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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/44—Polycarboxylic acids
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/001—Amines; Imines
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  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
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  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/10—Nitrogen as only ring hetero atom
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric

Definitions

• This invention relates to methods for biosynthesizing adipic acid, 6- aminohexanoic acid, hexamethylenediamine, caprolactam, and 1,6-hexanediol using one or more isolated enzymes such as ⁇ -ketothiolases, dehydrogenases, reductases, hydratases, monooxygenases, ⁇ -hydroxylases and transaminases or using one or more isolated enzymes such as ⁇ -ketothiolases, dehydrogenases, reductases, hydratases, monooxygenases, ⁇ -hydroxylases and transaminases or using one or more isolated enzymes such as ⁇ -ketothiolases, dehydrogenases, reductases, hydratases, monooxygenases, ⁇ -hydroxylases and transaminases or using one or more isolated enzymes such as ⁇ -ketothiolases, dehydrogenases, reductases, hydratases, monooxygenases,
• Nylons are polyamides that are generally synthesized by the condensation polymerisation of a diamine with a dicarboxylic acid. Similarly, nylons may be produced by the condensation polymerisation of lactams. A ubiquitous nylon is nylon 6,6, which is produced by reaction of hexamethylenediamine (HMD) and adipic acid. Nylon 6 is produced by a ring opening polymerisation of caprolactam. Therefore, adipic acid, hexamethylenediamine and caprolactam are important intermediates in the production of nylons (Anton & Baird, Polyamides Fibers, Encyclopedia of Polymer Science and Technology, 2001).
• adipic acid and caprolactam are produced via air oxidation of cyclohexane.
• the air oxidation of cyclohexane produces, in a series of steps, a mixture of cyclohexanone (K) and cyclohexanol (A), designated as KA oil.
• K cyclohexanone
• A cyclohexanol
• Nitric acid oxidation of KA oil produces adipic acid (Musser, Adipic acid, Ullmann's
• Caprolactam is produced from cyclohexanone via its oxime and subsequent acid rearrangement (Fuchs, Kieczka and Moran, Caprolactam, Ullmann's Encyclopedia of Industrial Chemistry, 2000)
• HMD hexamethylenediamine
• Biocatalysis is the use of biological catalysts, such as enzymes, to perform biochemical transformations of organic compounds.
• the dicarboxylic acid, adipic acid is converted efficiently as a carbon source by a number of bacteria and yeasts via ⁇ -oxidation into central metabolites, ⁇ - oxidation of adipate to 3-oxoadipate faciliates further catabolism via, for example, the ortho-cleavage pathway associated with aromatic substrate degradation.
• the catabolism of 3-oxoadipyl-CoA to acetyl-CoA and succinyl-CoA by several bacteria and fungi has been characterised comprehensively (Harwood and Parales, Annual Review of Microbiology, 1996, 50, 553 - 590). Both adipate and 6-aminohexanoate are intermediates in the catabolism of caprolactam, finally degraded via 3-oxoadipyl- CoA to central metabolites.
• the efficient synthesis of the six carbon aliphatic backbone precursor is a key consideration in synthesizing C6 building blocks prior to forming terminal functional groups, such as carboxyl, amine or hydroxyl groups, on the C6 aliphatic backbone.
• This document is based at least in part on the discovery that it is possible to construct biochemical pathways for producing a six carbon chain aliphatic backbone precursor, in which two functional groups, e.g., carboxyl, amine or hydroxyl, can be formed, leading to the synthesis of one or more of adipic acid, 6-aminohexanoic acid, hexamethylenediamine, caprolactam, and 1,6-hexanediol (hereafter "C6 building blocks”).
• C6 building blocks 1,6-hexanediol
• the C6 aliphatic backbone for conversion to a C6 building block can be formed from acetyl-CoA via two cycles of CoA-dependent carbon chain elongation using either NADH or NADPH dependent enzymes. See FIG. 1 and FIG. 2.
• the enzyme in the CoA-dependent carbon chain elongation pathway generating the C6 aliphatic backbone catalyzes irreversible enzymatic steps.
• the terminal carboxyl groups can be enzymatically formed using an acyl-CoA hydrolase, an aldehyde dehydrogenase, a 6-oxohexanoate dehydrogenase or a cytochrome P450/oi-hydroxylase. See FIG. 3 and FIG. 4.
• the terminal amine groups can be enzymatically formed using an ⁇ -transaminase or a diamine transaminase. See FIG. 5 and FIG. 6.
• the terminal hydroxyl group can be enzymatically formed using a cytochrome P450, a monooxygenase, or an alcohol dehydrogenase. See FIG. 7 and FIG. 8.
• this document features a method for biosynthesizing one or more products selected from the group consisting of adipic acid, 6-aminohexanoic acid, hexamethylenediamine, caprolactam, and 1,6-hexanediol.
• the method includes enzymatically synthesizing a six carbon chain aliphatic backbone (e.g., hexanoyl- CoA) and enzymatically forming, in one or more steps, two terminal functional groups selected from the group consisting of carboxyl, amine, and hydroxyl groups in the backbone to directly produce the product or producing the product in a subsequent step.
• the two terminal functional groups can be the same or can be different.
• Hexanoyl-CoA can be enzymatically synthesized from acetyl-CoA via two cycles of CoA-dependent carbon chain elongation using either NADH or NADPH dependent enzymes.
• Hexanoyl-CoA can be formed by conversion of hex-2-enoyl- CoA by an enoyl-CoA reductase classified under EC 1.3.1.44, EC 1.3.1.38, or EC 1.3.1.8 such as the gene product of ter or tdter.
• Hex-2-enoyl-CoA can be formed by conversion of (S) 3-hydroxyhexanoyl-CoA by a trans-2-enoyl-CoA hydratase classified under EC 4.2.1.17 or by conversion of (R) 3-hydroxyhexanoyl-CoA by a trans-2-enoyl-CoA hydratase classified under EC 4.2.1.119.
• the trans-2-enoyl-CoA hydratase can be the gene product of crt.
• (S) 3-hydroxyhexanoyl-CoA can be formed by conversion of 3-oxohexanoyl-CoA by a 3 -hydroxy acyl-CoA dehydrogenase classified under EC 1.1.1.35 such as the 3 -hydroxy acyl-CoA dehydrogenase encoded by fadB.
• the 3-oxohexanoyl-CoA can be formed by conversion of butanoyl-CoA by an acetyl-CoA C-acyltransferase classified under EC 2.3.1.16 such as that encoded by bktB.
• Butanoyl-CoA can be formed by conversion of crotonyl-CoA by an enoyl-CoA reductase classified under EC 1.3.1.44, EC 1.3.1.38, or EC 1.3.1.8.
• Crotonyl-CoA can be formed by conversion of (S) 3-hydroxybutanoyl-CoA by a trans-2-enoyl-CoA hydratase classified under EC 4.2.1.17.
• the (S) 3-hydroxybutanoyl-CoA can be formed by conversion of acetoacetyl-CoA by a 3-hydroxybutyryl-CoA dehydrogenase classified under EC 1.1.1.157 such as a 3-hydroxybutyryl-CoA dehydrogenase is encoded by hbd.
• the acetoacetyl-CoA can be formed by conversion of acetyl-CoA by an acetyl-CoA C-acyltransferase classified under EC 2.3.1.9 such as that encoded by atoB or phaA.
• the acetoacetyl-CoA can be formed by conversion of malonyl-CoA by an acetoacetyl-CoA synthase classified under EC 2.3.1.194.
• the malonyl-CoA can be formed by conversion of acetyl-CoA by an acetyl-CoA carboxylase classified under EC 6.4.1.2.
• the trans-2-enoyl-CoA hydratase can be the gene product oiphaJ.
• the (R) 3-hydroxyhexanoyl-CoA can be formed by conversion of 3- oxohexanoyl-CoA by a 3-oxoacyl-CoA reductase classified under EC 1.1.1.100 such as that encoded by fabG.
• the crotonyl-CoA can be formed by conversion of (R) 3- hydroxybutanoyl-CoA by a trans-2-enoyl-CoA hydratase classified under EC 4.2.1.1 19.
• (R) 3-hydroxybutanoyl-CoA can be formed by conversion of acetoacetyl- CoA by an acetoacyl-CoA reductase classified under EC 1.1.1.36 such as that encoded by phaB.
• the method can include producing hexanoate by forming a first terminal carboxyl group in hexanoyl CoA using an acyl- CoA hydrolase and an aldehyde dehydrogenase, or a thioesterase.
• the acyl-CoA hydrolase or thioesterase can be encoded by YciA, tesB or Acotl 3.
• Hexanoate can be produced by forming a first terminal carboxyl group in hexanal by an aldehyde dehydrogenase classified under EC 1.2.1.4. Hexanal can be formed by conversion of hexanoyl-CoA by a butanal dehydrogenase classified under EC 1.2.1.57.
• the methods can include converting hexanoate to adipic acid, 6-aminohexanoic acid, hexamethylenediamine, ⁇ caprolactam or 1,6 hexanediol with one or more enzymatic conversions, wherein one of the enzymatic conversions introduces the second terminal functional group.
• the method can include converting hexanoate to 6-hydroxyhexanoate using a cytochrome P450/ ⁇ -hydroxylase such as from the family CYP153 such as CYP 152A6.
• 6- hydroxyhexanoate can be converted to adipate semialdehyde using (i) an alcohol dehydrogenase such as encoded by YMR318C, (ii) a 6-hydroxyhexanoate dehydrogenase such as that encoded by ChnD, or (iii) a cytochrome P450/ ⁇ - hydroxylase.
• an alcohol dehydrogenase such as encoded by YMR318C
• a 6-hydroxyhexanoate dehydrogenase such as that encoded by ChnD
• a cytochrome P450/ ⁇ - hydroxylase a cytochrome P450/ ⁇ - hydroxylase
• adipic acid can be produced by forming the second terminal functional group in adipate semialdehyde using (i) an aldehyde dehydrogenase classified under EC 1.2.1.3, (ii) a 6-oxohexanoate dehydrogenase classified under EC 1.2.1.63 such as that encoded by ChnE or iii) a cytochrome P450/ ⁇ -hydroxylase.
• 6-aminohexanoic acid can be produced by forming the second terminal functional group in adipate semialdehyde using an ⁇ -transaminase classified under EC 2.61.18, EC 2.6.1.19 or EC 2.6.1.48.
• caprolactam can be produced from 6- aminohexanoic acid using a lactamase classified under EC 3.5.2.-.
• the amide bond associated with caprolactam is the result of first having a terminal carboxyl group and terminal amine group to form the bond.
• 6-aminohexanoic acid can be converted to 6- aminohexanal using a carboxylate reductase classified under EC 1.2.99.6 such as that encoded by car alongside the gene product of npt, or GriC & GriD.
• hexamethylenediamine can be produced by forming a second terminal functional group in 6-aminohexanal using a diamine transaminase classified under EC 2.6.1.29 or EC 2.6.1.82.
• 1,6 hexandiol can be produced by forming the second terminal functional group in 6-hydroxyhexanal using an alcohol dehydrogenase classified under EC 1.1.1. -(1,2,21, 184).
• 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, CO2/H2, 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 bacterial genus Escherichia such as Escherichia coli; from the bacterial genus Clostridia such as Clostridium ljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; from the bacterial genus Corynebacteria such as Corynebacterium glutamicum; from the bacterial genus Cupriavidus such as Cupriavidus necator or Cupriavidus metallidurans ; from the bacterial genus
• Pseudomonas such as Pseudomonas fluorescens, Pseudomonas putida or
• Pseudomonas oleavorans from the bacterial genus Delftia such as Delftia acidovorans; from the bacterial genus Bacillus such as Bacillus subtillis; from the bacterial genus Lactobacillus such as Lactobacillus delbrueckii; or from the bacterial genus Lactococcus such as Lactococcus lactis.
• Such prokaryotes also can be sources of genes for constructing recombinant host cells described herein that are capable of producing C6 building blocks.
• the host microorganism is a eukaryote (e.g., a fungus such as a yeast).
• the eukaryote can be from the fungal genus Aspergillus such as Aspergillus niger; from the yeast genus Saccharomyces such as
• Saccharomyces cerevisiae from the yeast genus Pichia such as Pichia pastoris; from the yeast genus Yarrowia such as Yarrowia lipolytica; from the yeast genus
• Issatchenkia such as Issathenkia orientalis
• yeast genus Debaryomyces such as Debaryomyces hansenii
• Arxula such as Arxula
• adenoinivorans or from the yeast genus Kluyveromyces such as Kluyveromyces lactis.
• Such eukaryotes also can be sources of genes for constructing recombinant host cells described herein that are capable of producing C6 building blocks.
• concentrations of one or more C6 building blocks is improved through continuous cultivation in a selective environment.
• the host microorganism's biochemical network is attenuated or augmented to (1) ensure the intracellular availability of acetyl-CoA, (2) create an NADH or NADPH imbalance that may only be balanced via the formation of one or more C6 building blocks, (3) prevent degradation of central metabolites, central precursors leading to and including C6 building blocks and (4) ensure efficient efflux from the cell.
• a non-cyclical cultivation strategy is used to achieve anaerobic, micro-aerobic, or aerobic cultivation conditions.
• a cyclical cultivation strategy is used to alternate between anaerobic and aerobic cultivation conditions.
• the cultivation strategy includes limiting nutrients, such as limiting nitrogen, phosphate or oxygen.
• one or more C6 building blocks are produced by a single type of microorganism, e.g., a recombinant host containing one or more exogenous nucleic acids, using a non-cyclical or cyclical fermentation strategy.
• one or more C6 building blocks are produced by co- culturing more than one type of microorganism, e.g., two or more different recombinant hosts, with each host containing a particular set of exogenous nucleic acids, using a non- cyclical or cyclical fermentation strategy.
• one or more C6 building blocks are produced by successive fermentations, where the broth or centrate from the preceding fermentation is fed to a succession of fermentations as a source of feedstock, central metabolite or central precursor ; finally producing the C6 building block.
• This document also features a recombinant host comprising at least one exogenous nucleic acid encoding, for example, one or more of a formate
• dehydrogenase dehydrogenase, enoyl-CoA reductase, trans-2-enoyl-CoA hydratase, 3- hydroxybutyryl-CoA dehydrogenase, acetyl-CoA C-acyltransf erase, acetoacyl-CoA reductase, acetyl-CoA synthetase, acetyl-CoA carboxylase, a malic enzyme, puridine nucleotide transhydrogenase, glyceraldehydeSP-dehydrogenase, acyl-CoA hydrolase, aldehyde dehydrogenase, thioesterase, cytochrome P450/ ⁇ -hydroxylase, alcohol dehydrogenase, 6-hydroxyhexanoate dehydrogenase, 6-oxohexanoate dehydrogenase, diamine transaminase, propionyl-CoA synthetase
• FIG. 1 is a schematic of an exemplary biochemical pathway leading to hexanoyl-CoA using NADH-dependent enzymes and with acetyl-CoA as a central metabolite.
• FIG. 2 is a schematic of an exemplary biochemical pathway leading to hexanoyl-CoA using NADPH-dependent enzymes and with acetyl-CoA as a central metabolite.
• FIG. 3 is a schematic of exemplary biochemical pathways leading to hexanoate using hexanoyl-CoA as a central metabolite.
• FIG. 4 is a schematic of exemplary biochemical pathways leading to adipic acid using hexanoate as a central precursor.
• FIG. 5 is a schematic of exemplary biochemical pathways leading to 6- aminhexanoate using hexanoate as a central precursor.
• FIG. 6 is a schematic of an exemplary biochemical pathway leading to hexamethylenediamine using 6-aminhexanoate as a central precursor.
• FIG. 7 is a schematic of an exemplary biochemical pathway leading to 6- hydroxyhexanoate using hexanoate as a central precursor.
• FIG. 8 is a schematic of an exemplary biochemical pathway leading to 1,6- hexanediol using 6-hydroxyhexanoate as a central precursor.
• this document provides enzymes, non-natural pathways, cultivation strategies, feedstocks, host microorganisms and attenuations to the host's biochemical network, which generates a six carbon chain aliphatic backbone from central metabolites into which two terminal functional groups may be formed leading to the synthesis of adipic acid, caprolactam, 6-aminohexanoic acid, hexamethylenediamine or 1,6-hexanediol (referred to as "C6 building blocks” herein).
• the term “central precursor” is used to denote a key metabolite in a pathway leading to the synthesis of C6 building blocks.
• 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 one or more C6 building blocks 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 genera, or can be from multiple sources, i.e., different species or genera.
• 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. Endogenous genes of the engineered hosts also can be disrupted to prevent the formation of undesirable metabolites or prevent the loss of intermediates in the pathway through other enzymes acting on such intermediates. Engineered hosts can be referred to as recombinant hosts or recombinant host cells.
• recombinant hosts can include nucleic acids encoding one or more of a ⁇ -ketothiolases , dehydrogenases, reductases, hydratases, monooxygenases, ⁇ -hydroxylase or transaminases as described in more detail below.
• C6 building blocks 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
• the reactions of the pathways described herein can be performed in one or more host strains (a) naturally expressing one or more relevant enzymes,(b) genetically engineered to express one or more relevant enzymes, or (c) naturally expressing one or more relevant enzymes and genetically engineered to express one or more relevant enzymes.
• relevant enzymes can be extracted from of the above types of host cells and used in a purified or semi-purified form.
• extracts include lysates (e.g. cell lysates) that can be used as sources of relevant enzymes.
• all the steps can be performed in host cells, all the steps can be performed using extracted enzymes, or some of the steps can be performed in cells and others can be performed using extracted enzymes.
• the C6 aliphatic backbone for conversion to C6 building blocks can be formed from acetyl-CoA via two cycles of CoA-dependent carbon chain elongation using either NADH or NADPH dependent enzymes.
• a CoA-dependent carbon chain elongation cycle comprises an acetyl- CoA C-acyltransferase, which converts acetyl-CoA to acetoacetyl-CoA and converts butanoyl-CoA to 3-oxohexanoyl-CoA, or an acetyl-CoA carboxylase, which converts acetyl-CoA to malonyl-CoA & an acetoacetyl-CoA synthase, which converts malonyl-CoA to acetoacetyl-CoA, a 3-hydroxybutyrl-CoA dehydrogenase, which converts acetoacetyl-CoA to 3-hydroxybutanoyl CoA or 3-oxoacyl-CoA reductase/3- hydroxyacyl-CoA dehydrogenase, which converts 3-oxohexanoyl-CoA to 3- hydroxyhexano
• an acetyl-CoA C-acyltransferase may be classified under EC 2.3.1.9, such as the gene product oiatoB or phaA, or classified under EC 2.3.1.16, such as the gene product oi bktB.
• ⁇ -ketothiolase encoded by atoB or phaA accepts acetyl-CoA as substrates, forming butanoyl-CoA (Haywood et al, FEMS Microbiology Letters, 1988, 52, 91- 96; Slater et al, Journal of Bacteriology, 1998, 180(8), 1979 - 1987).
• the ⁇ -ketothiolase encoded by bktB from Cupriavidus necator accepts acetyl- CoA and butanoyl-CoA as substrates, forming the CoA-activated C6 aliphatic backbone (Haywood et al, FEMS Microbiology Letters, 1988, 52, 91-96; Slater et al, Journal of Bacteriology, 1998, 180(8), 1979 - 1987).
• an acetyl-CoA carboxylase may be classified under EC
• an acetoacetyl-CoA synthase may be classified under EC 2.3.1.194.
• acetoacetyl-CoA synthase may be used as an irreversible substitute for the gene product of phaA in the carbon chain elongation associated with polyhydroxybutyrate synthesis (Matsumoto et al, Biosci. Biotechnol. Biochem., 2011, 75(2), 364 - 366).
• a 3-hydroxyacyl-CoA dehydrogenase may be classified under EC 1.1.1.35, such as the gene product oifadB, or a 3 -hydroxy butyryl-CoA dehydrogenase may be classified under EC 1.1.1.157, such as the gene product of hbd, or an acetoacetyl-CoA reductase may be classified under EC 1.1.1.36, such as the gene product oiphaB (Liu & Chen, Appl. Microbiol. Biotechnol, 2007, 76(5), 1153 - 1159; Shen et al, Appl. Environ. Microbiol, 2011, 77(9), 2905 - 2915; Budde et al, Journal of Bacteriology, 2010, 192(20), 5319 - 5328).
• a 3-oxoacyl-CoA reductase may be classified under EC 1.1.1.100, such as the gene product oifabG (Budde et al, Journal of Bacteriology, 2010, 192(20), 5319 - 5328; Nomura et al, Appl. Environ. Microbiol, 2005, 71(8), 4297 - 4306) .
• an enoyl-CoA hydratase may be classified under EC 4.2.1.17, such as the gene product of crt, or classed under EC 4.2.1.119, such as the gene product ofp/zaJ(Shen et ⁇ ., ⁇ . Environ. Microbiol, 2011, 77(9), 2905 - 2915; Fukui et al, Journal of Bacteriology, 1998, 180(3), 667 - 673).
• a trans-2-enoyl-CoA reductase may be classified under EC 1.3.1.38, EC 1.3.1.8 or EC 1.3.1.44, such as the gene product of fer or tdter (Nishimaki et al, J. Biochem., 1984, 95, 1315 - 1321; Shen et al, Appl. Environ. Microbiol, 2011, 77(9), 2905 - 2915); Bond-Watts et al, Biochemistry, 2012, 51, 6827 - 6837 ).
• the terminal carboxyl groups can be enzymatically formed using an acyl-CoA hydrolase, an aldehyde dehydrogenase, a 6- oxohexanoate dehydrogenase or a cytochrome P450/ co-hydroxylase.
• the first terminal carboxyl group leading to the synthesis of a C6 building block is enzymatically formed in hexanoyl-CoA by an acyl-CoA hydrolase or thioesterase classified under EC 3.1.2.-, resulting in the production of hexanoate.
• the acyl-CoA hydrolase or thioesterase can be the gene product of YciA, tesB ox AcotB (Cantu et al., Protein Science, 2010, 19, 1281 - 1295; Zhuang et al.,
• the first terminal carboxyl group leading to the synthesis of a C6 building block is enzymatically formed in hexanal by an aldehyde dehydrogenase classified under EC 1.2.1.4 (Ho & Weiner, Journal of Bacteriology, 2005, 187(3), 1067 - 1073), resulting in the production of hexanoate.
• the second terminal carboxyl group leading to the synthesis of adipic acid is enzymatically formed in adipate semilaldehyde by an aldehyde dehydrogenase classified under EC 1.2.1.3 (Guerrillot & Vandecasteele, Eur. J.
• the second terminal carboxyl group leading to the synthesis of adipic acid is enzymatically formed in adipate semialdehyde by a 6- oxohexanoate dehydrogenase classified under EC 1.2.1.63, such as the gene product of ChnE (Iwaki et al., Appl. Environ. Microbiol., 1999, 65(11), 5158 - 5162).
• the second terminal carboxyl group leading to the synthesis of adipic acid is enzymatically formed in adipate semilaldehyde by a cytochrome P450 (Sanders et al., Journal of Lipid Research, 2005, 46(5), 1001-1008; Sanders et al., The FASEB Journal, 2008, 22(6), 2064 - 2071).
• the utility of ⁇ -oxidation in introducing carboxyl groups into alkanes has been demonstrated in the yeast Candida tropicalis, leading to the synthesis of adipic acid (Okuhara et al, Agr. Boil. Chem., 1971, 35(9), 1376 - 1380).
• terminal amine groups can be enzymatically formed using a ⁇ -transaminase or a diamine transaminase.
• the first terminal amine group leading to the synthesis of 6-aminohexanoic acid is enzymatically formed in adipate semialdehyde by a ⁇ - transaminase classified under EC 2.6.1.18, such as obtained from Vibrio fluvialis or Chromobacterium violaceum, or classified under EC 2.6.1.19, such as obtained from Streptomyces griseus, or classified under 2.6.1.48, such as obtained from Clostridium viride.
• EC 2.6.1.18 such as obtained from Vibrio fluvialis or Chromobacterium violaceum
• EC 2.6.1.19 such as obtained from Streptomyces griseus
• 2.6.1.48 such as obtained from Clostridium viride.
• the reversible ⁇ -transaminase from Chromobacterium violaceum has demonstrated activity accepting 6-aminohexanoic acid as amino donor, thus forming the first terminal amine group in adipate seminaldehyde (Kaulmann et al., Enzyme and Microbial Technology, 2007, 41, 628 - 637).
• Clostridium viride has demonstrated activity for the conversion of 6-aminohexanoate to adipate semialdehyde (Barker et al., The Journal of Biological Chemistry, 1987, 262(19), 8994 - 9003).
• the second terminal amine group leading to the synthesis of hexamethylenediamine is enzymatically formed in 6-aminohexanal by a diamine transaminase classified under EC 2.6.1.29 or classified under EC 2.6.1.82, such as the gene product of YgjG.
• the gene product of ygjG accepts a broad range of diamine carbon chain length substrates, such as putrescine, cadaverine and spermidine (Samsonova et al., BMC Microbiology, 2003, 3:2)
• the diamine transaminase from E.coli strain B has demonstrated activity for 1,5 diaminopentane and 1,7 diaminoheptane, with 1,6 diaminohexane (HMD) expected to be active (Kim, The Journal of Chemistry, 1963, 239(3), 783 - 786).
• the terminal hydroxyl group can be enzymatically forming using a cytochrome P450, a monooxygenase or an alcohol dehydrogenase.
• the first terminal hydroxyl group leading to the synthesis of C6 building blocks is enzymatically formed in hexanoate by a monooxygenase, a cytochrome P450 or a ⁇ -hydroxylase such as from the CYP153 family such as
• CYP153A6 (Van Beilen & Funhoff, Current Opinion in Biotechnology, 2005, 16, 308 - 314; Koch et al., Appl. Environ. Microbiol., 2009, 75(2), 337-344; Nieder and Shapiro, Journal of Bacteriology, 1975, 122(1), 93 - 98).
• the second terminal hydroxyl group leading to the synthesis of 1 ,6 hexanediol is enzymatically formed in 6-hydroxyhexanal by an alcohol dehydrogenase classified under EC 1.1.1.- (e.g., 1, 2, 21, or 184).
• an alcohol dehydrogenase classified under EC 1.1.1.- e.g., 1, 2, 21, or 184.
• hexanoyl-CoA is synthesized from the central metabolite, acetyl-CoA, by conversion of acetyl-CoA to acetoacetyl-CoA by acetoacetyl-CoA thiolase (EC 2.3.1.9), such as the gene product oi atoB or phaA, or by acetyl-CoA carboxylase (EC 6.4.1.2) & acetoacetyl-CoA synthase (EC 2.3.1.194) via malonyl-CoA; followed by conversion of acetoacetyl-CoA to (S) 3- hydroxybutanoyl-CoA by a 3 -hydroxy butyryl-CoA dehydrogenase (EC 1.1.1.157) such as the gene product oihbd; followed by conversion of (S) 3-hydroxybutanoyl- CoA to crotonyl-CoA by enoyl-CoA hydrat
• hexanoyl-CoA is synthesized from the central metabolite, acetyl-CoA, by conversion of acetyl-CoA to acetoacetyl-CoA by acetoacetyl-CoA thiolase (EC 2.3.1.9) , such as the gene product oi atoB or phaA, or by acetyl-CoA carboxylase (EC 6.4.1.2) & acetoacetyl-CoA synthase (EC 2.3.1.194) via malonyl-CoA; followed by conversion of acetoacetyl-CoA to (R) 3- hydroxybutanoyl-CoA by a 3-acetoacetyl-CoA reductase (EC 1.1.1.36) such as the gene product oiphaB; followed by conversion of (R) 3-hydroxybutanoyl-CoA to crotonyl-CoA by enoyl-CoA hydrat
• hexanoate is synthesized from the central metabolite, hexanoyl-CoA, by conversion of hexanoyl-CoA to hexanoate by acyl-CoA hydrolase or thioesterase (EC 3.1.2.-) such as the gene product of YciA, tesB or Acotl3.
• hexanoate is synthesized from the central metabolite, hexanoyl-CoA, by conversion of hexanoyl-CoA to hexanal by butanal dehydrogenase (EC 1.2.1.57); followed by conversion of hexanal to hexanoate by aldehyde dehydrogenase (EC 1.2.1.4). See FIG. 3.
• adipic acid is synthesized from the central precursor, hexanoate, by conversion of hexanoate to 6-hydroxyhexanoate by monooxygenase or cytochrome P450 such as from the CYP153 family such as CYP153A6; followed by conversion of 6-hydroxyhexanoate to adipate semialdehyde by alcohol dehydrogenase (EC 1.1.1.2) such as the gene product of YMR318C; followed by conversion of adipate semialdehyde to adipic acid by 6-oxohexanoate dehydrogenase (EC 1.2.1.63). See FIG. 4.
• the alcohol dehydrogenase encoded by YMR318C has broad substrate specificity, including the oxidation of C6 alcohols.
• adipic acid is synthesized from the central precursor, hexanoate, by conversion of hexanoate to 6-hydroxyhexanoate by monooxygenase or cytochrome P450 such as from the CYP153 family such as CYP153A6; followed by conversion of 6-hydroxyhexanoate to adipate semialdehyde by 6-hydroxyhexanoate dehydrogenase (EC 1.1.1.258) such as the gene product of ChnD (Iwaki et al., Appl. Environ. Microbiol., 1999, 65(11), 5158 - 5162); followed by conversion of adipate semialdehyde to adipic acid by aldehyde dehydrogenase (EC 1.2.1.3). See FIG. 4.
• adipic acid is synthesized from the central precursor, hexanoate, by conversion of hexanoate to 6-hydroxyhexanoate by monooxygenase or cytochrome P450 such as from the CYP153 family such as CYP153A6; followed by conversion of 6-hydroxyhexanoate to adipate semialdehyde by cytochrome P450 (Sanders et al., Journal of Lipid Research, 2005, 46(5), 1001-1008; Sanders et al., The FASEB Journal, 2008, 22(6), 2064 - 2071); followed by conversion of adipate semialdehyde to adipic acid by cytochrome P450. See FIG. 4. 4.5.4 Pathway using hexanoate as central precursor to 6- aminohexanoate and e-caprolactam
• 6-aminohexanoate is synthesized from the central precursor, hexanoate, by conversion of hexanoate to 6-hydroxyhexanoate by monooxygenase or cytochrome P450 such as from the CYP153 family such as
• CYP153A6 followed by conversion of 6-hydroxyhexanoate to adipate semialdehyde by alcohol dehydrogenase (EC 1.1.1.2) or 6-hydroxyhexanoate dehydrogenase (EC 1.1.1.258); followed by conversion of adipate semialdehyde to 6-aminohexanoate by ⁇ - transaminase (EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.48). See FIG. 5.
• e-caprolactam is synthesized from the central precursor, hexanoate, by conversion of hexanoate to 6-hydroxyhexanoate by monooxygenase or cytochrome P450 such as from the CYP153 family such as CYP153A6; followed by conversion of 6-hydroxyhexanoate to adipate semialdehyde by alcohol dehydrogenase (EC 1.1.1.2) or 6-hydroxyhexanoate dehydrogenase (EC 1.1.1.258); followed by conversion of adipate semialdehyde to 6-aminohexanoate by ⁇ -transaminase (EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.48); followed by conversion of 6-aminohexanoate to e- caprolactam by hydrolase (EC 3.5.2.-). See FIG. 5.
• hexamethylenediamine is synthesized from the central precursor, 6-aminohexanoate, by conversion of 6-aminohexanoate to 6-aminohexanal by carboxylate reductase (EC 1.2.99.6) such as the gene product of car alongside the gene product of npt or the gene product of GriC & GriD (Suzuki et al., J. Antibiot., 2007, 60(6), 380 - 387); followed by conversion of 6-aminohexanal to hexamethylenediamine by diamine transaminase (EC 2.6.1.29, EC 2.6.1.82). See FIG. 6.
• the carboxylate reductase encoded by the gene product of car and enhancer npt has broad substrate specificity, including terminal difunctional C4 and C5 carboxylic acids (Venkitasubramanian et al., Enzyme and Microbial Technology, 2008, 42, 130— 137).
• 6-hydroxyhexanoate is synthesized from the central precursor, hexanoate, by conversion of hexanoate to 6-hydroxyhexanoate by by monooxygenase or cytochrome P450 such as from the CYP153 family such as
• 1,6 hexanediol is synthesized from the central precursor, 6-hydroxyhexanoate, by conversion of 6-hydroxyhexanoate to 6-hydroxyhexanal by carboxylate reductase (EC 1.2.99.6) such as the gene product of car alongside the gene product of npt; followed by conversion of 6-hydroxyhexanal to 1 ,6 hexanediol by an alcohol dehydrogenase (e.g., EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) (Liu et al., Microbiology, 2009, 155, 2078 - 2085).
• carboxylate reductase EC 1.2.99.6
• an alcohol dehydrogenase e.g., EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184
• one or more C6 building blocks are biosynthesized in a recombinant host using anaerobic, aerobic or micro-aerobic cultivation conditions.
• a non-cyclical or a cyclical cultivation strategy can be used to achieve the desired cultivation conditions.
• a non-cyclical strategy can be used to achieve anaerobic, aerobic or micro-aerobic cultivation conditions.
• a cyclical cultivation strategy can be used to alternate between anaerobic cultivation conditions and aerobic cultivation conditions.
• the cultivation strategy entails nutrient limitation either via nitrogen, phosphate or oxygen limitation.
• one or more C6 building blocks are produced by a single microorganism via a non-cyclical or cyclical fermentation strategy.
• one or more C6 building blocks are produced by co- culturing more than one microorganism via a non-cyclical or cyclical fermentation strategy.
• one or more C6 building blocks are produced by successive fermentations, where the broth or centrate from the preceding fermentation is fed to a succession of fermentations as feedstock; finally producing the C6 building block.
• a cell retention strategy using, for example, ceramic hollow fiber membranes is employed to achieve and maintain a high cell density during either fed-batch or continuous fermentation.
• the principal carbon source fed to the fermentation in the synthesis of C6 building block 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.
• fermentable sugars such as monosaccharides and disaccharides derived from cellulosic, hemicellulosic, cane and beet molasses, cassava, corn and other argricultural sources has been demonstrated for several microorganism such as Escherichia coli, Corynebacterium glutamicum and
• Lactobacillus delbrueckii and Lactococcus lactis see, e.g., Hermann et al, Journal of Biotechnology, 2003, 104, 155 - 172; Wee et al, Food Technol. Biotechnol, 2006, 44(2), 163 - 172; Ohashi et al, Journal of Bioscience and Bioengineering, 1999, 87(5), 647 - 654).
• the non-biological feedstock is or derives from natural gas, syngas, CO2/H2, methanol, ethanol, non-volatile residue (NVR) or a caustic wash waste stream from cyclohexane oxidation processes.
• C0 2 and H 2 which may be derived from natural gas and other chemical and petrochemical sources, has been demonstrated for Cupriavidus necator (Prybylski et al., Energy, Sustainability and Society, 2012, 2: 11).
• the host is a prokaryote.
• the prokaryote can be 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 delbrueckii; or from the genus Lactococcus such as Lactococcus lactis.
• Corynebacteria such as Corynebacterium glutamicum
• Cupriavidus such as Cupriavidus necator or Cupriavidus metallidurans
• prokaryotes also can be a source of genes to construct recombinant host cells described herein that are capable of producing C6 building blocks.
• the host 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 C6 building blocks.
• 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.
• This document provides host cells of any of the genera and species listed and genetically engineered to express one or more (e.g., two, three, four, five, six, seven, eight, nine, 10, eleven, twelve or more) recombinant forms of any of the enzymes recited in the document.
• the host cells can contain exogenous nucleic acids encoding enzymes catalyzing one or more of the steps of any of the pathways described herein.
• the enzymes in the pathways outlined in section 4.5 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 4.5 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 a C6 building block.
• 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 a C6 building block.
• concentrations of a C6 building block is improved through continuous cultivation in a selective environment.
• the host microorganism's biochemical network is attenuated or augmented to (1) ensure the intracellular availability of acetyl-CoA, (2) create an NADH or NADPH imbalance that may only be balanced via the formation of C6 building blocks, (3) prevent degradation of central metabolites, central precursors leading to and including C6 building blocks and (4) ensure efficient efflux from the cell.
• a phosphotransacetylase generating acetate such as pta is attenuated (Shen et ⁇ , ⁇ . Environ. Microbiol, 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 ⁇ , ⁇ . Environ. Microbiol, 201 1, 77(9), 2905 - 2915).
• genes encoding the degradation of phophoenolpyruvate to succinate such as frdBC are attenuated (see, e.g., Shen et al, 201 1, supra).
• a gene encoding the degradation of acetyl-CoA to ethanol such as the alcohol dehydrogenase encoded by adhE is attenuated (Shen et al., 20 ⁇ ⁇ , supra).
• a gene encoding the degradation of pyruvate to ethanol such as pyruvate decarboxylase is attenuated.
• a gene encoding the generation of isobutanol such as a 2-oxoacid decarboxylase is attenuated.
• acetyl-CoA synthetase such as the gene product of acs is overexpressed in the microorganism (Satoh et al, Journal of Bioscience and
• carbon flux is directed into the pentose phosphate cycle by attenuating glucose-6-phosphate isomerase (EC 5.3.1.9).
• a formate dehydrogenase gene is overexpressed in the host organism (Shen et al, 201 1, supra).
• 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 zwfi ' s 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 flip is overexpressed in the host organisms (Becker et al, Journal of Biotechnology , 2001 , 132, 99 - 109).
• triose phosphate isomerase (EC 5.3.1.1) is attenuated.
• a glucose dehydrogenase such as the gene product oigdh is overexpressed in the host organism (Satoh et al, Journal of Bioscience and Bioengineering, 2003, 95(4), 335 - 341).
• enzymes facilitating the conversion of NADPH to NADH are attenuated, such as the NADH generation cycle that may be generated via inter-conversion of glutamate dehydrogenases in EC 1.4.1.2 (NADH-specific) and EC 1.4.1.4 (NADPH-specific).
• glutamate dehydrogenases (EC 1.4.1.3) that utilize both NADH and NADPH are co-factors are attenuated.
• membrane-bound cytochrome P450l o-hydroxylases are solubilized via truncation of the N-terminal region that anchors the P450 to the endoplasmic reticulum (Scheller et al., The Journal of Biological Chemistry, 1994, 269(17), 12779 0 12783).
• the polymer synthase enzymes can be attenuated in the host strain.
• a propionyl-CoA synthetase such as the gene product of PrpE-RS is overexpressed in the microorganism (Rajashekhara & Watanabe, FEBS Letters, 2004, 556, 143 - 147).
• ⁇ -oxidation enzymes degrading central metabolites and central precursors leading to and including C6 building blocks are attenuated.
• enzymes activating C6 building blocks via Coenzyme A esterification such as CoA-ligases are attenuated.
• the efflux of a C6 building block 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 a C6 building block.
• Genome-scale Flux Balance Analysis was undertaken using the genome-scale model for Saccharomyces cerevisiae designated ⁇ 904 (Mo et al, BMC Systems Biology, 2009, 3(37), 1-17).
• the IMM904 model was extended by including ⁇ -oxidation pathways as outlined in published work for eukaryotic organisms (Sanders et al., Journal of Lipid Research, 2005, 46(5), 1001 - 1008). Furthermore, the ⁇ -oxidation reactions in the peroxisome of the ⁇ 904 model were extended and relevant membrane transport reactions were included. The inactivation of a fumarate reductase was required during validation of the extended model to align model fluxes with experimental flux data.
• Allowance was made for the membrane transport of hexanoic acid and adipic acid to and from the extracellular media.
• the stoichiometric balance of NADH in the production of 1-butanol in E.coli is far more efficient when overexpressing brmate dehydrogenase (Shen et al, Appl. Environ. Microbiol, 2011, 77(9), 2905 - 2915).
• the ⁇ 904 model includes formate dehydrogenase, but lacked pyruvate formate lyase activity, which was included into the Saccharomyces cerevisiae model.
• the metabolic engineering workbench, Optflux was used to search the solution space associated with the biochemical network for attenuation strategies that (1) produce hexanoate anaerobically from glucose, followed by (2) production of adipate aerobically from the extracellular hexanoate, whilst co-feeding glucose as carbon and energy source.
• the optimization trials found four beneficial attenuations; viz. (1) attenuating glucose-6-phosphate isomerase, directing flux into the pentose phosphate cycle; (2) attenuating pyruvate decarboxylase or alcohol dehydrogenase, preventing ethanol production; (3) attenuating 2-oxoacid decarboxylase, preventing isobutanol production and (4) inactivating ⁇ -oxidation, preventing central precursor, central metabolite and adipic acid degradation.
• In-silico attenuation of the biochemical network using a validated model determined that a cultivation strategy, cycling between anaerobic and aerobic conditions, produces predominantly adipic acid from the fed glucose.
• Saccharomyces cerevisiae The ⁇ 904 model outlined in Example 1 was utilized to assess the non- cyclical production of adipic acid using glucose as carbon and energy source under micro-aerobic, substrate oxidation and growth limiting cultivation conditions.
• oxidoreductase preventing 2-hydroxybutyrate production; (4) attenuating DL gfycerol-3-phosphatase, preventing glycerol production and (5) attenuating malate dehydrogenase, preventing inter-conversion of NADH to NADPH.
• the resulting S. cerevisiae mutant produces adipate as the most advantageous means of balancing NADH to maximize biomass growth, producing adipate with a molar yield of 0.71 [(mol adipate)/(mol glucose)].
• Example 1 The ⁇ 904 model outlined in Example 1 was utilized to assess the non- cyclical production of adipic acid using glucose as carbon and energy source under aerobic cultivation and growth limiting conditions.

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