. The (R) 3 -hydroxy -pentanoate can be produced by converting (R) 3-hydroxypentanoyl-CoA using a thioesterase classified under EC 3.1.2.-. The (R) 3- hydroxypentanoyl-CoA can be produced by converting 3-oxopentanoyl-CoA using an acetoacety -CoA reductase classified under EC 1.1.1.36. The 3-oxopentanoyl-CoA can be produced by converting propanoyl-CoA using an acetyl-CoA C-acyltransferase classified under EC 2.3.1.16.

[0055] The 2,4-pentadienoyl-[acp] can be produced by forming a first vinyl group in pent-2-enoyl-acp using an acyl-[acp] dehydrogenase. The 2,4-pentadienoyl-CoA can be produced by forming a first vinyl group in (i) 5-hydroxypentanoyl-CoA using a 5- hydroxyvaleryl-CoA dehydratase classified under EC 4.2.1.- or (ii) pent-3-enoyl-CoA using a 2,4-dienoyl coenzyme A reductase classified under EC 1.3.1.34. The 5- hydroxyvaleryl-CoA dehydratase can originate from Clostridium viride.

[0056] The crotonyl-CoA can be produced by forming a first vinyl group in (i) glutaconyl-CoA using a glutaconyl-CoA decarboxylase classified under EC 4.1.1.70,

(ii) 4-hydroxybutryl-CoA using a 4-hydroxybutanoyl-CoA dehydratase classified under EC 4.2.1.120 and a vinylacetyl-CoA isomerase classified under EC 5.3.3.3, or

(iii) ( ) 3-hydroxybutanoyl-CoA using an enoyl-CoA hydratase classified under EC 4.2.1.119.

[0057] The 3-buten-2-ol can be produced by forming a first vinyl group in 2-butanol using a desaturase or a monooxygenase.

[0058] The second vinyl group is enzymatically formed in (R) 3-hydroxypent-4- enoate by a mevalonate diphosphate decarboxylase (MDD). The MDD can be classified under EC 4.1.1.33. The MDD can include a minimum of four serine residues within five residues either side of the catalytic arginine residue of the catalytic cleft. The MDD can be from the genus Streptococcus or Staphylococcus.

[0059] The second vinyl group can be enzymatically formed in either 2-buten-l-ol diphosphate or 3-buten-2-ol diphosphate by an isoprene synthase (ISPS). The second vinyl group can be enzymatically formed in either 3-buten-2-ol or 2-buten-l-ol by a dehydratase in enzyme class EC 4.2.1.- such as a linalool dehydratase (EC

4.2.1.127), a kievitone hydrase (EC 4.2.1.95), an oleate hydratase (EC 4.2.1.53) or a carotenoid 1 ,2-hydratase (EC 4.2.1.131).

[0060] The pent-2-enoyl-[acp] can be produced by converting (R) 3- hydroxypentanoyl-[acp] using a 3-Hydroxyacy -facpJ dehydratase classified under EC 4.2.1.59. The (R) 3-hydroxypentanoyl-[acp] can be produced by converting 3- oxopentanoyl-[acp] using a 3-oxoacyl-facpJ reductase classified under EC 1.1.1.100.

[0061] 3-oxopentanoyl-[acp] can be produced by converting propanoyl-CoA using a beta-ketoacyl-facpj synthase I classified under EC 2.3.1.41 and an acyl-transferase such as tcsA.

[0062] The pent-2-enoyl-[acp] can be produced by converting pent-2-enoyl-CoA using an acyl transferase. The pent-2-enoyl-CoA can be produced by converting (R) 3-hydroxypentanoyl-CoA using an enoyl-CoA hydratase classified under EC

4.2.1.119. The (R) 3-hydroxypentanoyl-CoA can be produced by converting 3- oxopentanoyl-CoA using an acetoacetyl-CoA reductase classified under EC 1.1.1.36.

[0063] The 3-oxopentanoyl-CoA can be produced by converting propanoyl-CoA using an acetyl-CoA C-acyltransferase classified under EC 2.3.1.16.

[0064] The pent-3-enoyl-CoA can be produced by converting pent-2-enoyl-CoA using an isomerase classified under EC 5.3.3.8.

[0065] The 5-hydroxypentanoyl-CoA can be produced by converting either (i) 5- hydroxypentanoate using 5-hydroxypentanoate CoA-transferase classified under EC 2.8.3.14 or (ii) pentanoyl-CoA using a cytochrome P450 such as the gene product of CYP153A6. The 5-hydroxypentanoate can be produced by converting 5- oxopentanoate using a 5 -hydroxy valerate dehydrogenase such as the gene product of cpnD or the dehydrogenase from Clostridium viride. The 5-oxopentanoate can be produced by converting 5-aminovalerate using a 5-aminovalerate transaminase classified under EC 2.6.1.48. The 5-aminovalerate can be produced by converting D- proline using a Ό-proline reductase classified under EC 1.21.4.1. D-proline can be produced by converting L-proline using a proline racemase classified under EC 5.1.1.4. L-proline can be produced by converting (S)-l-Pyrroline-5-carboxylate using a pyrroline-5-carboxylate reductase classified under EC 1.5.1.2. (S)-l-Pyrroline-5- carboxylate can be produced by spontaneous conversion of L-glutamate 5- semialdehyde. L-glutamate 5-semialdehyde can be produced by converting L- glutamyl-5-phosphate using a glutamate-5-semialdehyde dehydrogenase classified under EC 1.2.1.41. The L-glutamyl-5-phosphate can be produced by converting L- glutamate using glutamate 5-kinase classified under EC 2.7.2.11.

[0066] The pentanoyl-CoA can be produced by converting pent-2-enoyl-CoA using a trans-2-enoyl-CoA reductase classified under EC 1.3.1.38.

[0067] Glutaconyl-CoA can be produced by converting 2-hydroxyglutaryl-CoA using a dehydratase classified under EC 4.2.1.- The 2-hydroxyglutaryl-CoA can be produced by converting 2-hydroxyglutarate using a glutaconate CoA-transferase classified under EC 2.8.3.12. The 2-hydroxyglutarate can be produced by converting 2-oxoglutarate using a 2-hydroxyglutarate dehydrogenase classified under EC 1.1.99.2. The 3-hydroxybutanoyl-CoA can be produced by converting acetoacetyl- CoA using 3-hydroxybutyryl-CoA dehydrogenase classified under EC 1.1.1.36. The acetoacetyl-CoA can be produced by converting acetyl-CoA using acetyl-CoA C- acetyltransferase classified under EC 2.3.1.9.

[0068] The 4-hydroxybutyryl-CoA can be produced by converting 4-hydroxybutyrate using a CoA-transferase such as the gene product of Ck-cat2. The 4-hydroxybutyrate can be produced by converting succinate semialdehyde using a 4-hydroxybutyrate dehydrogenase classified under EC 1.1.1.61. The succinate semialdehyde can be produced by converting succinyl-CoA using a succinate-semialdehyde dehydrogenase classified under EC 1.2.1.76.

[0069] The 2-butanol can be produced by converting butanone using a (R)-speciflc secondary alcohol dehydrogenase classified under EC 1.1.1.B4. The butanone can be produced by converting 2,3 butanediol using a propanediol dehydratase classified under EC 4.2.1.28. The 2,3 butanediol can be produced by converting (R)-acetoin using a (R,R)-butanediol dehydrogenase classified under EC 1.1.1.4. (R)-acetoin can be produced by converting 2-acetolactate using an acetolactate decarboxylase classified under EC 4.1.1.5. The 2-acetolactate can be produced by converting pyruvate using an acetolactate synthase classified under EC 2.2.1.6. The (R) 3- hydroxypent-4-enoate can be produced by converting 3-hydroxypent-4-enoyl-CoA using a thioesterase classified under EC 3.1.2.-. The 3-hydroxypent-4-enoyl-CoA can be produced by converting 2,4-pentadienoyl-CoA using an enoyl-CoA dehydratase 2 classified under EC 4.2.1. The 2,4-pentadienoyl-CoA can be produced by converting

2- hydroxypent-4-enoyl-CoA using a 2-Hydroxyisocaproyl-CoA dehydratase such as the gene products of the initiator HadI and HadBC. The 2-hydroxypent-4-enoyl-CoA can be produced by converting 2-hydroxypent-4-enoate using a CoA-transferase such the gene product of GctAB. The 2-hydroxypent-4-enoate can be produced by converting 2-oxopent-4-enoate using a (R)-2-hydroxyisocaproate dehydrogenase such as the gene product of LdhA from Clostridium difficile.

[0070] The (R)-hydroxypent-4-enoate can be produced by converting (R) 3- hydroxypent-4-enoyl-CoA using a thioesterase classified under EC 3.1.2.-. The (R)

3- hydroxypent-4-enoyl-Cft4 can be produced by converting 3-oxopent-4-enoyl-CoA using an acetoacetyl-CoA reductase classified under EC 1.1.1.36. The 3-oxopent-4- enoyl-CoA can be produced by converting propenoyl-CoA using a β-ketothiolase classified under EC 2.3.1.16. The ( )-hydroxypent-4-enoate can be produced by converting (R) 3-hydroxypent-4-enoyl-CoA using a thioesterase classified under EC 3.1.2.-. The (R) 3-hydroxypent-4-enoyl-CoA can be produced by converting (R)-3- hydroxypen-4-enoyl-[acp] using a (R)-3-hydroxyacyl-ACP:CoA transacylase such as the gene product of phaG. (R)-3-hydroxypen-4-enoyl-[acp] can be produced by converting 2,4 pentadienoyl-[acp] using a 3-hydroxyacyl-facyl-carrier-proteinJ dehydratase classified under EC 4.2.1.59. (R) 3-hydroxypent-4-enoyl-CoA can be produced by converting 2,4-pentadienoyl-CoA using an enoyl-CoA dehydratase 2 classified under EC 4.2.1.119.

[0071] 2-buten-l-ol diphosphate can be produced by converting 2-buten-l-ol phosphate using a phosphomevalonate kinase classified under EC 2.7.4.2 or using a diphosphokinase classified under EC 2.7.6.-. The 2-buten-l-ol phosphate can be produced by converting 2-buten- 1 -ol using a mevalonate kinase classified under EC 2.7.1.36. The 2-buten-l-ol can be produced by converting 2-buten-l-al using an allyl- alcohol dehydrogenase classified under EC 1.1.1.54. The 2-buten- 1 -al can be produced by converting crotonic acid using a long-chain-aldehyde dehydrogenase classified under EC 1.2.1.48. Crotonic acid can be produced by converting crotonyl- CoA using a succinate-CoA ligase classified under EC 6.2.1.5.

[0072] The 2-buten-l-ol diphosphate can be produced by converting 2-buten-l-ol using a diphosphokinase classified under EC 2.7.6.- such as a thiamine diphosphokinase classified under EC 2.7.6.2.

[0073] The 3-buten-2-ol diphosphate can be produced by converting 3-buten-2-ol using a diphosphokinase classified under EC 2.7.6.- or 3-buten-2-ol phosphate using a phosphomevalonate kinase classified under EC 2.7.4.2. The 3-buten-2-ol phosphate can be produced by converting 3-buten-2-ol using mevalonate kinase classified under EC 2.7.1.36.

[0074] In any of the methods described herein, the method can be performed using isolated enzymes, using cell lysates comprising the enzymes, or using a recombinant host. The recombinant host can be anaerobically, micro-aerobically or aerobically cultivated. Recombinant host cells can be retained in ceramic hollow fiber membranes to maintain a high cell density during fermentation. The principal carbon source fed to the fermentation can derive from biological or non-biological feedstocks. For example, the biological feedstock is or derives from

monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin such as levulinic acid and furfural, lignin, triglycerides such as glycerol and fatty acids, agricultural waste or municipal waste. The non-biological feedstock is or derives from either natural gas, syngas, CO ₂ /H ₂ , methanol, ethanol, non-volatile residue (NVR) or caustic wash waste stream from cyclohexane oxidation processes.

[0075] The host microorganism can be a prokaryote from the genus Escherichia such as Escherichia coli; from the genus Clostridia such as Clostridium ljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; from the genus Corynebacteria such as Corynebacterium glutamicum; from the genus Cupriavidus such as Cupriavidus necator or Cupriavidus metallidurans ; from the genus Pseudomonas such as Pseudomonas fluorescens, Pseudomonas putida or Pseudomonas oleavorans; from the genus Delftia such as Delftia acidovorans; from the genus Bacillus such as Bacillus subtillis; from the genus Lactobacillus such as Lactobacillus delbrueckii; or from the genus Lactococcus such as Lactococcus lactis. The host microorganism can be a eukaryote from the genus Aspergillus such as Aspergillus niger; from the genus Saccharomyces such as Saccharomyces cerevisiae; from the genus Pichia such as Pichia pastoris; from the genus Yarrowia such as Yarrowia lipolytica; from the genus Issatchenkia such as Issathenkia orientalis; from the genus Debaryomyces such as Debaryomyces hansenii; from the genus Arxula such as Arxula adenoinivorans; or from the genus Kluyveromyces such as Kluyveromyces lactis.

[0076] In the recombinant hosts described herein, the enzymes catalyzing the hydrolysis of propionyl-CoA and acetyl-CoA can be attenuated; the enzymes consuming propanoyl-CoA via the methyl-citrate cycle can be attenuated; the enzymes consuming propanoyl-CoA to pyruvate can be attenuated; the enzymes consuming propanoyl-CoA to malonyl-CoA can be attenuated; a feedback-resistant threonine deaminase can be genetically engineered into the host organism; the β- ketothiolases catalyzing the condensation of acetyl-CoA to acetoacetyl-CoA such as the gene products oiAtoB or phaA can be attenuated; the polymer synthase enzymes in a host strain that naturally accumulates polyhydroxyalkanoates can be attenuated; a gene encoding a phosphotransacetylase, such as pta, can be attenuated; a gene encoding an acetate kinase degrading propanoate, such as ack, is attenuated; a gene encoding the degradation of pyruvate to lactate can be attenuated; a gene encoding the degradation of phophoenolpyruvate to succinate such as fr lBC is attenuated; a gene encoding the degradation of acetyl-CoA to ethanol such as adhE can be attenuated; the enzymes catalyzing anaplerotic reactions supplementing the citric acid cycle intermediates can be amplified; a puridine nucleotide transhydrogenase gene such as UdhA can be overexpressed; a glyceraldehyde-3P-dehydrogenase gene such as GapN can be overexpressed in the host organisms; a malic enzyme gene such as maeA or maeB is overexpressed in the host organism; a glucose-6-phosphate dehydrogenase gene such as zwf is overexpressed in the host organism; a fructose 1, 6 diphosphatase gene such as fl>p is overexpressed in the host organism; the efflux of butadiene across the cell membrane to the extracellular media can be enhanced or amplified by genetically engineering structural modifications to the cell membrane; or the efflux of butadiene across the cell membrane to the extracellular media is enhanced or amplified by genetically engineering an increase to any associated transporter activity for butadiene; oxygenases degrading butadiene to toxic intermediates such as 1,2- epoxy-3-butene and l,2:3,4-diepoxybutane are attenuated in the host organism.

[0077] In any of the methods described herein, the thioesterase can be the gene product of tesB; the acetoacetyl-CoA reductase can be the gene product of phaB; the acetyl-CoA C-acyltransferase can be the gene product of BhtB; the enoyl-CoA hydratase can be the gene product of phaJ; the desaturase can be the gene product of MdpJ; the cytochrome P450 can be a gene product of the CYP4 family; the beta- ketoacyl-[acp] synthase I can be the gene product of tcsB; the acyl-transferase can be the gene product of tcsA.

[0078] Also provided by the document is a method of converting a butenol into butadiene . The method includes contacting 3-buten-2-ol with a linalool dehydratase, such that 1,3 -butadiene is produced. The linalool dehydratase can be that classified under EC 4.2.1.127.

[0079] 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.

[0080] 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

[0081] FIG 1 is a schematic overview of the principal enzyme activities leading to 1 ,3 butadiene from C4 aldehydes and ketones, C4 hydroxy-aldehydes and diketones, butenes, butenals or unsaturated ketones, butenols, butanediols, C5 alkenols, and activated butenols.

[0082] FIG. 2 is a schematic of biochemical pathways leading to butadiene using 2- oxopent-4-enoate as a central precursor.

[0083] FIG. 3 is a schematic of biochemical pathways leading to butadiene using propenoyl-CoA as a central precursor.

[0084] FIG. 4 is a schematic of biochemical pathways leading to butadiene using 3- hydroxy-4-pentenoate as a central precursor.

[0085] FIG. 5 is a schematic of biochemical pathways leading to butadiene using 2,4- pentadienoyl-[acp] as a central precursor.

[0086] FIG. 6 is a schematic of biochemical pathways leading to butadiene using 2,4- pentadienoyl-CoA as a central precursor.

[0087] FIG. 7 is a schematic of biochemical pathways leading to butadiene using crotonyl-CoA as a central precursor.

[0088] FIG. 8 is a schematic of biochemical pathways leading to butadiene using 3- buten-2-ol as a central precursor. [0089] FIG. 9 is a schematic of biochemical pathways to synthesize butadiene using mevahnate diphosphate decarboxylase.

[0090] FIG. 10 is a schematic of biochemical pathways to synthesize butadiene using isoprene synthase.

[0091] FIG. 11 is a schematic of biochemical pathways to synthesize butadiene using dehydratases.

[0092] FIG. 12 is the structure of alternate substrates accepted by MDD, (a) is 3- hydroxy-5-diphosphatepentanoic acid and (b) is 3-hydroxy-3-methyl-butyrate.

[0093] FIG. 13 is the amino acid sequences for MDD enzymes from Saccharomyces cerevisiae (Uniprot Accession No. P32377, SEQ ID NO:l), Staphyloccocus epidermidis (Uniprot Accession No. Q7CCL9, SEQ ID NO:2), and Streptococcus pneumonia (Uniprot Accession No. B8ZLF3, SEQ ID NO:3), highlighting the conserved residues within the catalytic cleft of the enzyme in bold.

DETAILED DESCRIPTION

[0094] In particular, this document provides enzymes, non-natural pathways, cultivation strategies, feedstocks, host microorganisms and attenuations to the host's biochemical network, which generates two terminal vinyl groups in four and five carbon chain metabolites leading to the synthesis of 1 ,3 butadiene (referred to as "butadiene" herein) from central precursors or central metabolites. As used herein, the term "central precursor" is used to denote a key metabolite in a pathway leading to the synthesis of butadiene. The term "central metabolite" is used herein to denote a metabolite that is produced in all microorganisms to support growth.

[0095] As such, host microorganisms described herein can include endogenous pathways that can be manipulated such that butadiene can be produced. In an endogenous pathway, the host microorganism naturally expresses all of the enzymes catalyzing the reactions within the pathway. A host microorganism 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 host. Within an engineered pathway, the enzymes can be from a single source, i.e., from one species, or can be from multiple sources, i.e., different species. Nucleic acids encoding the enzymes described herein have been identified from various organisms and are readily available in publicly available databases such as GenBank or EMBL. Engineered hosts 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 pathways described herein.

Endogenous genes of the engineered hosts also can be disrupted to prevent the formation of undesirable metabolites or prevent the loss of intermediates in the pathway through other enzymes acting on such intermediates. Engineered hosts can be referred to as recombinant hosts or recombinant host cells. Thus, as described herein recombinant hosts can include nucleic acids encoding one or more of a decarboxylase, a dehydrogenase, a desaturase, a monooxygenase, an acyl [acyl carrier protein (acp)] dehydrogenase, a dehydratase, or a hydratase as described in more detail below.

[0096] In addition, the production of butadiene can be performed in vitro using the isolated enzymes described herein, using a lysate (e.g., a cell lysate) from a host microorganism as a source of the enzymes, or using a plurality of lysates from different host microorganisms as the source of the enzymes.

[0097] FIG. 1 provides an overview of the principal enzyme activities that can be used to produce butadiene from various four or five carbon molecules, including C4 aldehydes and ketones, C4 hydroxy-aldehydes and diketones, butenes, butenals or unsaturated ketones, butenols, butanediols, C5 alkenols, and activated butenols.

4.1 Enzymes generating the first terminal vinyl group in the biosynthesis of butadiene

[0098] As depicted in FIGs. 2-8, the first vinyl group can be formed in 4- oxalocrotonate, 2-hydroxymuconate semialdehyde, 2-hydroxy-6-oxonona-2,4-diene- 1,9-dioate, propanoyl-CoA, lactoyl-CoA, 3-hydroxypropionyl-CoA, (R) 3-hydroxy- pentanoate, pent-2-enoyl-[acp], 5-hydroxypentanoyl-CoA (via 5-hydroxy-pent-2- enoyl-CoA), pent-3-enoyl-CoA 4-hydroxybutyryl-CoA, glutaconyl-CoA, (R) 3- hydroxybutanoyl-CoA or 2-butanol to produce such compounds as 2-oxopent-4- enoate, propenoyl-CoA, (R) 3-hydroxypent-4-enoate, (R) 3-hydroxypent-4-enoyl- [acp], 2,4-pentadienoyl-CoA, crotonyl-CoA, and 3-buten-2-ol. [0099] In some embodiments, the first vinyl group leading to the synthesis of butadiene is enzymatically formed in 4-oxalocrotonate, 2-hydroxymuconate semialdehyde, or 2-hydroxy-6-oxonona-2,4-diene-l,9-dioate by 4-oxalocrotonate decarboxylase (EC 4.1.1.77), 2-hydroxymuconate-semialdehyde hydrolase (EC 3.7.1.9) or 2-hydroxy-6-oxonona-2,4-dienedioate hydrolase (EC 3.7.1.14) to produce 2-oxopent-4-enoate. See, e.g., FIG. 2.

[00100] In some embodiments, the first vinyl group leading to the synthesis of butadiene is enzymatically formed in propanoyl-CoA, lactoyl-CoA, 3- hydroxypropionyl-CoA by butyryl-CoA dehydrogenase (EC 1.3.8.1), medium-chain acyl-CoA dehydrogenase (EC 1.3.8.7), lactoyl-CoA dehydratase (EC 4.2.1.54) or 3- hydroxypropionyl-CoA dehydratase (EC 4.2.1.116) to produce propenoyl-CoA. See, e.g., FIG. 3.

[00101] In some embodiments, the first vinyl group leading to the synthesis of butadiene is enzymatically formed in (R) 3 -hydroxy -pentanoate by a desaturase or monooxygenase such as the gene product of MdpJ or cytochrome P450 such as the gene product of the CYP4 family to produce (R) 3-hydroxypent-4-enoate . See, e.g., FIG. 4.

[00102] The gene product of the monooxygenase, MdpJ, desaturates the terminal of the ethyl group adjacent to a secondary alcohol (Schafer et al, Applied and Environmental Microbiology, 2012, 78(24)).

[00103] The gene product of the cytochrome P450 CYP4 family has displayed specificity for terminal desaturation rather than ω-hydroxylation of the C5 carboxylic acid, valproic acid (Rettie et al, Biochemistry, 1995, 34, 7889 - 7895).

[00104]

[00105] In some embodiments, the first vinyl group leading to the synthesis of butadiene is enzymatically formed in pent-2-enoyl-[acp] by an acyl-[acp] dehydrogenase such as the gene product of TcsD to produce 2, 4 pentdienoyl-[acp]. See, e.g., FIG. 5.

[00106] The gene product of the acyl-[acp] dehydrogenase TcsD desaturates the terminal methylene of pent-2-enoyl-[acp] to 2,4-pentadienoyl-[acp] (Mo et al, J. Am. Chem. Soc, 2011, 133(4), 976 - 985). [00107] In some embodiments, the first vinyl group leading to the synthesis of butadiene is enzymatically formed in 5-hydroxypentanoyl-CoA (via 5-hydroxy-pent- 2-enoyl-CoA by a 5-hydroxyvaleryl-CoA dehydratase (EC 4.2.1.-) or 2, 4-dienoyl coenzyme A reductase (EC 1.3.1.34) to produce 2,4-pentadienoyl-CoA. See, e.g., FIG. 6.

[00108] The dehydration of 5 -hydroxy valerate by 5-hydroxyvaleryl-CoA dehydratase to 2,4 pentadienoyl-CoA has been characterized from Clostridium viride (Eikmanns and Buckel, Eur. J. Biochem., 1991, 197, 661 - 668).

[00109] In some embodiments, the first vinyl group leading to the synthesis of butadiene is enzymatically formed in 3-hydroxybutanoyl-CoA, 4-hydroxybutyryl- CoA or glutaconyl-CoA by an enoyl-CoA hydratase an by an enoyl-CoA hydratase (EC 4.2.1.119) such as the gene product iphaJ, a 4-hydroxybutanoyl-CoA dehydratase (EC 4.2.1.120) or a glutaconyl-CoA decarboxylase (EC 4.1.1.70) to produce crotonyl-CoA. See, e.g., FIG. 7.

[00110] The reversible dehydratase, 4-hydroxybutanoyl-CoA dehydratase, has been characterised has been characterized in several Clostridium species, such as Clostridium kluyveri, providing for a route to crotonyl-CoA via the central metabolite, succinate (Scherf et al, Arch. Microbiol, 1994, 161(3), 239 - 245; Sherf and Buckel, Eur. J. Biochem., 1993, 215, 421 - 429).

[00111] The biotin-dependent decarboxylase, glutaconyl-CoA decarboxylase, maintains the position of the substrate's vinyl group after decarboxylation, providing a route to crotonyl-CoA via the central metabolite, 2-oxoghitarate ( erstin et al., The EMBO Journal, 2003, 22(14), 3493 - 3502).

[00112] In some embodiments, the first vinyl group leading to the synthesis of butadiene is formed in 2-butanol by a desaturase or a monooxygenase such as the gene product of MdpJ or cytochrome P450 such as the gene product of the CYP4 family to produce 3-buten-2-ol. See, e.g., FIG. 8. 4.2 Enzymes generating the second terminal vinyl group in the biosynthesis of butadiene

[00113] As depicted in FIGs. 9-11, the second vinyl group can be

enzymatically formed using a mevalonate diphosphate decarboxylase (MDD), an isoprene synthase (ISPS), or a dehydratase.

[00114] In some embodiments, the second vinyl group leading to the synthesis of butadiene is formed by a mevalonate diphosphate decarboxylase (MDD), an enzyme classified under EC 4.1.1.33. See, e.g., FIG. 9.

[00115] In some embodiments, the second vinyl group leading to the synthesis of butadiene is enzymatically formed by an isoprene synthase (ISPS), an enzyme classified under 4.2.3.27. See, e.g., FIG. 10.

[00116] In some embodiments , the second vinyl group leading to the synthesis of butadiene is enzymatically formed by a dehydratase in enzyme class EC 4.2.1.-, particularly linalool dehydratase (EC 4.2.1.127), kievitone hydratase (EC 4.2.1.95), oleate hydratase (EC 4.2.1.53) or carotenoid 1 ,2-hydratase (EC 4.2.1.131). See, e.g., FIG. 1 1.

[00117] Linalool may be regarded as 3-buten-2-ol substituted with an isohexenyl R-group at the alpha position. The dehydration of linalool to myrcene is favored thermodynamically and likely proceeds via deprotonation, where the R-group has no mechanistic role (Bordkorb et ah, J. Biol. Chem., 2010, 285(40), 30436 - 30442).

[00118] Oleate hydratase converts long chain unsaturated fatty acid, oleic acid, to (R)-10-hydroxystearate. However, in screening 165 homologues to the oleate hydratase sequence of E. meningoseptica, several accepted isobutanol as substrate forming isobutene (Bianca et al, Appl. Microbiol Biotechnol., 2012, 93, 1377 - 1387).

4.3 Biochemical pathways

4.3.1 Pathways using 2-oxopent-4-enoate as central precursor to butadiene

[00119] Pathways leading to the production of 2-oxopent-4-enoate from aromatic compounds such as protocatechuate (Kasai et al, J. Bacteriol, 2009,

191(21), 6758 - 6768), catechol (He and Spain, J. Bacteriol, 1998, 180(9), 2502 - 2506), anthranilate (Muraki et al., Applied and Environmental Microbiology, 2003, 69(3), 1564 - 1572) and 3-phenylpropionic acid (Ferrandez et al, J. Bacteriol, 1997, 179(8), 2573 - 2581) are well characterized. Carbon flux from the central metabolites may be directed to these degradation pathways via 3 -dehydroshikimate by 3- dehydroshikimate dehydratase (EC 4.2.1.118), via chorismate by anthranilate synthase (EC 4.1.3.27), and via L-phenylalanine by phenylalanine ammonia lyase (EC 4.3.1.24) and 2-enoate reductase (EC 1.3.1.31).

[00120] In some embodiments, 2-oxopent-4-enoate is synthesized from the central metabolite, chorismate, by conversion to anthranilate by anthranilate synthase (EC 4.1.3.27); followed by conversion to catechol by anthranilate 1,2-dioxygenase (EC 1.14.12.1); followed by conversion to 2-hydroxymuconate semialdehyde by catechol 2,3-dioxygenase (EC 1.13.11.2); followed by conversion to 2-oxopent-4- enoate by 2-hydroxymuconate-semialdehyde hydrolase (EC 3.7.1.9). In addition, 2- hydroxymuconate semialdehyde can be converted to 2-hydroxymuconate by aminomuconate semialdehyde dehydrogenase (EC 1.2.1.32), 2-hydroxymuconate can be converted to 4-oxalocrotonate by 2-hydroxymuconate tautomerase (EC 5.3.2.6), and 4-oxalocrotonate can be converted to 2-oxopent-4-enoate 4-oxalocrotonate decarboxylase (EC 4.1.1.77). See, e.g., FIG. 2.

[00121] In some embodiments, 2-oxopent-4-enoate is synthesized from the central metabolite, 3 -dehydroshikimate, by conversion to protocatechuate by 3- dehydroshikimate dehydratase (EC 4.2.1.1 18); followed by conversion to catechol by protocatechuate decarboxylase (EC 4.1.1.63); followed by conversion to 2- hydroxymuconate semialdehyde by catechol 2, 3-dioxygenase (EC 1.13.11.2);

followed by conversion to 2-oxopent-4-enoate by 2-hydroxymuconate-semialdehyde hydrolase (EC 3.7.1.9) or by aminomuconate semialdehyde dehydrogenase (EC 1.2.1.32), 2-hydroxymuconate tautomerase (EC 5.3.2.6) and 4-oxalocrotonate decarboxylase (EC 4.1.1.77). See, e.g., FIG. 2.

[00122] In some embodiments, 2-oxopent-4-enoate is synthesized from the central metabolite, 3 -dehydroshikimate, by conversion to protocatechuate by 3- dehydroshikimate dehydratase (EC 4.2.1.1 18); followed by conversion to 5-carboxy- 2-hydroxymuconate-6-semialdehyde by protocatechuate 2,3-dioxygenase such as the gene product oipraA; followed by conversion to 2-hydroxymuconate semialdehyde by 5-carboxy-2-hydroxymuconate-6-semialdehyde decarboxylase such as the gene product oipraH; followed by conversion to 2-oxopent-4-enoate by 2- hydroxymuconate-semialdehyde hydrolase (EC 3.7.1.9) or by aminomuconate semialdehyde dehydrogenase (EC 1.2.1.32), 2-hydroxymuconate tautomerase (EC 5.3.2.6) and 4-oxalocrotonate decarboxylase (EC 4.1.1.77). See, e.g., FIG. 2.

[00123] In some embodiment, 2-oxopent-4-enoate is synthesized from the central metabolite, L-phenylalanine, by conversion to E-cinnamate by phenylalanine ammonia-lyase (EC 4.3.1.24); followed by conversion to 3 -phenyl-propionate by 2- enoate reductase (EC 1.3.1.31); followed by conversion to cis-3-(carboxy-ethyl)-3,5- cyclo-hexadiene- l,2-diol by 3-phenylpropanoate dioxygenase (EC 1.14.12.19); followed by conversion to 2,3-dihydroxyphenylpropionoate by 3-(cis-5, 6- dihydroxycyclohexa-l,3-dien-l-yl)propanoate dehydrogenase (EC 1.3.1.87); followed by conversion to 2-hydroxy-6-oxonona-2,4-diene- l,9-dioate by 3- carboxyethylcatechol 2, 3-dioxygenase (EC 1.13.11.16); followed by conversion to 2- oxopent-4-enoate by 2-hydroxy-6-oxonona-2,4-dienedioate hydrolase (EC 3.7.1.14). See, e.g., FIG. 2.

[00124] In some embodiments, butadiene is synthesized from 2-oxopent-4- enoate by conversion to 2-hydroxypent-4-enoate by (R)-2-hydroxyisocaproate dehydrogenase such as the gene product ofLdhA; followed by conversion to 2- hydroxypent-4-enoyl-CoA by CoA transferase such as the gene product of GctAB; followed by conversion to 2,4-pentadienoyl-CoA by 2-Hydroxyisocaproyl-CoA dehydratase such as the gene products of the initiator HadI and HadBC; followed by conversion to (R)-3-hydroxypent-4-enoyl-CoA by enoyl-CoA dehydratase 2 (EC 4.1.1.11 ); followed by conversion to (R)-3-hydroxypent-4-enoate by a thioesterase (EC 3.1.2.-) such as the gene product of tesB; followed by conversion to butadiene by mevalonate diphosphate decarboxylase (EC 4.1.1.33). See, e.g., FIG. 9.

[00125] (R)-2-hydroxyisocaproate dehydrogenase (gene product oiLdhA) accepts 2-oxopentanoate and 2-oxohexanoate as substrates (Kim, On the enzymatic mechanism of 2-hydroxyisocaproyl-CoA dehydratase from Clostridium difficile, 2004, Ph.D. dissertation, Philipps-Universitat, Marburg, 2004). 2-oxopentanoate is a near substrate analogue of 2-oxopent-4-enoate. [00126] Glutaconate CoA-transferase (GctAB) is a promiscuous enzyme accepting carbon chains ranging from 3 to 6 carbons in length, that are branched and unbranched, alpha-substituted and unsubstituted monocarboxylic and dicarboxylic acids (see, e.g., Buckel et al, Eur. J. Biochem., 1981, 118, 315 - 321). 2- hydroxypent-4-enoic acid has comparable structure and functional groups where CoA activation is required for the activity of 2-Hydroxyisocaproyl-CoA dehydratase.

[00127] 2-Hydroxyisocaproyl-CoA dehydratase (Hadl & HadBC) accepts the substrate analogue 2-hydroxypent-4-enoyl-CoA as substrate, synthesizing 2,4- pentadienoyl-CoA (Kim et al, Nature Letters, 2008, 452, 239 - 243).

[00128] The hydrolysis of short and medium carbon chain acyl-CoA substrates has been demonstrated using the gene product of tesB (Liu et al. , Appl. Microbiol. Biotechnol, 2007, 76, 811 - 818). The thioesterase II gene product oitesB hydrolyses ( )-3-hydroxypentanoyl-CoA efficiently (Martin and Prather, Journal of

Biotechnology, 2009, 139, 61 - 67).

4.3.2 Pathways to propanoyl-CoA as precursor leading to central precursors to butadiene

[00129] In some embodiments, propanoyl-Coenzyme A (CoA) is a precursor leading to central precursors in the synthesis of butadiene (see, e.g., FIG. 3).

[00130] In some embodiments, propanoyl-CoA is synthesized from the central metabolite, succinyl-CoA, by conversion of succinyl-CoA to (2R)-methylmalonyl- CoA by methylmalonyl-CoA mutase (EC 5.4.99.2); followed by conversion to (2S)- methylmalonyl-CoA by methylmalonyl-CoA epimerase (EC 5.1.99.1); followed by conversion to propanoyl-CoA by methylmalonyl-CoA carboxy transferase (EC 2.1.3.1) or methylmalonyl-CoA decarboxylase (EC 4.1.1.41). See e.g., FIG. 3.

[00131] In some embodiments, propanoyl-CoA is synthesized from the central metabolite, L-threonine, by conversion of L-threonine to 2-oxobutyrate by threonine ammonia lyase (EC 4.3.1.19); followed by conversion to propanoyl-CoA by 2- ketobutyrate formate-lyase such as the gene product of tdcE (EC 2.3.1.-) (see, Tseng et al, Microbial Cell Factories, 2010, 9:96). See, e.g., FIG. 3. [00132] The intracellular accumulation of propanoyl-CoA from L-threonine as a precursor to other products has been demonstrated (Tseng et al. , Microbial Cell Factories, 2010, 9:96).

[00133] In some embodiments, propanoyl-CoA is synthesized from 1,2- propanediol by conversion to propanal by propanediol dehydratase (EC 4.2.1.28); followed by conversion to propanoyl-CoA by CoA-dependent propionaldehyde dehydrogenase such as the gene product of pduP (see Luo et al., Bioresource Technology, 2012, 103, 1- 6) See, e.g., FIG. 3.

[00134] The intracellular accumulation of propanoyl-CoA from 1,2 propanediol has been reported (Luo et al, Bioresource Technology, 2012, 103, 1- 6).

[00135] In some embodiments, propanoyl-CoA is synthesized from the carbon source, levulinic acid, by conversion of levulinic acid to levulinyl-CoA by acyl-CoA synthetase or ligase (EC 6.2.1.-); followed by conversion to propanoyl-CoA by a transferase in EC 2.3.1.- (Jaremko and Yu, Journal of Biotechnology, 2011, 155, 2011, 293 - 298). See, e.g., FIG. 3.

[00136] In some embodiments, propanoyl-CoA is synthesized from the central metabolite, pyruvate, by conversion of pyruvate to L-lactate by L-lactate

dehydrogenase (EC 1.1.1.27); followed by conversion to lactoyl-CoA by proprionate CoA-transferase (EC 2.8.3.1); followed by conversion to propenoyl-CoA by lactoyl- CoA dehydratase (EC 4.2.1.54); followed by conversion to propanoyl-CoA by butyryl-CoA dehydrogenase (EC 1.3.8.1) or medium-chain acyl-CoA dehydrogenase (EC 1.3.8.7). See, e.g., FIG. 3.

[00137] In some embodiments, propanoyl-CoA is synthesized from the central metabolite, malonyl-CoA, by conversion of malonyl-CoA to malonate semialdehyde by malonyl-CoA reductase (EC 1.2.1.75); followed by conversion to 3- hydroxypropionate by 3-hydroxypropionate dehydrogenase (EC 1.1.1.59); followed by conversion to 3-hydroxypropionyl-CoA by 3-hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4); followed by conversion to propenoyl-CoA by 3-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116); followed by conversion to propanoyl-CoA by butyryl- CoA dehydrogenase (EC 1.3.8.1) or medium-chain acyl-CoA dehydrogenase (EC 1.3.8.7). See, e.g., FIG. 3. 4.3.3 Pathways using propenoyl-CoA as central precursor to butadiene

[00138] In some embodiments, propenoyl-CoA is synthesized from propanoyl- CoA by butyryl-CoA dehydrogenase (EC 1.3.8.1) or medium-chain acyl-CoA dehydrogenase (EC 1.3.8.7). See, e.g., FIG. 3.

[0013 ] In some embodiments, propenoyl-CoA is synthesized from the central metabolite, pyruvate, by conversion of pyruvate to L-lactate by L-lactate

dehydrogenase (EC 1.1.1.27); followed by conversion to lactoyl-CoA by proprionate CoA-transferase (EC 2.8.3.1); followed by conversion to propenoyl-CoA by lactoyl- CoA dehydratase (EC 4.2.1.54). See, e.g., FIG. 3.

[00140] In some embodiments, propenoyl-CoA is synthesized from the central metabolite, malonyl-CoA, by conversion to malonate semialdehyde by malonyl-CoA reductase (EC 1.2.1.75); followed by conversion to 3-hydroxypropionate by 3- hydroxy propionate dehydrogenase (EC 1.1.1.59); followed by conversion to 3- hydroxypropionyl-CoA by 3-hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4); followed by conversion to propenoyl-CoA by 3-hydroxypropiony -CoA dehydratase (EC 4.2.1.116). See, e.g., FIG. 3.

[00141] In some embodiments, butadiene is synthesized from propenoyl-CoA by conversion to 3-oxopent-4-enoyl-CoA by β-ketothiolase such as EC 2.3.1.16; followed by conversion to (R)-3-hydroxypent-4-enoyl-CoA by acetoacetyl-CoA reductase (EC 1.1.1.36) such as the gene product oiphaB; followed by conversion to (R)-3-hydroxypent-4-enoate by a thioesterase (EC. 3.1.2.-) such as the gene product of tesB; followed by conversion to butadiene by mevalonate diphosphate decarboxylase (EC 4.1.1.33). See, e.g., FIG. 9.

4.3.4 Pathway using (R) 3-hydroxypent-4-enoate as central precursor to butadiene

[00142] In some embodiments, (R) 3-hydroxypent-4-enoate is synthesized from propanoyl-CoA by conversion to 3-oxopentanoyl-CoA by acetyl-CoA C- acyltransferase (EC 2.3.1.16); followed by conversion to (R) 3-hydroxypentanoyl- CoA by acetoacetyl-CoA reductase (EC 1.1.1.36) such as the gene product of phaB; followed by conversion to (R) 3-hydroxypent-4-enoyl-CoA by a thioesterase such as the gene product oitesB (EC 3.1.2.-); followed by conversion to (R) 3-hydroxypent- 4-enoate by a desaturase such as the gene product of MdpJ or cytochrome P450 such as the gene product of the CYP4 family. See, e.g., FIG. 4.

[00143] Terminal desaturation of carboxylic acids by cytochrome P450 enzymes in the CYP4 family has been elucidated. CYP4B 1 desaturates the twelve carbon chain length fatty acid lauric acid by removing the ω- 1 hydrogen at the terminal (Guan et al, Chemico-Biology Interactions , 1998, 110, 103 - 121).

[00144] In some embodiments, butadiene is synthesized from (R) 3- hydroxypent-4-enoate by mevalonate diphosphate decarboxylase (EC 4.1.1.33). See, e.g., FIG. 9.

4.3.5 Pathway using 2,4-pentadienoyl-[acp] as central precursor to butadiene

[00145] In some embodiments, (R) 3-hydroxypent-4-enoyl-[acp] is synthesized from propanoyl-CoA by conversion of propanoyl-CoA to 3-oxopentanoyl-CoA by acetyl-CoA C-acyltransferase (EC 2.3.1.16); followed by conversion to (R) 3- hydroxypentanoyl-CoA by 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.36) such as the gene product of phaB; followed by conversion to pent-2-enoyl-CoA by enoyl-CoA hydratase (EC 4.2.1.119) such as the gene product oiphaJ; followed by conversion to pent-2-enoyl-[acp] in reaction with the gene product of an acyl transferase such as tcsA; followed by conversion to (R) 2,4-pentadienoyl-[acp] by an acyl-facpj dehydrogenase such as the gene product of TcsD. See, e.g., FIG. 5.

[00146] In some embodiments, (R) 3-hydroxypent-4-enoyl-[acp] is synthesized from propanoyl-CoA by conversion of propanoyl-CoA to 3-oxopentanoyl-[acp] by a Beta-ketoacyl-facpJ synthase I (EC 2.3.1.41) such as tcsB and an acyl-transferase such as tcsA; followed by conversion to (R) 3-hydroxypentanoyl-CoA by 3-oxoacyl- [acyl-carrier-protein] reductase (EC 1.1.1.100); followed by conversion to pent-2- enoyl-[acp] by 3-Hydroxyacyl-facpJ dehydratase (EC 4.2.1.59); followed by conversion to 2,4-pentadienoyl-[acp] by acyl-facpj dehydrogenase such as the gene product of TcsD. See, e.g., FIG. 5.

[00147] In some embodiments, butadiene is synthesized from (R)-3- hydroxypent-4-enoyl-[acp] by conversion to (R)-3-hydroxypent-4-enoyl-CoA by (R)- 3-hydroxyacyl-facpj ':CoA transacylase such as the gene product oiphaG; followed by conversion to (R)-3-hydroxypent-4-enoate by a thioesterase such as the gene product of tesB; followed by conversion to butadiene by mevalonate diphosphate decarboxylase (EC 4.1.1.33). See, e.g., FIG. 9.

[00148] The gene product of phaJ (EC 4.2.1.119) is a key enzyme for providing short and medium chain R-specific 3-hydroxyacyl-CoA monomers from fatty acid synthesis to polyhydroxyalkanoate synthase enzymes (Chung and Rhee, Biosci. Biotechnol. Biochem., 2012, 76(3), 613 - 616; Tsuge et al., International Journal of Biological Macromolecules, 2003, 31, 195 - 205).

[00149] Utilizing 4-pentenoic acid as a carbon source to polyhydroxyalkanoate- producing bacteria produces (R)-3-hydroxypent-4-enoate via beta-oxidation.

Accordingly, 4-pentenoic acid is converted to 2,4-pentadienoyl-CoA which is made available to polymer synthase enzymes after hydration to (R)-3-hydroxypent-4-enoate by R-specific enoyl-CoA dehydrase activity (Ulmer et al, Macromolecules, 1994, 27, 1675 - 1679).

4.3.6 Pathway using 2,4 pentadienoyl-CoA as central precursor to butadiene

[00150] In some embodiments, 2,4-pentadienoyl-CoA is synthesized from propanoyl-CoA by conversion of propanoyl-CoA to 3-oxo-pentanoyl-CoA by an acetyl-CoA C-acyltransferase (EC 2.3.1.16) such as the gene product of bktB; followed by conversion to (R) 3-hydroxypentanoyl-CoA by a 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.36) such as the gene product of phaB; followed by conversion to pent-2-enoyl-CoA by an enoyl-CoA hydratase (EC 4.2.1.119) such as the gene product of phaJ; followed by conversion to pent-3-enoyl-CoA by an isomerase (EC 5.3.3.8); followed by conversion to 2,4,-pentadienoyl-CoA by a 2,4- dienoyl coenzyme A reductase (EC 1.3.1.34). See, e.g., FIG. 6.

[00151] In some embodiments, 2,4-pentadienoyl-CoA is synthesized from propanoyl-CoA by conversion of propanoyl-CoA to 3-oxo-pentanoyl-CoA by an acetyl-CoA C-acyltransferase (EC 2.3.1.16) such as the gene product of bktB;

followed by conversion to (R) 3-hydroxypentanoyl-CoA by a 3 -hydrox acyl-CoA dehydrogenase (EC 1.1.1.36) such as the gene product of phaB; followed by conversion to pent-2-enoyl-CoA by an enoyl-CoA hydratase (EC 4.2.1.119) such as the gene product oiphaJ; followed by conversion to pent-3-enoyl-CoA by an isomerase (EC 5.3.3.8); followed by conversion to 2,4,-pentadienoyl-CoA by a 2,4- dienoyl coenzyme A reductase (EC 1.3.1.34). See, e.g., FIG. 6.

[00152] In some embodiments, 2,4-pentadienoyl-CoA is synthesized from propanoyl-CoA by conversion of propanoyl-CoA to 3-oxo-pentanoyl-CoA by an acetyl-CoA C-acyltransferase (EC 2.3.1.16) such as the gene product oi bktB;

followed by conversion to (R) 3-hydroxypentanoyl-CoA by a 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.36) such as the gene product oiphaB; followed by conversion to 2E-pentenoyl-CoA by an enoyl-CoA hydratase (EC 4.2.1.119) such as the gene product oiphaJ; followed by conversion to pentanoyl-CoA by a trans-2- enoyl-CoA reductase such as EC 1.3.1.38; followed by conversion to 5- hydroxypentanoyl-CoA by a cytochrome P450 monooxygenase such as the gene product of CYP 153A6; followed by conversion to 2,4,-pentadienoyl-CoA by a 5- hydroxyvaleryl-CoA dehydratase (EC 4.2.1.-) (e.g., from Clostridium viride). See, e.g., FIG. 6.

[00153] In some embodiments, 2,4-pentadienoyl-CoA is synthesized from the central metabolite, L-glutamic acid, by conversion of L-glutamic acid to L-glutamyl- 5-phosphate by a glutamate 5-kinase (EC 2.7.2.1 1); followed by conversion to L- glutamate-5-semialdehyde by a glutamate-5-semialdehyde dehydrogenase (EC 1.2.1.41); followed by spontaneous conversion to (S)-l-pyrroline-5-carboxylate; followed by conversion to L-proline by a pyrroline-5-carboxylate reductase (EC 1.5.1.2); followed by conversion to D-proline by aproline racemase (EC 5.1.1.4); followed by conversion to 5-aminovalerate by a Ό-proline reductase (EC 1.21.4.1); followed by conversion to 5-oxopentanoate by a 5-aminovalerate transaminase (EC 2.6.1.48); followed by conversion to 5-hydroxypentanoate by a 5-hydroxyvalerate dehydrogenase such as the gene product of cpnD or a dehydrogenase from

Clostridium viride; followed by conversion to 5-hydroxypentanoyl-CoA by a 5- hydroxypentanoate CoA-transferase (EC 2.8.3.14); followed by conversion to 2,4- pentadienoyl-CoA by a 5-hydroxyvaleryl-CoA dehydratase (EC 4.2.1.-) (e.g., from Clostridium viride). See, e.g., FIG. 6.

[00154] In some embodiments, butadiene is synthesized from 2,4-pentadienoyl- CoA by conversion of 2,4-pentadienoyl-CoA to (R)-3-hydroxypent-4-enoyl-CoA by an enoyl-CoA dehydratase 2 (EC 4.2.1.1 19) such as the gene product oiphaJ; followed by conversion to (R)-3-hydroxypent-4-enoate by a thioesterase such as the gene product oi tesB; followed by conversion to butadiene by a mevalonate diphosphate decarboxylase (EC 4.1.1.33). See, e.g., FIG. 9. .

[00155] The gene product iphaJ(EC 4.2.1.119) is a key enzyme for providing short and medium chain R-specific 3-hydroxyacyl-CoA monomers from fatty acid synthesis to polyhydroxyalkanoate synthase enzymes (Chung and Rhee, Biosci. Biotechnol. Biochem., 2012, 76(3), 613 - 616; Tsuge et al, International Journal of Biological Macromolecules, 2003, 31, 195 - 205).

[00156] Utilizing 4-pentenoic acid as a carbon source to polyhydroxyalkanoate- producing bacteria produces (R)-3-hydroxypent-4-enoate via beta-oxidation.

Accordingly, 4-pentenoic acid is converted to 2,4-pentadienoyl-CoA, which is made available to polymer synthase enzymes after hydration to (R)-3-hydroxypent-4-enoate by R-specific enoyl-CoA dehydrase activity (see, e.g., Ulmer et ah, Macromolecules, 1994, 27, 1675 - 1679).

4.3.7 Pathway using crotonyl-CoA as central precursor to butadiene

[00157] In some embodiments, crotonyl-CoA is synthesized from the central metabolite, acetyl-CoA, by conversion of acetyl-CoA to acetoacetyl-CoA by an acetyl-CoA C-acetyltransferase (EC 2.3.1.9) such as the gene product of atoB or phaA; followed by conversion to (R) 3-hydroxybutanoyl-CoA by a 3-hydroxybutyry - CoA dehydrogenase (EC 1.1.1.36) such as the gene product oiphaB; followed by conversion to crotonyl-CoA by an enoyl-CoA hydratase (EC 4.2.1.1 19) such as the gene product oiphaJ. See, e.g., FIG. 7.

[00158] In some embodiments, crotonyl-CoA is synthesized from the central metabolite, succinyl-CoA, by conversion of succinyl-CoA to succinate semialdehyde by a succinate-semialdehyde dehydrogenase (EC 1.2.1.76); followed by conversion to 4-hydroxybutyrate by a 4-hydroxybutyrate dehydrogenase (EC 1.1.1.61); followed by conversion to 4-hydroxybutyryl-CoA by a CoA-transferase such as the gene product of Ck-cat2; followed by conversion to crotonyl-CoA by a 4-hydroxybutanoyl-CoA dehydratase (EC 4.2.1.120) and a vinylacetyl-CoA isomerase (EC 5.3.3.3). See, e.g., FIG. 7.

[00159] In some embodiments, crotonyl-CoA is synthesized from the central metabolite, 2-oxo-glutarate, by conversion of 2-oxo-glutarate to 2-hydroxyglutarate by a 2-hydroxyglutarate dehydrogenase (EC 1.1.99.2); followed by conversion to 2- hydroxyglutaryl-CoA by a glutaconate CoA-transferase (EC 2.8.3.12); followed by conversion to glutaconyl-CoA by a dehydrase (EC 4.2.1.-); followed by conversion to crotonyl-CoA by a glutaconyl-CoA decarboxylase (EC 4.1.1.70). See, e.g., FIG. 7.

[00160] In some embodiments, butadiene is synthesized from crotonyl-CoA by conversion to crotonic acid by a succinate-CoA ligase (EC 6.2.1.5); followed by conversion to 2-buten-al by a long-chain-aldehyde dehydrogenase (EC 1.2.1.48); followed by conversion to 2-buten-l-ol by an allyl-alcohol dehydrogenase (EC 1.1.1.54); followed by conversion to 2-buten-l-ol phosphate by a mevalonate kinase (EC 2.7.1.36); followed by conversion to 2-buten-l-ol diphosphate by a

phosphomevalonate kinase (EC 2.7.4.2); followed by conversion to butadiene by an isoprene synthase (EC 4.2.3.27). See, e.g., FIG. 10.

[00161] In some embodiments, butadiene is synthesized from crotonyl-CoA by conversion to crotonic acid by a succinate-CoA ligase (EC 6.2.1.5); followed by conversion to 2-buten-al by a long-chain-aldehyde dehydrogenase (EC 1.2.1.48); followed by conversion to 2-buten-l-ol by an allyl-alcohol dehydrogenase (EC 1.1.1.54); followed by conversion to 2-buten-l-ol diphosphate by a

diphosphotransferases such as a thiamine diphosphokinase (EC 2.7.6.2); followed by conversion to butadiene by an isoprene synthase (EC 4.2.3.27). See, e.g., FIG. 10.

[00162] In some embodiments, butadiene is synthesized from crotonyl-CoA by conversionof crotonyl-CoA to crotonic acid by a succinate-CoA ligase (EC 6.2.1.5); followed by conversion to 2-buten-al by a long-chain-aldehyde dehydrogenase (EC 1.2.1.48); followed by conversion to 2-buten-l-ol by an allyl-alcohol dehydrogenase (EC 1.1.1.54); followed by conversion to butadiene by a dehydratase in enzyme class EC 4.2.1.-, such as linalool dehydratase (EC 4.2.1.127), kievitone hydratase (EC 4.2.1.95), oleate hydratase (EC 4.2.1.53) or carotenoid 1,2-hydratase (EC 4.2.1.131). See, e.g., FIG. 1 1.

4.3.8 Pathway using 3-buten-2-ol as central precursor to butadiene

[00163] In some embodiments, 3-buten-2-ol is synthesized from the central metabolite, pyruvate, by conversion of pyruvate to 2-acetolactate by an acetolactate synthase (EC 2.2.1.6); followed by conversion to (R)-acetoin by an acetolactate decarboxylase (EC 4.1.1.5); followed by conversion to 2,3 butanediol by a (R,R)- butanediol dehydrogenase (EC 1.1.1.4); followed by conversion to butanone by a propanediol dehydratase (EC 4.2.1.28); followed by conversion to 2-butanol by a (R)- specific secondary alcohol dehydrogenase (EC 1.1.1.B4); followed by conversion to 3-buten-2-ol by a desaturase or a monooxygenase such as the gene product ofMdpJ or cytochrome P450 in, for example, the CYP4 family. See, e.g., FIG. 8.

[00164] In some embodiments, butadiene is synthesized from 3-buten-2-ol by conversion to 3-buten-2-ol phosphate by a mevalonate kinase (EC 2.7.1.36); followed by conversion to 3-buten-2-ol diphosphate by a phosphomevalonate kinase (EC 2.7.4.2); followed by conversion to butadiene by an isoprene synthase (EC 4.2.3.27). See, e.g., FIG. 10.

[00165] In some embodiments, butadiene is synthesized from 3-buten-2-ol by conversion to 3-buten-2-ol diphosphate by a diphosphotransferases such as a thiamine diphosphokinase (EC 2.7.6.2); followed by conversion to butadiene by an isoprene synthase (EC 4.2.3.27). See, e.g., FIG. 10.

[00166] In some embodiments, butadiene is synthesized from 3-buten-2-ol by a dehydratase in enzyme class EC 4.2.1.-, such as a linalool dehydratase (EC

4.2.1.127), a kievitone hydratase (EC 4.2.1.95), an oleate hydratase (EC 4.2.1.53) or a carotenoid 1,2-hydratase (EC 4.2.1.131),. See, e.g., FIG. 1 1.

4.4 Cultivation strategy

[00167] In some embodiments, butadiene is biosynthesized in a recombinant host using a fermentation strategy that can include anaerobic, micro-aerobic or aerobic cultivation of the recombinant host.

[00168] Pathways in the synthesis of butadiene that incorporate enzymes requiring molecular oxygen and enzymes characterized in vitro as being oxygen sensitive require a micro-aerobic cultivation strategy maintaining a low dissolved oxygen concentration, whilst maintaining sufficient oxygen transfer to prevent substrate oxidation controlled conditions (Chayabatra & Lu-Kwang, Appl. Environ. Microbiol, 2000, 66(2), 493 0 498). [00169] In some embodiments, a cell retention strategy using, for example, ceramic hollow fiber membranes is employed to achieve and maintain a high cell density during either fed-batch or continuous fermentation in the synthesis of butadiene.

[00170] In some embodiments, the principal carbon source fed to the fermentation in the synthesis of butadiene derives from biological or non-biological feedstocks.

[00171] In some embodiments, the biological feedstock is, includes, or derives from, monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin such as levulinic acid and furfural, lignin, triglycerides such as glycerol and fatty acids, agricultural waste or municipal waste.

[00172] The efficient catabolism of crude glycerol stemming from the production of biodiesel has been demonstrated in several microorganisms such as Escherichia coli, Cupriavidus necator, Pseudomonas oleavorans, Pseudomonas putida and Yarrowia lipolytica (Lee et αΙ., ΑρρΙ. Biochem. Biotechnol, 2012, 166, 1801 - 1813; Yang et al, Biotechnology for Biofuels, 2012, 5: 13; Meijnen et al, Appl. Microbiol. Biotechnol, 2011, 90, 885 - 893).

[00173] The efficient catabolism of lignocellulosic-derived levulinic acid has been demonstrated in several organisms such as Cupriavidus necator and

Pseudomonas putida in the synthesis of 3-hydroxyvalerate via the precursor propanoyl-CoA (Jaremko and Yu, Journal of Biotechnology, 201 1, 155, 2011, 293 - 298; Martin and Prather, Journal of Biotechnology, 2009, 139, 61 - 67).

[00174] The efficient catabolism of lignin-derived aromatic compounds such benzoate analogues has been demonstrated in several microorganisms such as Pseudomonas putida, Cupriavidus necator (Bugg et al. , Current Opinion in Biotechnology, 2011, 22, 394 - 400; Perez-Pantoja et al, FEMS Microbiol Rev., 2008, 32, 736 - 794).

[00175] The efficient utilization of agricultural waste, such as olive mill waste water has been demonstrated in several microorganisms, including Yarrowia lipolytica (Papanikolaou et al, Bioresour. Technol, 2008, 99(7), 2419 - 2428).

[00176] The efficient utilization of fermentable sugars such as

monosaccharides and disaccharides derived from cellulosic, hemicellulosic, cane and beet molasses, cassava, corn and other agricultural sources has been demonstrated for several microorganism such as Escherichia coli, Corynebacterium glutamicum and Lactobacillus delbrueckii and Lactococcus lactis (see, e.g., Hermann et al, Journal of Biotechnology, 2003, 104, 155 - 172; Wee et al, Food Techno!. BiotechnoL, 2006, 44(2), 163 - 172; Ohashi et al, Journal of Bioscience and Bioengineering, 1999, 87(5), 647 - 654).

[00177] The efficient utilization of furfural, derived from a variety of agricultural lignocellulosic sources, has been demonstrated for Cupriavidus necator (Li et al, Biodegradation, 2011, 22, 1215 - 1225).

[00178] In some embodiments, the non-biological feedstock is or derives from natural gas, syngas, CO ₂ /H ₂ , methanol, ethanol, non-volatile residue (NVR) or a caustic wash waste stream from cyclohexane oxidation processes.

[00179] The efficient catabolism of methanol has been demonstrated for the methylotropic yeast Pichia pastoris.

[00180] The efficient catabolism of ethanol has been demonstrated for Clostridium kluyveri (Seedorf et al, Proc. Natl Acad. Sci. USA, 2008, 105(6) 2128 - 2133).

[00181] The efficient catabolism of CO ₂ and ¾, which may be derived from natural gas and other chemical and petrochemical sources, has been demonstrated for Cupriavidus necator (Prybylski et al, Energy, Sustainability and Society, 2012, 2: 11).

[00182] The efficient catabolism of syngas has been demonstrated for numerous microorganisms, such as Clostridium ljungdahlii and Clostridium autoethanogenum (Kopke et al, Applied and Environmental Microbiology, 2011, 77(15), 5467 - 5475).

[00183] The efficient catabolism of the non-volatile residue waste stream from cyclohexane processes has been demonstrated for numerous microorganisms, such as Delftia acidovorans and Cupriavidus necator (Ramsay et al, Applied and

Environmental Microbiology, 1986, 52(1), 152 - 156).

[00184] In some embodiments, the host microorganism is a prokaryote. For example, the prokaryote can be from the genus Escherichia such as Escherichia coli; from the genus Clostridia such as Clostridium ljungdahlii, Clostridium

autoethanogenum or Clostridium kluyveri; from the genus Corynebacteria such as Corynebacterium glutamicum; from the genus Cupriavidus such as Cupriavidus necator or Cupriavidus metallidurans; from the genus Pseudomonas such as Pseudomonas fluorescens, Pseudomonas putida or Pseudomonas oleavorans; from the genus Delftia such as Delftia acidovorans; from the genus Bacillus such as Bacillus subtillis; from the genus Lactobacillus such as Lactobacillus delbrueckii; or from the genus Lactococcus such as Lactococcus lactis. Such prokaryotes also can be a source of genes to construct recombinant host cells described herein that are capable of producing butadiene.

[00185] In some embodiments, the host microorganism is a eukaryote. For example, the eukaryote can be from the genus Aspergillus such as Aspergillus niger; from the genus Saccharomyces such as Saccharomyces cerevisiae; from the genus Pichia such as Pichia pastoris; or from the genus Yarrowia such as Yarrowia lipolytica; from the genus Issatchenkia such as Issathenkia orientalis; from the genus Debaryomyces such as Debaryomyces hansenii; from the genus Arxula such as Arxula adenoinivorans; or from the genus Kluyveromyces such as Kluyveromyces lactis. Such eukaryotes also can be a source of genes to construct recombinant host cells described herein that are capable of producing butadiene.

4.5 Metabolic engineering

[00186] The present document provides methods involving less than all the steps described for all the above pathways. Such methods can involve, for example, one, two, three, four, five, six, seven, eight, nine, ten, or more of such steps. Where less than all the steps are included in such a method, the first step can be any one of the steps listed.

[00187] Furthermore, recombinant hosts described herein can include any combination of the above enzymes such that one or more of the steps, e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more of such steps, can be performed within a recombinant host.

[00188] In addition, this document recognizes that where enzymes have been described as accepting CoA-activated substrates, analogous enzyme activities associated with [acp]-bound substrates exist that are not necessarily in the same enzyme class. [00189] Also, this document recognizes that where enzymes have been described accepting (R)-enantiomers of substrate, analogous enzyme activities associated with (S)-enantiomer substrates exist that are not necessarily in the same enzyme class.

[00190] This document also recognizes that where an enzyme is shown to accept a particular co-factor, such as NADPH, or co-substrate, such as acetyl-CoA, many enzymes are promiscuous in terms of accepting a number of different co-factors or co-substrates in catalyzing a particular enzyme activity. Also, this document recognizes that where enzymes have high specificity for e.g., a particular co-factor such as NADH, an enzyme with similar or identical activity that has high specificity for the co-factor NADPH may be in a different enzyme class.

[00191] In some embodiments, the enzymes in the pathways outlined in section 4.3 are the result of enzyme engineering via non-direct or rational enzyme design approaches with aims of improving activity, improving specificity, reducing feedback inhibition, reducing repression, improving enzyme solubility, changing stereo- specificity, or changing co-factor specificity.

[00192] In some embodiments, the enzymes in the pathways outlined in section 4.3 are gene dosed, i.e., overexpressed, into the resulting genetically modified organism via episomal or chromosomal integration approaches.

[00193] In some embodiments, genome-scale system biology techniques such as Flux Balance Analysis are utilized to devise genome scale attenuation or knockout strategies for directing carbon flux to butadiene.

[00194] Attenuation strategies include, but are not limited to; the use of transposons, homologous recombination (double cross-over approach), mutagenesis, enzyme inhibitors and RNAi interference.

[00195] In some embodiments, fluxomic, metabolomic and transcriptomal data are utilized to inform or support genome-scale system biology techniques, thereby devising genome scale attenuation or knockout strategies in directing carbon flux to butadiene.

[00196] In some embodiments requiring intracellular availability of propanoyl- CoA or propenoyl-CoA for butadiene synthesis, enzymes catalyzing the hydrolysis of propionyl-CoA and acetyl-CoA can be attenuated in the host organism. [00197] In some embodiments requiring the intracellular availability of propanoyl-CoA or propenoyl-CoA for butadiene synthesis, enzymes consuming propanoyl-CoA via the methyl-citrate cycle are attenuated in the host organism (Upton and Mckinney, Microbiology, 2007, 153, 3973 - 3982).

[00198] In some embodiments requiring the intracellular availability of propanoyl-CoA or propenoyl-CoA for butadiene synthesis, enzymes consuming propanoyl-CoA to pyruvate are attenuated in the host organism.

[00199] In some embodiments requiring the intracellular availability of propanoyl-CoA or propenoyl-CoA for butadiene synthesis, enzymes consuming propanoyl-CoA to malonyl-CoA are attenuated in the host organism.

[00200] In some embodiments requiring the intracellular availability of propanoyl-CoA or propenoyl-CoA via L-threonine as central metabolite for butadiene synthesis, a feedback-resistant threonine deaminase is genetically engineered into the host organism (Tseng et al, Microbial Cell Factories, 2010, 9:96).

[00201] In some embodiments requiring condensation of acetyl-CoA and propanoyl-CoA/propenoyl-CoA for butadiene synthesis, the β-ketothiolases catalyzing the condensation of acetyl-CoA to acetoacetyl-CoA such as the gene products iAtoB or phaA can be attenuated.

[00202] In some embodiments using hosts that naturally accumulate polyhydroxyalkanoates, the polymer synthase enzymes can be attenuated in the host strain.

[00203] In some embodiments requiring the intracellular availability of acetyl- CoA for butadiene synthesis, a host that is deficient (e.g., attenuated level of activity) in one or more enzymes in the acetate synthesis pathway can be used. For example, a host that is deficient in a phosphotransacetylase (encoded by the pta gene) can be used (Shen et al, Appl. Environ. Microbio., 201 1, 77(9), 2905 - 2915).

[00204] In some embodiments requiring the intracellular availability of acetyl- CoA for butadiene synthesis, a gene in an acetate synthesis pathway encoding an acetate kinase, such as ack, is attenuated.

[00205] In some embodiments requiring the intracellular availability of acetyl- CoA for butadiene synthesis, a gene encoding the degradation of pyruvate to lactate such as IdhA is attenuated (Shen et al, Appl. Environ. Microbio., 2011, 77(9), 2905 - 2915).

[00206] In some embodiments requiring the intracellular availability of acetyl- CoA for butadiene synthesis, a gene encoding the degradation of phophoenolpyruvate to succinate such as frdBC is attenuated (see, e.g., Shen et al., 201 1, supra).

[00207] In some embodiments requiring the intracellular availability of acetyl- CoA for butadiene synthesis, a gene encoding the degradation of acetyl-CoA to ethanol such as adhE is attenuated (Shen et al, 2011, supra).

[00208] In some embodiments requiring the intracellular availability of L- glutamate for butadiene synthesis, the enzymes catalyzing anaplerotic reactions supplementing the citric acid cycle intermediates are amplified.

[00209] In some embodiments using MDD to enzymatically form the second vinyl group into butadiene, the thioesterase // gene product oi tesB hydrolyses (R)-3- hydroxypent-4-enoyl-CoA to (R)-3-hydroxypent-4-enoate.

[00210] In some embodiments, where pathways require excess NADPH co- factor in the synthesis of butadiene, a puridine nucleotide transhydrogenase gene such as UdhA is overexpressed in the host organisms (Brigham et al, Advanced Biofuels and Bioproducts, 2012, Chapter 39, 1065 - 1090).

[00211] In some embodiments, where pathways require excess NADPH co- factor in the synthesis of butadiene, a glyceraldehyde-3P-dehydrogenase gene such as GapN is overexpressed in the host organisms (Brigham et al, 2012, supra).

[00212] In some embodiments, where pathways require excess NADPH co- factor in the synthesis of butadiene, a malic enzyme gene such as maeA or maeB is overexpressed in the host organisms (Brigham et al, 2012, supra).

[00213] In some embodiments, where pathways require excess NADPH co- factor in the synthesis of butadiene, a glucose-6-phosphate dehydrogenase gene such as zwf ^' is overexpressed in the host organisms (Lim et al, Journal of Bioscience and Bioengineering, 2002, 93(6), 543 - 549).

[00214] In some embodiments, where pathways require excess NADPH co- factor in the synthesis of butadiene, a fructose 1,6 diphosphatase gene such as fbp is overexpressed in the host organisms (Becker et al., Journal of Biotechnology, 2007, 132, 99 - 109). [00215] In some embodiments, the efflux of butadiene across the cell membrane to the extracellular media is enhanced or amplified by genetically engineering structural modifications to the cell membrane or increasing any associated transporter activity for butadiene.

[00216] In some embodiments, oxygenases degrading butadiene to toxic intermediates such as l,2-epoxy-3 jutene and l,2:3,4-diepoxybutane are attenuated in the host organism (see, e.g., Sweeney et ah, Carcinogenesis, 1997, 18(4), 611 - 625).

[00217] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1

Enzyme activity of mevalonate diphosphate decarboxylase accepting 3- hydroxypent-4-enoic acid as substrate

[00218] The his-tagged MDD genes from Saccharomyces cerevisiae,

Staphyloccocus epidermidis and Streptococcus pneumonia were cloned and expressed in E. coli in a shake flask culture containing Luria Broth media.

[00219] The pellet from each of the induced shake flask cultures was harvested by centrifugation, and then the pellet was resuspended and lysed. The cell debris was separated from the supernatant via centrifugation and filtered using a 0.2 μπι filter. The MDD enzymes were purified from the supernatant using Ni-affmity

chromatography, concentrated and buffer exchanged via ultrafiltration into 50 mM Tris buffer (pH = 7.5), 100 mM NaCl, and 5 % (v/v) glycerol using a 10 kDa polyethersulfone membrane.

[00220] Native enzyme activity was confirmed in a buffer composed of 50 mM Tris-HCl (pH = 7.5), 100 mM NaCl, 5 % (v/v) glycerol, 10 mM MgCl ₂ , 15 mM ATP and 5 mM of the native substrate mevalonate diphosphate (from Sigma Aldrich) at 30 °C. The enzyme activity assay reaction was initiated by adding \0 μΐ _^ of each purified MDD enzyme to the assay buffer containing the substrate. All three MDD enzymes accepted mevalonate diphosphate as substrate as confirmed via LC-MS. [00221] Non-native enzyme activity assays were undertaken in a buffer composed of 50 mM Tris HC1 (pH = 7.5), 100 mM NaCl, 5 % (v/v) glycerol, 10 mM MgC¾, 15 mM ATP and 4 mM of the non-native racemic substrate, 3-hydroxypent-4- enoic acid (purity >95%, from Epison Chimie) at 30 °C. Non-native activity assay were undertaken in 2 mL septum-sealed vials, thereby allowing butadiene accumulation in the headspace. The reaction was initiated by adding \0 μΐ _^ of each purified MDD enzyme variant to the assay buffer containing the substrate.

[00222] The three MDD enzymes from Saccharomyces cerevisiae,

Staphyloccocus epidermidis and Streptococcus pneumonia had similar

chromatograms and spectra for non-native enzyme activity assays using 3- hydroxypent-4-enoic acid as substrate. [23] The retention time for the butadiene standard and the assay samples were within 2 %. The ratio of the MS ion peak areas from the butadiene standard and the MS ion peak areas of the samples agree to within 20 %. Also, the ion peak areas were above the limit of quantitation for the GC/MS.

[00223] The MDD enzymes from Saccharomyces cerevisiae, Staphyloccocus epidermidis and Streptococcus pneumonia accepted 3-hydroxypent-4-enoic acid as substrate, synthesizing butadiene.

[00224]

Example 2

Amino acid residues increasing the activity of mevalonate diphosphate decarboxylase in accepting 3-hydroxypent-4-enoic acid as substrate

[00225] FIG. 13 provides the amino acid sequences for the MDD enzymes from Saccharomyces cerevisiae, Staphyloccocus epidermidis and Streptococcus pneumonia, with the conserved residues within the catalytic cleft of the enzyme in bold.

[00226] Using the total protein concentration and the purity from densitometry, the enzyme concentration for the purified MDD from S. cerevisiae was 385 μg/mL and for the purified MDD from S. pneumonia, it was 88 μg/mL.

[00227] Given the incomplete conversion of 3-hydroxypent-4-enoic acid as non-native substrate, the specific conversion of MDD from S. cerevisiae was 809 [(peak area for m z 54 ion)/^g MDD)] and MDD from S. pneumonia 's was 3200 [(peak area for m/z 54 ion) jig MDD)]. The specific conversion of MDD from S. pneumonia is thus approximately four times greater than the specific conversion of MDD from S. cerevisiae. The specific conversion of MDD from S. epidermidis lies between the specific conversions of MDD from S. pneumonia and S. cerevisiae (not calculated).

[00228] The amino acid residues in the region of the catalytic arginine residue at R158 in S. cerevisiae, R144 in S. epidermidis and R144 in S. pneumonia reveal a trend of increasing serine density within 5 amino acid residues. R158 in S. cerevisiae has 3 serine residues within 5 amino acid residues (residues 153, 155, and 159), R144 in S. epidermidis has 4 serine residues within 5 amino acid residues (residues 139, 141, 143, and 145) and R144 in S. pneumonia has 5 serine residues within 5 amino acid residues (residues 139, 141, 142, 143, and 145). See, e.g, FIG. 13.

[00229] MDD's activity in accepting 3-hydroxypent-4-enoic acid as non-native substrate increases as the serine density increases within the region of the catalytic arginine residue of the catalytic cleft. [00230]

Example 3

Enzyme activity of isoprene synthase accepting trans-2-butenylpyrophosphate as substrate

[00231] The his-tagged isoprene synthase (ISPS) gene from Populus alba was cloned and expressed in E. coli in a shake flask culture containing Luria Broth media.

[00232] The pellet from each of the induced shake flask cultures was harvested by centrifugation, and then the pellet was resuspended and lysed. The cell debris was separated from the supernatant via centrifugation and filtered through a 0.2 μιη filter. The ISPS enzyme variants were purified from the supernatant using Ni-affmity chromatography, concentrated and buffer exchanged into 50 mM Tris buffer (pH = 7.5), 100 mM NaCl and 5% (v/v) glycerol using a 10 kDa polyethersulfone membrane.

[00233] Native enzyme activity was confirmed in a buffer composed of 50 mM Tris'HCl (pH = 7.5), 100 mM NaCl, 5% (v/v) glycerol, 20 mM MgCl ₂ and 5 mM of the native substrate, dimethylallyl diphosphate from Sigma- Aldrich, at 30 °C. The native activity assay was undertaken in 2 mL septum-sealed vials, thereby allowing isoprene accumulation in the headspace. The enzyme activity assay reaction was initiated by adding 10 μΐ _^ of each purified ISPS enzyme to the assay buffer containing the substrate. ISPS from P. alba accepted dimethylallyl diphosphate as substrate as confirmed via GC-MS.

[00234] Non-native enzyme activity assays were undertaken in a buffer composed of 50 mM Tris ^» HCl (pH = 7.5), 100 mM aCl, 5 % (v/v) glycerol, 20 mM MgC¾ and 5 mM of the non-native substrate, trans-2-butenylpyrophosphate (purity > 90 %) from DALTON Pharma Services, at 30 °C. Non-native activity assay were undertaken in 2 mL septum-sealed vials, thereby allowing butadiene accumulation in the headspace. The enzyme activity assay reaction was initiated by adding 10 μΤ of the purified ISPS enzyme to the assay buffer containing the substrate.

[00235] The retention time for the butadiene standard and the assay samples are within 2 %. The ratio of the MS ion peak areas from the butadiene standard and the MS ion peak areas of the samples agree to within 20 %. Also, the ion peak areas were above the limit of quantitation for the GC/MS. [00236] The ISPS enzymes from Populus alba accepted trans-2- butenylpyrophosphate as substrate, synthesising butadiene.

[00237]

Example 4

Enzyme activity of linalool dehydratase using 3-buten-2-ol as substrate

[00238] The his-tagged linalool dehydratase (EC 4.2.1.127) from

Castellaniella defragrans was cloned into a pARZ4 vector and transformed into E. coli BL21. The resulting strain was cultivated and induced using 1 [M] IPTG (isopropyifhio- -gaiactoside) in a shake flask culture containing Luria Broth media and kanamycin selection pressure.

[00239] The cells from each of the induced shake flask cultures were harvested and pelleted by centrifugation. The cell pellet was resuspended and the cells were lysed. The cell debris was separated from the supernatant via centrifugation and filtered using a 0.2 μιη filter. The enzyme was purified from the filtered supernatant using Ni-affinity chromatography and concentrated and buffer exchanged using a Vivaspin 15R Centrifugal Concentrator and Hi-trap Desalting column into 80 mM Tris buffer (pH = 9).

[00240] Non-native enzyme activity assays were undertaken in a buffer containing 11 mM of 3-buten-2-ol at 25 °C. The activity assays were undertaken in 2 mL septum-sealed vials, thereby allowing butadiene accumulation in the headspace. The reaction was initiated by adding 1 mL of purified enzyme to the assay buffer containing the substrate.

[00241] The headspace was sampled for butadiene analysis by GC-MS (gas chromatography-mass spectrometry). The retention time for the butadiene standard and the assay samples were within 2 %. The ratio of the MS ion peak areas from the butadiene standard and the MS ion peak areas of the samples agree to within 20 %. Also, the ion peak areas were above the limit of quantitation for the GC-MS.

[00242] These findings show that linalool dehydratase (EC 4.2.1.127) accepts 3-buten-2-ol as a substrate, thereby synthesizing butadiene. OTHER EMBODIMENTS

[00243] 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.