Methods for biosynthesizing 3-oxopent-4-enoyl-coa

Abstract

This document describes biochemical pathways that include the production of 3-oxopent-4-enoyl-CoA by condensation of acryloyl-CoA and acetyl-CoA using a β-ketothiolase with a SER-HIS-HIS catalytic triad. These pathways described herein rely on enzymes such as, inter alia, dehydrogenases, dehydratases and β-ketothiolases.

1 : A method of 3-oxopent-4-enoyl-CoA synthesis, the method comprising condensing acryloyl-CoA and acetyl-CoA using a protein with β-ketothiolase activity. 
     
     
         2 : The method of  claim 1 , wherein at least one subunit of the protein is a polypeptide, or is derived from a polypeptide, encoded by an archaeon gene. 
     
     
         3 : The method of  claim 2 , wherein the archaeon gene is a haloarchaeon gene. 
     
     
         4 : The method according to  claim 1 , wherein the protein comprises a SER-HIS-HIS catalytic triad associated with its catalytic mechanism. 
     
     
         5 : The method according to  claim 1 , wherein the protein is a heterodimer and wherein one or both of the two polypeptides of the heterodimer are:
 full-length gene products of the  Haloferax mediterranei  HFX_6004 (bktBα) and HFX_6003 (bktBβ) genes;   biologically active fragments of the gene products; or   biologically active variants of the full-length gene products or the biologically active fragments.   
     
     
         6 : The method according to  claim 5 , wherein one or both of the polypeptides is an engineered polypeptide having greater than 50% homology to the corresponding polypeptide chain of the BktB β-ketothiolase of  Haloferax mediterranei.    
     
     
         7 : The method according to  claim 1 , wherein the acryloyl-CoA is produced using a butyryl-CoA dehydrogenase (EC 1.3.8.1), a medium-chain acyl-CoA dehydrogenase (EC 1.3.8.7), a 2-methylacyl-CoA dehydrogenase (EC 1.3.99.12), lactoyl-CoA dehydratase (EC 4.2.1.54) or a 3-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116). 
     
     
         8 : The method according to  claim 1 , wherein the method comprises a fermentation process using a host cell expressing the protein. 
     
     
         9 : The method according to  claim 8 , wherein the host cell is a prokaryote or a eukaryote. 
     
     
         10 : The method according to  claim 9 , wherein the prokaryote is a bacterium of the genus  Escherichia, Clostridia, Corynebacteria, Cupriavidus, Pseudomonas, Bacillus  or  Rhodococcus.    
     
     
         11 : The method according to  claim 9 , wherein the eukaryote is a fungus of the genus  Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces Arxula  or  Kluyveromyces.    
     
     
         12 : The method according to  claim 9 , wherein the prokaryote is an archaeon of the genus  Methanocaldococcus, Haloferax, Metallosphera, Methanobacterium  or  Pyrococcus.    
     
     
         13 : The method according to  claim 8 , wherein the fermentation process comprises anaerobic, micro-aerobic or aerobic cell cultivation. 
     
     
         14 : The method according to  claim 8 , wherein cell retention strategies using, for example, ceramic hollow fibre membranes are employed to achieve and maintain a high cell density during fermentation. 
     
     
         15 : The method according to  claim 8 , wherein the principal carbon source fed to the fermentation derives from biological or non-biological feedstocks. 
     
     
         16 : The method according to  claim 15 , 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. 
     
     
         17 : The method according to  claim 15 , 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. 
     
     
         18 : The method according to  claim 9 , wherein the prokaryote is a bacterium selected from  Escherichia coli; Clostridium ljungdahlii, Clostridium autoethanogenum  or  Clostridium kluyveri; Corynebacterium glutamicum; Cupriavidus necator  or  Cupriavidus metallidurans; Pseudomonas fluorescens  or  Pseudomonas putida; Bacillus subtillis ; or  Rhodococcus equi.    
     
     
         19 : The method according to  claim 9 , wherein the eukaryote is a fungus selected from  Aspergillus niger; Saccharomyces cerevisiae; Pichia pastoris; Yarrowia lipolytica; Issathenkia orientalis; Debaryomyces hansenii; Arxula adenoinivorans ; or  Kluyveromyces lactis.    
     
     
         20 : The method according to  claim 9 , wherein the prokaryote is selected from  Methanocalcococcus jannaschii; Haloferax mediterranei; Metallosphera sedula; Methanobacterium thermoautotrophicum ; or  Pyrococcus abyssi.