Materials and methods for the biosynthesis of seven carbon chemicals in the presence of methanol oxidation

Abstract

This disclosure describes methods for regulating the biosynthesis of pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, 7-aminoheptanol, or 1,7-heptanediol by channeling increased flux through the biosynthesis pathway to obtain an intermediate required for growth of the host microorganism.

1 . A method for regulating biosynthesis of a product chosen from pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, 7-aminoheptanol, and 1,7-heptanediol, or salts and derivatives thereof, using a pathway having a pimeloyl-ACP intermediate, the method comprising converting methanol to formate via at least one spontaneous enzymatic reaction, wherein the formate is used in the conversion of tetrahydrofolate to N 10 -formyl-tetrahydrofolate. 
     
     
         2 . The method of  claim 1 , wherein:
 the methanol is produced during BioH enzyme activity; and/or   the method comprises the step of downregulating the activity of FolD.   
     
     
         3 . The method of  claim 2 , wherein:
 BioH removes the methyl group from pimeloyl-ACP methyl ester during conversion of pimeloyl-ACP methyl ester to pimeloyl-ACP; and/or   the step of downregulating the activity of FolD comprises a step of attenuating folD.   
     
     
         4 . (canceled) 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 3 , wherein
 the method comprises the step of cloning in a formate-tetrahydrofolate ligase (fhs);   the method comprises the step of downregulating the activity of PflB and TdcE;   the method comprises a step of cloning in an alcohol dehydrogenase (adh);   the method comprises a step of cloning in a S-(hydroxymethyl), glutathione dehydrogenase (frmA); and/or   the method comprises a step of cloning in a S-formylglutathione hydrolase (frmB).   
     
     
         7 . The method of  claim 6 , wherein:
 the formate-tetrahydrofolate ligase has at least 70%, at least 80%, or at least 90% sequence identity or homology to an amino acid sequence chosen from SEQ ID NOs: 18-22;   the alcohol dehydrogenase has at least 70%, at least 80%, or at least 90% sequence identity or homology to the amino acid sequence set forth in SEQ ID NO: 31;   the S-(hydroxymethyl) glutathione dehydrogenase has at least 70%, at least 80%, or at least 90% sequence identity or homology to an amino acid sequence chosen from SEQ ID NOs: 23-26; and/or   the S-formylglutathione hydrolase has at least 70%, at least 80%, or at least 90% sequence identity or homology to an amino acid sequence chosen from SEQ ID NOs: 27-30.   
     
     
         8 . (canceled) 
     
     
         9 . The method of  claim 6 , wherein:
 the step of downregulating the activity of PflB and TdcE comprises a step of deleting pflB and tdcE;   the alcohol dehydrogenase converts the methanol to formaldehyde;   the S-(hydroxymethyl) glutathione dehydrogenase converts S-hydroxymethylglutathione to S-formylgluathione; and/or   the S-formylglutathione hydrolase converts S-formylglutathione to formate.   
     
     
         10 - 18 . (canceled) 
     
     
         19 . The method of  claim 6 , wherein:
 the expression of adh, frmA, and frmB allows conversion of the methanol to formate; and   the biosynthesis of the product is increased.   
     
     
         20 . (canceled) 
     
     
         21 . The method of  claim 1 , wherein the product is chosen from salts and derivatives thereof of pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, 7-aminoheptanol, and 1,7-heptanediol. 
     
     
         22 . The method of  claim 1 , wherein the method is performed in a recombinant host. 
     
     
         23 . The method of  claim 22 , wherein:
 the host is subjected to a cultivation strategy under aerobic, anaerobic, micro-aerobic, or mixed oxygen/denitrification cultivation conditions;   the host is cultured under conditions of nutrient limitation;   the host is retained using a ceramic hollow fiber membrane to maintain a high cell density during fermentation; and/or   the host's tolerance to high concentrations of a C7 building block is improved through continuous cultivation in a selective environment.   
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . The method of  claim 22 , wherein the principal carbon source fed to the fermentation derives from biological or non-biological feedstocks. 
     
     
         27 . The method of  claim 26 , 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/I isophthalic acid mixture waste streams.   
     
     
         28 . (canceled) 
     
     
         29 . (canceled) 
     
     
         30 . The method of  claim 29 , wherein:
 the host is a prokaryote chosen from the following genera:  Escherichia; Corynebacteria; Cupriavidus; Pseudomonas; Delftia; Bacillus; Lactobacillus; Lactococcus  and  Rhodococcus ; or   the host is a eukaryote chosen from the following genera:  Aspergillus; Saccharomyces; Pichia; Yarrowia; Issatchenkia; Debaryomyces; Arxula ; and  Kluyveromyces.      
     
     
         31 . The method of  claim 33 , wherein the host is  Escherichia coli.    
     
     
         32 . (canceled) 
     
     
         33 . The method of  claim 30 , wherein:
 the prokaryote is chosen from  Escherichia coli, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium kluyveri, Corynebacterium glutamicum, Cupriavidus necator, Cupriavidus metallidurans, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas oleavorans, Delftia acidovorans, Bacillus subtillis, Lactobacillus delbrueckii, Lactococcus lactis , and  Rhodococcus equi ; or   the eukaryote is chosen from  Aspergillus niger, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Issathenkia orientalis, Debaryomyces hansenii, Arxula adenoinivorans , and  Kluyveromyces lactis.      
     
     
         34 . (canceled) 
     
     
         35 . The method of  claim 22 , wherein:
 the host comprises one or more of the following attenuated enzymes: polyhydroxyalkanoate synthase; an acetyl-CoA thioesterase; an acetyl-CoA specific β-ketothiolase; a phosphotransacetylase forming acetate; an acetate kinase; a lactate dehydrogenase; a menaquinol-fumarate oxidoreductase; a 2-oxoacid decarboxylase producing isobutanol; an alcohol dehydrogenase forming ethanol; a triose phosphate isomerase; a pyruvate decarboxylase; a glucose-6-phosphate isomerase; a transhydrogenase dissipating the NADH or NADPH imbalance; an glutamate dehydrogenase dissipating the NADH or NADPH imbalance; a NADH/NADPH-utilizing glutamate dehydrogenase; a pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenase accepting C7 building blocks and central precursors as substrates; a glutaryl-CoA dehydrogenase; or a pimeloyl-CoA synthetase; and/or   the host overexpresses an acetyl-CoA synthetase; a 6-phosphogluconate dehydrogenase; a transketolase; a puridine nucleotide transhydrogenase; a formate dehydrogenase; a glyceraldehyde-3P-dehydrogenase; a malic enzyme; a glucose-6-phosphate dehydrogenase; a fructose 1.6 diphosphatase; a L-alanine dehydrogenase; a L-glutamate dehydrogenase specific to the NADH or NADPH used to generate a co-factor imbalance; a methanol dehydrogenase; a formaldehyde dehydrogenase; a diamine transporter a dicarboxylate transporter an S-adenosylmethionine synthetase; and/or a multidrug transporter.   
     
     
         36 . (canceled) 
     
     
         37 . A recombinant host for producing pimeloyl-ACP, wherein:
 the host comprises at least one exogenous nucleic acid encoding (i) a formate-tetrahydrofolate ligase, (ii) a S-(hydroxymethyl) glutathione dehydrogenase, and an (iii) a S-formylglutathione hydrolase; and   the host optionally further comprises at least one exogenous nucleic acid encoding an alcohol dehydrogenase.   
     
     
         38 . The recombinant host of  claim 37 , wherein:
 the host further comprises a deletion of folD; and/or   the host further comprises a deletion of pflB and tdcE.   
     
     
         39 . (canceled) 
     
     
         40 . (canceled) 
     
     
         41 . The recombinant host of  claim 37 , said host comprising at least one exogenous nucleic acid encoding one or more of a thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a glutaconate CoA-transferase, a reversible succinyl-CoA ligase, an acetylating aldehyde dehydrogenase, or a carboxylate reductase, and said host producing pimelic acid or pimelate semialdehyde. 
     
     
         42 . The recombinant host of  claim 37 , said host further comprising:
 at least one exogenous nucleic acid encoding a ω-transaminase, wherein the host produces 7-aminoheptanoate;   one or more of a 4-hydroxybutirate dehydrogenase, a 5-hydroxypentanoate dehydrogenase or a 6-hydroxyhexanoate dehydrogenase, said host producing 7-hydroxyheptanoic acid;   at least one exogenous nucleic acid encoding a ω-transaminase, a deacetylase, an N-acetyltransferase or an alcohol dehydrogenase, said host producing heptamethylenediamine; or   at least one exogenous nucleic acid encoding a (i) carboxylate reductase enhanced by a phosphopantetheinyl transferase or (ii) an alcohol dehydrogenase, said host producing 1,7-heptanediol.   
     
     
         43 - 53 . (canceled) 
     
     
         54 . A nucleic acid construct or expression vector comprising:
 a polynucleotide encoding a polypeptide having formate-tetrahydrofolate ligase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having formate-tetrahydrofolate ligase activity is selected from: (a) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 18; (b) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 19; (c) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 20; (d) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 21; and (e) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 22;   a polypeptide having alcohol dehydrogenase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having alcohol dehydrogenase is a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 31;   a polypeptide having S-(hydroxymethyl) glutathione dehydrogenase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having S-(hydroxymethyl) glutathione dehydrogenase activity is selected from: (a) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 23; (b) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 24; (c) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 25; and (d) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 26; or   a polypeptide having S-formylglutathione hydrolase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having S-formylglutathione hydrolase activity is selected from: (a) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 27; (b) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 28; (c) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 29; and (d) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 30.   
     
     
         55 - 58 . (canceled) 
     
     
         59 . A bio-derived, bio-based, or fermentation-derived product, wherein said product comprises:
 (i) a composition comprising at least one bio-derived, bio-based, or fermentation-derived compound produced or biosynthesized according to the methods of  claim 1 , or any combination thereof,   (ii) a bio-derived, bio-based, or fermentation-derived polymer comprising the bio-derived, bio-based, or fermentation-derived composition or compound of (i), or any combination thereof,   (iii) a bio-derived, bio-based, or fermentation-derived resin comprising the bio-derived, bio-based, or fermentation-derived compound or bio-derived, bio-based, or fermentation-derived composition of (i) or any combination thereof or the bio-derived, bio-based, or fermentation-derived polymer of (ii) or any combination thereof,   (iv) a molded substance obtained by molding the bio-derived, bio-based, or fermentation-derived polymer of (ii) or the bio-derived, bio-based, or fermentation-derived resin of (iii), or any combination thereof,   (v) a bio-derived, bio-based, or fermentation-derived formulation comprising the bio-derived, bio-based, or fermentation-derived composition of (i), bio-derived, bio-based, or fermentation-derived compound of (i), bio-derived, bio-based, or fermentation-derived polymer of (ii), bio-derived, bio-based, or fermentation-derived resin of (iii), or bio-derived, bio-based, or fermentation-derived molded substance of (iv), or any combination thereof, or   (vi) a bio-derived, bio-based, or fermentation-derived semi-solid or a non-semi-solid stream, comprising the bio-derived, bio-based, or fermentation-derived composition of (i), bio-derived, bio-based, or fermentation-derived compound of (i), bio-derived, bio-based, or fermentation-derived polymer of (ii), bio-derived, bio-based, or fermentation-derived resin of (iii), bio-derived, bio-based, or fermentation-derived formulation of (v), or bio-derived, bio-based, or fermentation-derived molded substance of (iv), or any combination thereof.