Biosynthesis of 1,3-butanediol

Abstract

This document describes biochemical pathways for producing 1,3-butanediol using a polypetide having β-ketothiolase activity to form a 3-oxo-5-hydroxypentanoyl-CoA intermediate that can be enzymatically converted to 1,3-butanediol, as well as recombinant hosts producing 1,3-butanediol.

1 : A method of producing 3-oxo-5-hydroxypentanoyl-CoA, said method comprising enzymatically converting 3-hydroxypropionyl-CoA to 3-oxo-5-hydroxypentanoyl-CoA using a polypeptide having β-ketothiolase activity classified under EC. 2.3.1.-. 
     
     
         2 : The method of  claim 1 , wherein said polypeptide having β-ketothiolase activity is classified under EC 2.3.1.16 or EC 2.3.1.174 and/or has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1, 2, 7 or 13. 
     
     
         3 - 4 . (canceled) 
     
     
         5 : The method of  claim 1 , further comprising enzymatically converting 3-oxo-5-hydroxypentanoyl-CoA to 1,3-butanediol using a thioesterase or a CoA transferase, a decarboxylase, and a secondary alcohol dehydrogenase. 
     
     
         6 : The method of  claim 5 , wherein said thioesterase is classified under EC 3.1.2.- and/or has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3 or 14. 
     
     
         7 . (canceled) 
     
     
         8 : The method of  claim 5 , wherein said CoA transferase is classified under EC 2.8.3.-, said decarboxylase is classified under EC 4.1.1.4, and said secondary alcohol dehydrogenase is classified under EC 1.1.1.B3, EC 1.1.1.B4, or EC 1.1.1.80. 
     
     
         9 : The method of  claim 8 , wherein said CoA transferase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 or 6, said decarboxylase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8 or 10, and said secondary alcohol dehydrogenase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4, 9, or 11. 
     
     
         10 - 13 . (canceled) 
     
     
         14 : A method for biosynthesizing 1,3-butanediol, said method comprising enzymatically synthesizing 3-oxo-5-hydroxypentanoyl-CoA from 3-hydroxypropionyl-CoA using a polypeptide having β-ketothiolase activity classified under EC. 2.3.1.- and enzymatically converting 3-oxo-5-hydroxypentanoyl-CoA to 1,3-butanediol. 
     
     
         15 : The method of  claim 14 , wherein said β-ketothiolase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NOs:1, 2, 7 or 13. 
     
     
         16 : The method of  claim 14 , wherein 3-oxo-5-hydroxypentanoyl-CoA is converted to 3-oxo-5-hydroxypentanoate using a CoA transferase or a thioesterase, 3-oxo-5-hydroxypentanoate is converted to 4-hydroxybutan-2-one using a decarboxylase, and 4-hydroxybutan-2-one is converted to 1,3-butanediol using a secondary alcohol dehydrogenase. 
     
     
         17 : The method of  claim 16 , wherein said thioesterase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3 or 14, said secondary alcohol dehydrogenase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:4, 9, or 11, said decarboxylase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8 or 10 and/or said CoA transferase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 or 6. 
     
     
         18 - 20 . (canceled) 
     
     
         21 : The method of  claim 1 , wherein said 3-hydroxypropionyl-CoA is enzymatically produced from malonyl-CoA. 
     
     
         22 : The method of  claim 21 , wherein 3-hydroxypropionyl-CoA is enzymatically produced from malonyl-CoA using one or more of a malonyl-CoA reductase, a 3-hydroxypropionate dehydrogenase, a CoA transferase, and a 3-hydroxypropionyl-CoA synthase. 
     
     
         23 : The method of  claim 22 , wherein said CoA transferase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7, 12 or 13. 
     
     
         24 : The method of  claim 1 , wherein said method is performed in a recombinant host. 
     
     
         25 : The method of  claim 24 , wherein said host is subjected to a cultivation strategy under aerobic, anaerobic or, micro-aerobic cultivation conditions, said host is cultured under conditions of nutrient limitation, said host is retained using a ceramic membrane and/or a principal carbon source fed to the fermentation derives from a biological or non-biological feedstock. 
     
     
         26 - 28 . (canceled) 
     
     
         29 : The method of  claim 25 , wherein the biological feedstock is, or derives from, monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid, formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, or municipal waste or the non-biological feedstock is, or derives from, natural gas, syngas, CO 2 /H 2 , methanol, ethanol, benzoate, non-volatile residue (NVR) caustic wash waste stream from cyclohexane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams. 
     
     
         30 - 32 . (canceled) 
     
     
         33 : The method of  claim 24 , wherein said host is a prokaryote from a genus selected from the group consisting of  Escherichia; Clostridia; Corynebacteria; Cupriavidus; Pseudomonas; Delfiia; Bacillus; Lactobacillus; Lactococcus ; and  Rhodococcus  or a eukaryote from a genus selected from the group consisting of  Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula , and  Kluyveromyces.    
     
     
         34 - 38 . (canceled) 
     
     
         39 : The method of  claim 24 , wherein said host comprises an attenuation to one or more of the following enzymes: polyhydroxyalkanoate synthase, an acetyl-CoA thioesterase, a phosphotransacetylase forming acetate, an acetate kinase, a lactate dehydrogenase, a menaquinol-fumarate oxidoreductase, an alcohol dehydrogenase forming ethanol, a triose phosphate isomerase, a pyruvate decarboxylase, a glucose-6-phosphate isomerase, NAD(P)H-consuming transhydrogenase, an NAD(P)H-specific glutamate dehydrogenase, and a NADH/NADPH-utilizing glutamate dehydrogenase. 
     
     
         40 : The method of  claim 24 , wherein said host overexpresses one or more genes encoding: an acetyl-CoA synthetase, a 6-phosphogluconate dehydrogenase; a transketolase; a puridine nucleotide transhydrogenase; a glyceraldehyde-3P-dehydrogenase; a malic enzyme; a glucose-6-phosphate dehydrogenase; a glucose dehydrogenase; a fructose 1,6 diphosphatase; and a formate dehydrogenase. 
     
     
         41 : A recombinant host comprising at least one exogenous nucleic acid encoding (i) a β-ketothiolase, (ii) a secondary alcohol dehydrogenase and one or more of (iii) a decarboxylase, and (iv) a thioesterase or a CoA transferase, said host producing 1,3-butanediol. 
     
     
         42 - 53 . (canceled)