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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
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  • C12Y—ENZYMES
  • C12Y401/00—Carbon-carbon lyases (4.1)
  • C12Y401/01—Carboxy-lyases (4.1.1)
  • C12Y401/01033—Diphosphomevalonate decarboxylase (4.1.1.33), i.e. mevalonate-pyrophosphate decarboxylase
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  • C12Y—ENZYMES
  • C12Y402/00—Carbon-oxygen lyases (4.2)
  • C12Y402/01—Hydro-lyases (4.2.1)
  • C12Y402/01127—Linalool dehydratase (4.2.1.127)
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y402/00—Carbon-oxygen lyases (4.2)
  • C12Y402/03—Carbon-oxygen lyases (4.2) acting on phosphates (4.2.3)
  • C12Y402/03027—Isoprene synthase (4.2.3.27)
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel

Definitions

• 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.
• isolated enzymes such as dehydrogenases, monooxygenases, desaturases, dehydratases, and decarboxylases
• 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).
• SBR styrene-butadiene-rubber
• PB polybutadiene
• SBL styrene-butadiene latex
• ABS acrylonitrile-butadiene-styrene resins
• nitrile rubber nitrile rubber
• adiponitrile adiponitrile
• 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).
• 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).
• Biocatalysis is the use of biological catalysts, such as enzymes, to perform biochemical transformations of organic compounds.
• 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).
• 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).
• MDD mevalonate diphosphate decarboxylase
• 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.
• ISPS isoprene synthase
• Isoprene synthase (EC 4.2.3.27) may thus be earmarked as a candidate enzyme in the synthesis of butadiene from non-native substrates.
• 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).
• microorganisms can generate vinyl groups in metabolites typically via dehydratase, ammonia lyase, desaturase, or decarboxylase activity.
• 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.
• 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.
• this document provides enzymes that can convert butadiene synthesis substrates into butadiene.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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).
• MDD mevalonate diphosphate decarboxylase
• FIG. 9 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).
• 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).
• ISPS isoprene synthase
• activated butenols 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).
• 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).
• 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).
• dehydratases accept hydroxylated substrates such as butenols.
• 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).
• 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).
• This document also provides methods of producing butadiene from a unsaturated hydroxylated four carbon molecule, comprising at least one biocatalytic step.
• the butenol can be activated to the corresponding butenol diphosphoester before conversion to butadiene (see, e.g., FIG. 1, reactions II & III).
• 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.
• 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.
• 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.
• 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.
• 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 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.
• This document also provides an enzyme from the enzyme class 4.2.1.-. which converts butanediols to butenol (FIG. 1 , reaction VIII).
• 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).
• 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.
• the butenol can be generated in situ as the enolate of the corresponding ketone or aldehyde such as 1- butanal or 2-butanone.
• 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.
• 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.
• 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.
• 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.
• 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
• 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.
• 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.
• 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.
• 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.-.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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,
• the 3-buten-2-ol can be produced by forming a first vinyl group in 2-butanol using a desaturase or a monooxygenase.
• the second vinyl group is enzymatically formed in (R) 3-hydroxypent-4- enoate by a mevalonate diphosphate decarboxylase (MDD).
• MDD mevalonate diphosphate decarboxylase
• 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.
• 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).
• ISPS isoprene synthase
• 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
• 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.
• 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.
• 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
• 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.
• the 3-oxopentanoyl-CoA can be produced by converting propanoyl-CoA using an acetyl-CoA C-acyltransferase classified under EC 2.3.1.16.
• the pent-3-enoyl-CoA can be produced by converting pent-2-enoyl-CoA using an isomerase classified under EC 5.3.3.8.
• 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 ⁇ -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.
• 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.
• 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.
• 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.
• 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.
• 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.-.
• 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 ⁇ -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.
• 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.
• 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.
• 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.
• 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.
• the biological feedstock is or derives from
• 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.
• 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 delbrueck
• 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.
• Aspergillus such as Aspergillus niger
• Saccharomyces such as Saccharomyces cerevisiae
• Pichia such as Pichia pastoris
• Yarrowia such as Yarrow
• 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 ⁇ - 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
• 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.
• 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.
• 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.
• FIG. 2 is a schematic of biochemical pathways leading to butadiene using 2- oxopent-4-enoate as a central precursor.
• FIG. 3 is a schematic of biochemical pathways leading to butadiene using propenoyl-CoA as a central precursor.
• FIG. 4 is a schematic of biochemical pathways leading to butadiene using 3- hydroxy-4-pentenoate as a central precursor.
• FIG. 5 is a schematic of biochemical pathways leading to butadiene using 2,4- pentadienoyl-[acp] as a central precursor.
• FIG. 6 is a schematic of biochemical pathways leading to butadiene using 2,4- pentadienoyl-CoA as a central precursor.
• FIG. 7 is a schematic of biochemical pathways leading to butadiene using crotonyl-CoA as a central precursor.
• FIG. 8 is a schematic of biochemical pathways leading to butadiene using 3- buten-2-ol as a central precursor.
• FIG. 9 is a schematic of biochemical pathways to synthesize butadiene using mevahnate diphosphate decarboxylase.
• FIG. 10 is a schematic of biochemical pathways to synthesize butadiene using isoprene synthase.
• FIG. 11 is a schematic of biochemical pathways to synthesize butadiene using dehydratases.
• 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.
• 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.
• 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.
• the term "central precursor” is used to denote a key metabolite in a pathway leading to the synthesis of butadiene.
• central metabolite is used herein to denote a metabolite that is produced in all microorganisms to support growth.
• host microorganisms described herein can include endogenous pathways that can be manipulated such that butadiene can be produced.
• 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.
• 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.
• 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.
• 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.
• a lysate e.g., a cell lysate
• 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.
• 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- [
• 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.
• 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.
• 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 .
• 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 .
• cytochrome P450 such as the gene product of the CYP4 family
• the gene product of the cytochrome P450 CYP4 family has displayed specificity for terminal desaturation rather than ⁇ -hydroxylation of the C5 carboxylic acid, valproic acid (Rettie et al, Biochemistry, 1995, 34, 7889 - 7895).
• 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.
• 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.
• 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.
• 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 ( réellein et al., The EMBO Journal, 2003, 22(14), 3493 - 3502).
• 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
• the second vinyl group can be any vinyl group.
• MDD mevalonate diphosphate decarboxylase
• ISPS isoprene synthase
• dehydratase a mevalonate diphosphate decarboxylase
• 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.
• MDD mevalonate diphosphate decarboxylase
• 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.
• ISPS isoprene synthase
• 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.
• 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).
• Oleate hydratase converts long chain unsaturated fatty acid, oleic acid, to (R)-10-hydroxystearate.
• isobutanol as substrate forming isobutene (Bianca et al, Appl. Microbiol Biotechnol., 2012, 93, 1377 - 1387).
• 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).
• 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).
• 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.
• 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);
• 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.
• 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)
• 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-dio
• 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 me
• (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.
• Glutaconate CoA-transferase 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.
• propanoyl-Coenzyme A is a precursor leading to central precursors in the synthesis of butadiene (see, e.g., FIG. 3).
• 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.
• 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.
• 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).
• 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.
• 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.
• propanoyl-CoA is synthesized from the central metabolite, pyruvate, by conversion of pyruvate to L-lactate by L-lactate
• 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
• 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.
• propenoyl-CoA is synthesized from the central metabolite, pyruvate, by conversion of pyruvate to L-lactate by L-lactate
• 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.
• butadiene is synthesized from propenoyl-CoA by conversion to 3-oxopent-4-enoyl-CoA by ⁇ -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.
• (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.
• CYP4B 1 desaturates the twelve carbon chain length fatty acid lauric acid by removing the ⁇ - 1 hydrogen at the terminal (Guan et al, Chemico-Biology Interactions , 1998, 110, 103 - 121).
• butadiene is synthesized from (R) 3- hydroxypent-4-enoate by mevalonate diphosphate decarboxylase (EC 4.1.1.33). See, e.g., FIG. 9.
• (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 transferase such as
• (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
• 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.
• 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).
• 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).
• 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 co
• 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;
• 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;
• 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 ⁇ -proline reductase (EC 1.21.4.1); followed by conversion to 5-oxopentanoate by a 5-aminovalerate transamin
• 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.
• 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. .
• 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).
• 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).
• 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.
• 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.
• 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.
• 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
• 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.
• 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.
• 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
• 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.
• 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.
• 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).
• butadiene is synthesized from 3-buten-2-ol by a dehydratase in enzyme class EC 4.2.1.-, such as a linalool dehydratase (EC).
• butadiene is biosynthesized in a recombinant host using a fermentation strategy that can include anaerobic, micro-aerobic or aerobic cultivation of the recombinant host.
• the principal carbon source fed to the fermentation in the synthesis of butadiene derives from biological or non-biological feedstocks.
• 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.
• 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.
• the host microorganism is a prokaryote.
• the prokaryote can be from the genus Escherichia such as Escherichia coli; from the genus Clostridia such as Clostridium ljungdahlii, Clostridium
• Such prokaryotes also can be a source of genes to construct recombinant host cells described herein that are capable of producing butadiene.
• the host microorganism is a eukaryote.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• Attenuation strategies include, but are not limited to; the use of transposons, homologous recombination (double cross-over approach), mutagenesis, enzyme inhibitors and RNAi interference.
• 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.
• a feedback-resistant threonine deaminase is genetically engineered into the host organism (Tseng et al, Microbial Cell Factories, 2010, 9:96).
• the ⁇ -ketothiolases catalyzing the condensation of acetyl-CoA to acetoacetyl-CoA such as the gene products iAtoB or phaA can be attenuated.
• the polymer synthase enzymes can be attenuated in the host strain.
• a host that is deficient e.g., attenuated level of activity
• a host that is deficient in a phosphotransacetylase encoded by the pta gene
• a host that is deficient in a phosphotransacetylase can be used (Shen et al, Appl. Environ. Microbio., 201 1, 77(9), 2905 - 2915).
• a gene in an acetate synthesis pathway encoding an acetate kinase, such as ack, is attenuated.
• 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).
• a gene encoding the degradation of phophoenolpyruvate to succinate such as frdBC is attenuated (see, e.g., Shen et al., 201 1, supra).
• the enzymes catalyzing anaplerotic reactions supplementing the citric acid cycle intermediates are amplified.
• the thioesterase // gene product oi tesB hydrolyses (R)-3- hydroxypent-4-enoyl-CoA to (R)-3-hydroxypent-4-enoate.
• 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).
• a glyceraldehyde-3P-dehydrogenase gene such as GapN is overexpressed in the host organisms (Brigham et al, 2012, supra).
• a malic enzyme gene such as maeA or maeB is overexpressed in the host organisms (Brigham et al, 2012, supra).
• 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).
• 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).
• 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.
• 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).
• Staphyloccocus epidermidis and Streptococcus pneumonia were cloned and expressed in E. coli in a shake flask culture containing Luria Broth media.
• 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 ⁇ filter.
• the MDD enzymes were purified from the supernatant using Ni-affmity
• the enzyme activity assay reaction was initiated by adding ⁇ 0 ⁇ ⁇ 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.
• 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 ⁇ ⁇ of each purified MDD enzyme variant to the assay buffer containing the substrate.
• the MDD enzymes from Saccharomyces cerevisiae, Staphyloccocus epidermidis and Streptococcus pneumonia accepted 3-hydroxypent-4-enoic acid as substrate, synthesizing butadiene.
• 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.
• the enzyme concentration for the purified MDD from S. cerevisiae was 385 ⁇ g/mL and for the purified MDD from S. pneumonia, it was 88 ⁇ g/mL.
• 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)
• 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.
• 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.
• ISPS isoprene synthase
• 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 ⁇ filter.
• 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 ⁇ ⁇ 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.
• 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 ⁇ of the purified ISPS enzyme to the assay buffer containing the substrate.
• 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.
• the ISPS enzymes from Populus alba accepted trans-2- butenylpyrophosphate as substrate, synthesising butadiene.
• 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 ⁇ filter.
• 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.
• 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.

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