Methods for biosynthesizing 1,3 butadiene

Abstract

This document describes biochemical pathways for producing butadiene by forming two vinyl groups in a butadiene synthesis substrate. These pathways described herein rely on enzymes such as, inter alia, a decarboxylating thioesterase, cytochrome P450, or dehydratases for the final enzymatic step.

1 . A method of butadiene synthesis, the method comprising introducing a first vinyl group into a first vinyl group acceptor compound using a dehydratase, a dehydrogenase, an isomerase, a synthase or a desaturase. 
     
     
         2 . A method of butadiene synthesis, the method comprising introducing a second vinyl group into a second vinyl group acceptor compound using a decarboxylating thioesterase, a decarboxylating cytochrome P450 or a dehydratase. 
     
     
         3 . The method of  claim 2 , further comprising, prior to the introduction of the second vinyl group, introducing a first vinyl group into a first vinyl group acceptor compound to produce the second vinyl group acceptor compound, using a dehydratase, a dehydrogenase, an isomerase, a synthase or a desaturase. 
     
     
         4 . The method according to  claim 2 , wherein
 the decarboxylating thioesterase introducing the second vinyl group is an engineered enzyme having greater than 70% homology to the decarboxylating thioesterase from  Lyngbya majuscula  (CurM TE),  Pseudomonas entomophila, H. ochraceum, Synechococcus  PCC 7002 , Cyanothece  PCC 7424 or  Cyanothece  PCC 7822;   the decarboxylating cytochrome P450 introducing the second vinyl group is an engineered enzyme having greater than 70% homology to the decarboxylating cytochrome P450 from  Jeotgalicoccus  sp. ATCC 8456; or   the dehydratase introducing the second vinyl group is an engineered enzyme having greater than 70% homology to linalool dehydratase (EC 4.2.1.127) from  Castellaniella defragrans.      
     
     
         5 . The method according to  claim 2 , where the decarboxylating thioesterase introducing the second vinyl group catalyses the hydrolysis of either 3-sulphorylpent-4-enoyl-[acp], 3-phosphopent-4-enoyl-[acp], 3-sulphorylpent-4-enoyl-CoA or 3-phosphopent-4-enoyl-CoA. 
     
     
         6 . The method of  claim 5 , wherein, following the introduction of the second vinyl group, the resulting compound undergoes spontaneous decarboxylation to butadiene. 
     
     
         7 - 8 . (canceled) 
     
     
         9 . The method according to  claim 3 , wherein the dehydratase (EC 4.2.1.—) enzyme introducing the first vinyl group is an engineered enzyme having greater than 70% homology to the 5-aminovaleryl-CoA dehydratase from  C. viride , linalool dehydratase (EC 4.2.1.127) or a dehydratase (EC 4.2.1.—) from species such as  Aquincola tertiaricarbonis  or  Methylibium petroleiphilum  PM1. 
     
     
         10 . The method according to  claim 1 , wherein
 the acyl-ACP dehydrogenase introducing the first vinyl group is an engineered enzyme having greater than 70% homology to the gene product of tcsD;   the desaturase/monooxygenase introducing the first vinyl group is an engineered enzyme have greater than 70% homology to the gene product of MdpJ or cytochrome P450 CYT4 family;   the synthase introducing the first vinyl group is an engineered enzyme have greater than 70% homology to 2-methyl-3-buten-2-ol synthase encoded by Tps-MBO1; or   the isomerase introducing the first vinyl group is an engineered enzyme having greater than 70% homology to the isomerase from  Pseudomonas putida  catalyzing the conversion of 2-methyl-3-buten-2-ol to 2-methyl-3-buten-1-ol.   
     
     
         11 - 13 . (canceled) 
     
     
         14 . The method according to  claim 2 , wherein the decarboxylating thioesterase converts 3-sulphorylpent-4-enoyl-[acp] or 3-phosphopent-4-enoyl-[acp] as substrate to butadiene. 
     
     
         15 . The method according to  claim 2 , wherein the decarboxylating cytochrome P450 converts pent-4-enoic acid to butadiene. 
     
     
         16 . The method according to  claim 15 , wherein the hydrogen peroxide co-substrate required for the conversion of pent-4-enoic acid to butadiene is provided by the activity of a primary amine oxidase. 
     
     
         17 . The method according to  claim 2 , wherein the dehydratase converts 3-buten-2-ol to butadiene. 
     
     
         18 . The method according to  claim 1 , wherein the isomerase converts 2-buten-1-ol to 3-buten-2-ol. 
     
     
         19 . The method of  claim 1 , wherein the method comprises a fermentation process using a host cell expressing an enzyme that catalyzes the introduction of a first vinyl group, an enzyme that catalyzes the introduction of a second vinyl group, or one or two enzymes that catalyze the introduction of a first and a second vinyl group. 
     
     
         20 . (canceled) 
     
     
         21 . The method according to  claim 19 , wherein the host cell is a prokaryote is of the genus  Escherichia, Clostridia, Corynebacteria, Cupriavidus, Pseudomonas, Bacillus  or  Rhodococcus  or a eukaryote of the genus  Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula  or  Kluyveromyces.    
     
     
         22 . (canceled) 
     
     
         23 . The method according to  claim 19 , wherein the fermentation process comprises anaerobic, micro-aerobic or aerobic cell cultivation. 
     
     
         24 . The method according to  claim 19 , wherein cell retention strategies using, for example, ceramic hollow fibre membranes are employed to achieve and maintain a high cell density during fermentation. 
     
     
         25 . The method according to  claim 19 , wherein the principal carbon source fed to the fermentation derives from biological or non-biological feedstocks. 
     
     
         26 . The method according to  claim 25 , where the biological feedstock is, or derives from, monosaccharides, disaccharides, hemicellulose such as levulinic acid and furfural, cellulose, lignocellulose, lignin, triglycerides such as glycerol and fatty acids, agricultural waste or municipal waste. 
     
     
         27 . The method according to  claim 25 , where the non-biological feedstock is, or derives from, natural gas, syngas, CO 2 /H 2 , methanol, ethanol, non-volatile residue (NVR), caustic wash from a cyclohexane oxidation processes, or other waste stream from the chemical or petrochemical industries.