EP2794897A2 - Bioconversion process for producing nylon-7, nylon-7,7 and polyesters - Google Patents

Bioconversion process for producing nylon-7, nylon-7,7 and polyesters

Info

Publication number
EP2794897A2
EP2794897A2 EP12816586.7A EP12816586A EP2794897A2 EP 2794897 A2 EP2794897 A2 EP 2794897A2 EP 12816586 A EP12816586 A EP 12816586A EP 2794897 A2 EP2794897 A2 EP 2794897A2
Authority
EP
European Patent Office
Prior art keywords
enzyme
coa
produced
catalyzes
acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP12816586.7A
Other languages
German (de)
English (en)
French (fr)
Inventor
Paul S. Pearlman
Changlin Chen
Adriana BOTES
Alex van Eck CONRADIE
Benjamin D. Herzog
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Invista Textiles UK Ltd
Original Assignee
Invista Technologies SARL Switzerland
Invista Technologies SARL USA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US2012/044984 external-priority patent/WO2013003744A2/en
Application filed by Invista Technologies SARL Switzerland, Invista Technologies SARL USA filed Critical Invista Technologies SARL Switzerland
Publication of EP2794897A2 publication Critical patent/EP2794897A2/en
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/26—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from polyamines and polycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • 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
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/50—Polycarboxylic acids having keto groups, e.g. 2-ketoglutaric acid

Definitions

  • Embodiments of the present invention relate to methods for the biosynthetic production of di- or trifunctional C7 alkanes in the presence of one or more isolated enzymes or in the presence of a recombinant host cell expressing one or more enzymes.
  • the di- or trifunctional C7 alkanes are useful as intermediates in the production, for example, of nylon- 7, nylon-7,x and/or nylon-x,7.
  • biotechnology also offers a way to utilize waste or low value streams such as glycerol from biodiesel production, or to transform petrochemical- derived feedstocks such as benzene, toluene, and polycyclic aromatic hydrocarbons (PAHs) to useful products in bioprocesses that are more benign to the environment than existing chemical processes.
  • waste or low value streams such as glycerol from biodiesel production
  • petrochemical- derived feedstocks such as benzene, toluene, and polycyclic aromatic hydrocarbons (PAHs)
  • This disclosure is based at least in part of the development of enzymatic systems and recombinant hosts for biosynthesizing di- or trifunctional C7 alkanes that are useful intermediates (monomers) for producing nylon-7, nylon-x,7, nylon-7,x, and polyesters.
  • intermediates useful in the production of nylon-7, and nylon- 7,x, nylon-x,7, and polyesters can be biosynthetically produced from renewable feedstocks, such as plant-derived sugars and glycerol, or from petrochemical-derived feedstocks such as benzene or cyclohexane carboxylate.
  • FIG. 1 is a schematic depicting the biotransformation of pimelic acid semilaldehyde to various difunctional C7 alkanes.
  • pimelic acid semialdehyde can be derived from pimeloyl-coenzyme A (CoA), pimeloyl-[acyl carrier protein (acp)], pimelic acid (also known as heptane 1,7-dioate), or a-ketosuberate.
  • pimeloyl-CoA, pimeloyl-[acp] and pimelic acid semialdehyde can be derived from a number of sources, including from a diverse number of naturally occurring metabolic pathways that form pimeloyl-CoA, pimeloyl-[acp] or pimelic acid semialdehyde as an intermediate in the naturally occurring metabolic pathway.
  • pimeloyl-CoA and pimelic acid also can be derived from the corresponding enoate 2-heptene-dioic acid or its corresponding CoA ester, 2-heptenedioyl- CoA.
  • these C7 2-enoates are derived from D,L-diaminopimelate or 2- oxopimelate.
  • the production of difunctional C7 alkanes proceeds through ⁇ -ketosuberate or a-aminosuberate, which can be derived from D,L-diaminopimelate.
  • the ⁇ -ketosuberate or ⁇ -aminosuberate is derived from a-ketopimelate.
  • the present document provides a variety of methods of converting compounds in order to produce intermediates useful for the manufacture of nylon-7, nylon- 7,7, and polyester. It is not required that all possible steps of any one method be performed and any of the methods can be terminated when a desired compound is obtained. Thus, any method can be terminated after the production of, for example, pimelic acid, 7-amino- heptanoic acid, enantholactam, 1,7-diaminoheptane, or 7-hydroxy-heptanoic acid.
  • a first method of converting a compound can include contacting a compound selected from 2,6 diaminopimelate (2,6 DAP) and a-amino-pimelate (AAP) with an enzyme that catalyzes the reductive deamination of 2,6 DAP to 6-amino-2-heptenedioic acid (6A2HA) or the reductive deamination of AAP to 2-heptene dioic acid (2 HDA), such that 6A2HA or 2 HDA is produced.
  • a compound selected from 2,6 diaminopimelate (2,6 DAP) and a-amino-pimelate (AAP) with an enzyme that catalyzes the reductive deamination of 2,6 DAP to 6-amino-2-heptenedioic acid (6A2HA) or the reductive deamination of AAP to 2-heptene dioic acid (2 HDA), such that 6A2HA or 2 HDA is produced.
  • the compound can be 2,6 DAP and the first method can further involve contacting the 6A2HA with an enzyme that catalyzes the enoate reduction of the 6A2HA to AAP, such that AAP is produced.
  • the AAP can be contacted with an enzyme that catalyzes the reductive deamination of the AAP to 2 HDA, such that 2 HDA is produced.
  • the 2 HDA can be contacted with an enzyme that catalyzes the enoate reduction of the 2 HDA to pimelic acid (PA), such that PA is produced.
  • PA pimelic acid
  • the PA can be contacted with an enzyme that catalyzes the carboxylic acid reduction of the PA to pimelic acid semialdehyde (PAS), such that PAS is produced.
  • PAS pimelic acid semialdehyde
  • the PAS can be contacted with an enzyme that catalyzes the semialdehyde amination of the PAS to 7-amino-heptanoic acid (7 AHA), such that 7 AHA is produced.
  • the 7 AHA can be contacted with an enzyme that catalyzes the amide hydrolysis of the 7 AHA to enantholactam (ENTL), such that ENTL is produced.
  • an enzyme that catalyzes the amide hydrolysis of the 7 AHA to enantholactam (ENTL), such that ENTL is produced.
  • a second method of converting compound can include, after performing all the steps of the first method up to the production of 7 AHA, contacting the 7 AHA with an enzyme that catalyzes the aldehyde dehydrogenation of the 7 AHA to 7-amino-heptanal (7 AHT), such that AHT is produced.
  • the 7 AHT can be contacted with an enzyme that catalyzes the transfer of an amino group to the 7 AHT to produce 1,7-diaminoheptane (1,7 DAH), such that 1,7 DAH is produced.
  • a third method of converting compound can include, after performing all the steps of the first method up to the production of 2 HDA, contacting the 2 HDA with an enzyme that catalyzes the transfer of Coenzyme A (CoA) to the 2 HDA to produce 2-heptene diacid-CoA (2 HDA-CoA), such that 2 HDA-CoA is produced.
  • CoA Coenzyme A
  • the 2 HDA-CoA can be contacted with an enzyme that catalyzes the enoate reduction of the 2 HDA-CoA to pimeloyl-CoA (PCoA), such that PCoA is produced.
  • PCoA pimeloyl-CoA
  • the PCoA can be contacted with an enzyme that catalyzes the thioester hydrolysis of the PCoA to PA, such that PA is produced.
  • the PA can be contacted with an enzyme that catalyzes the carboxylic acid reduction of the PA to PAS, such that PAS is produced.
  • the PAS can be contacted with an enzyme that catalyzes the semialdehyde amination of the PAS to 7 AHA, such that 7 AHA is produced.
  • the 7 AHA can be contacted with an enzyme that catalyzes the amide hydrolysis of the 7 AHA to ENTL, such that ENTL is produced.
  • a fourth method of converting a compound can include, after performing all the steps of the third method up to production of 7 AHA, contacting the 7 AHA with an enzyme that catalyzes the aldehyde dehydrogenation of the 7 AHA to 7 AHT, such that 7 AHT is produced.
  • the 7 AHT can be contacted with an enzyme that catalyzes the transfer of an amino group to the 7 AHT to produce 1,7 DAH, such that 1,7 DAH is produced.
  • a fifth method of converting a compound can include, after performing all the steps of the first method up to the production of AAP, contacting the AAP with an enzyme that catalyzes the transfer of an amino group to the AAP to produce a-keto-pimelate (AKP), such that AKP is produced.
  • AAP a-keto-pimelate
  • the AKP can be contacted with one or more enzymes that catalyze the ketone reduction of the AKP to a-hydroxy-pimelate (AHP), such that AHP is produced.
  • AHP a-hydroxy-pimelate
  • the AHP can be contacted with an enzyme that catalyzes the transfer of CoA to the AHP to produce a-hydroxy-pimelate-CoA (AHP-CoA), such that AHP-CoA is produced.
  • AHP-CoA a-hydroxy-pimelate-CoA
  • the AHP-CoA can be contacted with an enzyme that catalyzes the dehydration of the AHP-CoA to 2 HDA-CoA, such that 2 HDA-CoA is produced.
  • the 2 HDA-CoA can be contacted with an enzyme that catalyzes the enoate reduction of the 2 HDA-CoA to PCoA, such that PCoA is produced.
  • the PCoA can be contacted with an enzyme that catalyzes the thioester hydrolysis of the PCoA to PA, such that PA is produced.
  • the PA can be contacted with an enzyme that catalyzes the carboxylic acid reduction of the PA to PAS, such that PAS is produced.
  • the PAS with can be contacted with an enzyme that catalyzes the semialdehyde amination of the PAS to 7 AHA, such that 7 AHA is produced.
  • the 7 AHA can be contacted with an enzyme that catalyzes the amide hydrolysis of the 7 AHA to ENTL, wherein ENTL is produced.
  • a sixth method of converting a compound can include, after performing all the steps of the fifth method up to the production of 7 AHA, contacting the 7 AHA with an enzyme that catalyzes the aldehyde dehydrogenation of the 7 AHA to 7 AHT, such that 7 AHT is produced.
  • the 7 AHT can be contacted with an enzyme that catalyzes the transfer of an amino group to the 7 AHT to produce 1,7 DAH, such that 1,7 DAH is produced.
  • a seventh method of converting a compound can include, after performing all the steps of the fifth method up to the production of AHP, contacting the AHP with an enzyme that catalyzes the dehydration of the AHP to 2 HDA, such that 2 HDA is produced.
  • the 2 HDA can be contacted with an enzyme that catalyzes the enoate reduction of the 2 HDA to pimelic acid (PA), such that PA is produced.
  • PA pimelic acid
  • the PA can be contacted with an enzyme that catalyzes the carboxylic acid reduction of the PA to PAS, such that PAS is produced.
  • the PAS can be contacted with an enzyme that catalyzes the semialdehyde amination of the PAS to 7 AHA, such that 7 AHA is produced.
  • the 7 AHA can be contacted with an enzyme that catalyzes the amide hydrolysis of the 7 AHA to ENTL, such that ENTL is produced.
  • An eighth method of converting a compound can include, after performing all the steps of the fifth method up to the production of 7 AHA, contacting the 7 AHA with an enzyme that catalyzes the aldehyde dehydrogenation of the 7 AHA to 7 AHT, such that 7 AHT is produced.
  • the 7 AHT can be contacted with an enzyme that catalyzes the transfer of an amino group to the 7 AHT to produce 1,7 DAH, such that 1,7 DAH is produced.
  • a ninth method of converting a compound can include, after performing all the steps of the seventh method up to the production of 2 HDA, contacting the 2 HDA with an enzyme that catalyzes the transfer of CoA to 2 HDA to produce 2 HDA-CoA, such that 2 HDA-CoA is produced.
  • the 2 HDA-CoA can be contacted with an enzyme that catalyzes the enoate reduction of the 2 HDA-CoA to PCoA, such that PCoA is produced.
  • the PCoA can be contacted with an enzyme that catalyzes the thioester hydrolysis of the PCoA to PA, such that PA is produced.
  • the PA can be contacted with an enzyme that catalyzes the carboxylic acid reduction of the PA to PAS, such that PAS is produced.
  • the PAS can be contacted with an enzyme that catalyzes the semialdehyde amination of the PAS to 7 AHA, such that 7 AHA is produced.
  • the 7 AHA can be contacted with an enzyme that catalyzes the amide hydrolysis of the 7 AHA to ENTL, such that ENTL is produced.
  • a tenth method of converting a compound can include, after performing all the steps of the ninth method up to the production of 7 AHA, contacting the 7 AHA with an enzyme that catalyzes the aldehyde dehydrogenation of the 7 AHA to 7 AHT, such that 7 AHT is produced.
  • the 7 AHT can be contacted with an enzyme that catalyzes the transfer of an amino group to the 7 AHT to produce 1,7 DAH, such that 1,7 DAH is produced.
  • An eleventh method of converting a compound can include, after performing all the steps of the fifth method up to the production of AKP, contacting the AKP with an enzyme that catalyzes the a-keto acid chain elongation of the AKP to a-keto-suberate (AKS), such that AKS is produced.
  • AKS a-keto-suberate
  • the AKS can be contacted with an enzyme that catalyzes the transfer of an amino group to the AKS to produce a-amino suberate (AAS), such that AAS is produced.
  • AAS a-amino suberate
  • the AAS can be contacted with an enzyme that catalyzes the a-amino acid decarboxylation of the AAS to 7 AHA, such that 7 AHA is produced.
  • the 7 AHA can be contacted with an enzyme that catalyzes the amide hydrolysis of the 7 AHA to ENTL, such that ENTL is produced.
  • a twelfth method of converting a compound can include, after performing all the steps of the eleventh method up to the production of 7 AHA, contacting the 7 AHA with an enzyme that catalyzes the aldehyde dehydrogenation of the 7 AHA to 7 AHT, such that 7 AHT is produced.
  • a thirteenth method of converting a compound can include, after performing all the steps of the eleventh method up to the production of AKS, contacting the AKS with an enzyme that catalyzes the a-keto acid decarboxylation of the AKS to PAS, such that PAS is produced.
  • the PAS can be contacted with an enzyme that catalyzes the semialdehyde amination of the PAS to 7 AHA, such that 7 AHA is produced.
  • the 7 AHA can be contacted with an enzyme that catalyzes the amide hydrolysis of the 7 AHA to ENTL, such that ENTL is produced.
  • a fourteenth method of converting a compound can include, after performing all the steps of the thirteenth method up to the production of 7 AHA, contacting the 7 AHA with an enzyme that catalyzes the aldehyde dehydrogenation of the 7 AHA to 7 AHT, such that 7 AHT is produced.
  • the 7 AHT can be contacted with an enzyme that catalyzes the transfer of an amino group to the 7 AHT to produce 1,7 DAH, such that 1,7 DAH is produced.
  • a fifteenth method of converting a compound can include, after performing all the steps of the fifth method up to the production of PCoA, contacting the PCoA with an enzyme that catalyzes the reduction of the PCoA to PAS, such that PAS is produced.
  • the PAS can be contacted with an enzyme that catalyzes the semialdehyde amination of the PAS to 7 AHA, such that 7 AHA is produced.
  • the 7 AHA can be contacted with an enzyme that catalyzes the amide hydrolysis of the 7 AHA to ENTL, such that ENTL is produced.
  • a sixteenth method of converting a compound can include, after performing all the steps of the fifteenth method up to the production of 7 AHA, contacting the 7 AHA with an enzyme that catalyzes the aldehyde dehydrogenation of the 7 AHA to 7 AHT, such that 7 AHT is produced.
  • the 7 AHT can be contacted with an enzyme that catalyzes the transfer of an amino group to the 7 AHT to produce 1,7 DAH, such that 1,7 DAH is produced.
  • a seventeenth method of converting a compound can include, after performing all the steps of the ninth method up to the production of PCoA, contacting the PCoA with an enzyme that catalyzes the reduction of the PCoA to PAS, such that PAS is produced.
  • the PAS can be contacted with an enzyme that catalyzes the semialdehyde amination of the PAS to 7 AHA, such that 7 AHA is produced.
  • the 7 AHA can be contacted with an enzyme that catalyzes the amide hydrolysis of the 7 AHA to ENTL, such that ENTL is produced.
  • An eighteenth method of converting a compound can include, after performing all the steps of the seventeenth method up to the production of 7 AHA, contacting the 7 AHA with an enzyme that catalyzes the aldehyde dehydrogenation of the 7 AHA to 7 AHT, such that 7 AHT is produced.
  • the 7 AHT can be contacted with an enzyme that catalyzes the transfer of an amino group to the 7 AHT to produce 1 ,7 DAH, such that 1,7 DAH is produced.
  • a nineteenth method of converting a compound can include contacting a compound selected from 6A2HA and 2 HDA with an enzyme that catalyzes the enoate reduction of 6A2HA to AAP or for the enoate reduction of 2 HDA to PA, such that AAP or PA is produced. It is understood that, following the production of AAP or PA, any of the steps after the production of AAP or PA described above in the first to eighteenth methods can be performed.
  • a twentieth method of converting a compound can include contacting a compound selected from PCoA and pimeloyl [acp] (PACP) with an enzyme that catalyzes the thioesterase hydrolysis PCoA or PACP to PA, wherein PA is produced.
  • acp pimeloyl
  • the PA can be contacted with an enzyme that catalyzes the carboxylic acid reduction of the PA to PAS, such that PAS is produced.
  • the PAS can be contacted with an enzyme that catalyzes the semialdehyde amination of the PAS to 7 AHA, such that 7 AHA is produced.
  • the 7 AHA with an enzyme that catalyzes the amide hydrolysis of the 7 AHA to ENTL, such that ENTL is produced.
  • a twenty first method of converting a compound can include, after performing all the steps of the twentieth method up to the production of 7 AHA, contacting the 7 AHA with an enzyme that catalyzes the aldehyde dehydrogenation of the 7 AHA to 7 AHT, such that 7 AHT is produced.
  • the 7 AHT can be contacted with an enzyme that catalyzes the transfer of an amino group to the 7 AHT to produce 1 ,7 DAH, such that 1,7 DAH is produced.
  • a twenty second method of converting a compound can include contacting AKP with one or more enzymes that catalyze the ketone reduction of AKP to AHP, such that AHP is produced.
  • the AKP can have been produced by chain elongation of a-keto adipate or a-keto glutarate.
  • the AHP can then be converted to PA or to PCoA. It is understood that, following the production of PA or PCoA, any of the steps after the production of PA or PCoA described above in the first to eighteenth methods can be performed.
  • a twenty third method of converting a compound can include converting AKP to AKS by a-keto acid chain elongation.
  • the AKP can have been produced by chain elongation of a-keto adipate or a-keto glutarate. It is understood that, following the production of AKS, any of the steps after the production of AKS described above in the first to eighteenth methods can be performed.
  • a twenty fourth method of converting a compound can include contacting PAS with an enzyme that catalyzes the semialdehyde amination of PAS to 7 AHA, such that 7 AHA is produced.
  • the PAS can have been obtained by conversion from 2,6 DAP, AKG, PCoA, or PACP. It is understood that, following the production of AHA, any of the steps after the production of AHA described above in the first to eighteenth methods can be performed.
  • a twenty fifth method of converting a compound can include contacting a compound selected from PCoA and PACP with an enzyme that catalyzes the reduction of the compound to PAS, such that PAS is produced. It is understood that, following the production of PAS, any of the steps after the production of PAS described above in the first to eighteenth methods can be performed. Moreover, any of the steps described in the first to eighteenth and the twenty second method leading to the production of PCoA can be performed prior to the twenty fifth method.
  • a twenty sixth method of converting a compound can include contacting PAS with an enzyme that catalyzes the alcohol dehydrogenation of PAS to 7-hydroxy-heptanoic acid (7 HHA), such that 7 HHA is produced. It is understood that any of the steps described in the first to eighteenth and the twentieth, twenty second, twenty third, and twenty fifth methods leading to the production of PAS can be performed prior to the twenty sixth method.
  • the enzyme(s) can be used in purified form.
  • the enzyme(s) can be used in the form of a cell lysate or a partially purified cell lysate.
  • the enzyme(s) can be in a cell recombinantly expressing it/them.
  • an enzyme that catalyzes reductive deamination can include an ammonia lyase.
  • the ammonia lyase can be in EC 4.3.1, e.g., EC 4.3.1.1 ; EC 4.3.1.2; EC 4.3.1.3; EC 4.3.1.9; EC 4.3.1.12; EC 4.3.1.13; EC 4.3.1.14, EC 4.3.1.23 or EC 4.3.1.24.
  • an enzyme that catalyzes 2-enoate reduction can include an enoate reductase.
  • the enoate reductase can be in EC 1.3.1, e.g., EC 1.3.1.8; EC 1.3.1.9; EC 1.3.1.10, such as the gene product of Fabl; EC 1.3.1.31 ; EC 1.3.1.38; EC 1.3.1.39; EC 1.3.1.44 such as the gene product of ter or tdter (Nishimaki et al, J. Biochem., 1984, 95, 1315 - 1321 ; Shen et al, Appl. Environ.
  • the enoate reductase may also be in the EC 1.3, such as EC 1.3.8.1; EC 1.3.99.3; EC 1.3.99.B10 or Syntrophus aciditrophicus 2,3-didehydropimelyl-CoA reductase and homologs thereof.
  • the enoate reductases may act on iraws-2-enoyl-dicarboxylic acids, or thioesters of dicarboxylic acids such as 2-heptene dioic acid-CoA (trans-2,3- didehydropimlyl-CoA) or 2-heptene dioic acid-[acp] (?ra «s-2,3-didehydropimlyl-[acp]).
  • an enzyme that catalyzes carboxylic acid reduction can include a carboxylic acid reductase.
  • the carboxylic acid reductase can be in EC 1.2.99, e.g., EC 1.2.99.6.
  • the carboxylic acid reductase may also belong to the NAD-dependent non- acylating aldehyde dehydrogenases, such as ThnG in Sphingomonas and Cupriavidus spp..
  • an enzyme that catalyzes semialdehyde amination can include a ⁇ -transaminase.
  • the ⁇ -transaminase can be in EC 2.6.1, e.g., EC 2.6.1.18; EC 2.6.1.19; EC 2.6.1.11 ; EC 2.6.1.13; EC 2.6.1.39; EC 2.6.1.48; or EC 2.6.1.62.
  • an enzyme that catalyzes thioester hydrolysis of CoA thioesters can include a thioesterase, an acid-thiol ligase, or a CoA transferase.
  • the thioesterase can be in EC 3.1.2, e.g., such as the gene product of YciA, tesB or Acotl3; EC 3.1.2.2; EC 3.1.2.18; EC 3.1.2.19; or EC 3.1.2.20.
  • the acid-thiol ligase/CoA synthetase can be in EC 6.2.1, e.g., EC 6.2.1.3; EC 6.2.1.5, EC 6.2.1.14; or EC 6.2.1.23.
  • the CoA transferase can be in EC 2.8.3, e.g., EC 2.8.3.12; EC 2.8.3.13 or EC 2.8.3.14, or or the gene product of ThnH that catalyses the reversible transfer of CoA to pimelic acid.
  • the enzyme that catalyses thioester hydrolysis of acyl- carrier-protein ([ACP]) thioesters can include a acyl-[ACP] thioesterase from EC 3.1.2, e.g. EC 3.1.2.14 or EC 3.1.2.21, an acyl-[ACP]-synthtase from EC 6.2.1, e.g. EC 6.2.1.14, such as BioW; and EC 6.2.1.20.
  • an enzyme that catalyzes the transfer of an a- amino group can include an amino acid aminotransferase.
  • the amino acid aminotransferase can be a L- or D-selective aminotransferase in EC 2.6.1, e.g., EC 2.6.1.39; EC 2.6.1.42 or EC 2.6.1.21, or a 2-aminohexanoate aminotransferase from EC 2.6.1.67.
  • the enzyme that catalyses the transfer of an alpha amino group can also be a dehydrogenase from the class EC 1.4.1.-, such as glutamate dehydrogenase, which depending on the co-factor used belongs to EC 1.4.1.2; EC 1.4.1.3; or EC 1.4.1.4, or a diaminopimelate dehydrogenase from EC 1.4.1.16.
  • a dehydrogenase from the class EC 1.4.1.-, such as glutamate dehydrogenase, which depending on the co-factor used belongs to EC 1.4.1.2; EC 1.4.1.3; or EC 1.4.1.4, or a diaminopimelate dehydrogenase from EC 1.4.1.16.
  • an enzyme that catalyzes ketone reduction can be a carbonyl reductase in EC 1.1.1.- or 1.1.99,.
  • the carbonyl reductase can include EC 1.1.1.184; EC 1.1.1.79; EC 1.1.1. B3; EC 1.1.1. B4 or 2-hydroxyglutaryl dehydogenases/alpha ketogluterate reductases from EC 1.1.99.2. or EC 1.1.99.6
  • an enzyme that catalyzes a-keto acid decarboxylation can be an a-keto acid decarboxylase in EC 4.1.1, e.g. 4.1.1.1; 4.1.1.7; 4.1.1.72; or an acetolactate synthase in the class EC 2.2.1.6 or EC 2.2.2.6.
  • the enzyme that catalyses a-amino acid decarboxylation can be an a-amino acid decarboxylase from the class EC 4.1.1.-.
  • the a-amino acid decarboxylase can include EC 4.1.1.11 ; EC 4.1.1.15; EC 4.1.1.16; EC 4.1.1.18; EC 4.1.1.20, EC 4.1.1.45; and EC 4.1.1.86.
  • an enzyme that catalyzes reduction of the CoA ester of a dicarbylic acid to the corresponding semialdehyde can be a fatty-acyl-CoA reductase.
  • the fatty-acyl-CoA reductase can be in EC 1.2.1.-, e.g., EC 1.2.1.3; EC 1.2.1.10; EC 1.2.1.22; EC 1.2.1.50; EC 1.2.1.57 and EC 1.2.1.76.
  • an enzyme that catalyses the reduction of the [ACP] thioester to the corresponding semialdehyde can be a fatty- acyl-CoA reductase acting on CoA esters and [ACP] esters described above, or an acyl-acyl- carrier-protein reductase from e.g. EC 1.2.1.80.
  • an enzyme that catalyzes aldehyde dehydrogenation to the carboxylic acid can be an aldehyde dehydrogenase or an aldehyde oxidase.
  • the aldehyde dehydrogenase can be in EC 1.2.1, e.g., EC 1.2.1.3; EC 1.2.1.4 or EC 1.2.1.63.
  • the aldehyde dehydrogenase/ carboxylic acid reductase may also be in EC 1.2.99, e.g., EC 1.2.99.6.
  • the carboxylic acid reductase may also belong to the NAD-dependent non-acylating aldehyde dehydrogenases, such as ThnG in Sphingomonas and Cupriavidus spp..
  • the aldehyde oxidase can be in EC 1.2.3, e.g. 1.2.3.1.
  • an enzyme that catalyzes the ring closure of 7- aminoheptanoic acid to enantholactam can be an amidohydrolase of EC 3.5.2, e.g. 3.5.2.1 1.
  • an enzyme that catalyzes dehydration can be a hydro-lyase.
  • the hydro-lyase can be in EC 4.2.1, e.g., EC 4.2.1.2; EC 4.2.1.59; 4.2.1.61 ; 4.2.1.17 or 4.2.1.18.
  • the dehydratase may also be 2- hydroxyglutaryl-CoA dehydratase described in clostrida and fusobacteria to which an EC number had not yet been assigned, that is expressed with its activator (HgdCAB) or a 2- hydroxyacyl_CoA dehydratase of anaerobic bacteria.
  • enzymes that catalyze a-keto acid chain elongation can include one of more of the set of enzymes including AksA, AksD, AksE and AksF.
  • the AksA can be in EC 2.3.3, e.g., EC 2.3.3.13 or 2.3.3.14.
  • the AksD can be in EC 4.2.1, e.g., EC 4.2.1.36.
  • the AksF can be in EC 1.1.1, e.g., EC 1.1.1.87.
  • One or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 12 or more, 14 or more, 18, or more, or 20 or more) of the a-keto acid chain elongation enzymes can be from methanogenic bacteria.
  • an enzyme that catalyzes alcohol dehydrogenation can include an alcohol dehydrogenase.
  • the alcohol dehydrogenase can include EC. 1.1.1.1 or EC 1.1.1.2 or 1.1.1.21. It can also be, for example, adhA from Zymomonas mobilis, adhB from Zymomonas mobilis, butanol dehydrogenase from Clostridium acetobutylicum, Saccharomyces ADHIV, and ADH6 from S. cerevisiae.
  • the enzyme that catalyzes alcohol dehydrogenation can be an alcohol oxidase.
  • the alcohol oxidase can be in class EC 1.1.3.13.
  • PCoA or PACP used in the methods of the document can be derived from acetyl- CoA or benzoyl-CoA or from any metabolic pathway such as biotin biosynthesis, benzoate degradation, cyclohexane carboxylate pathway, or condensation of malonyl-CoA olr fatty acid ⁇ -oxidation.
  • the acetyl-CoA can be derived from renewable feedstocks comprising cellulosic feedstocks, sugars, glycerol, or fatty acids.
  • the acetyl-CoA can be derived from SynGas, methane or methanol.
  • the benzoyl-CoA can be derived from poly cyclic aromatic hydrocarbons.
  • PAS can be derived from the tetralin degradation pathway.
  • the 2,6 DAP used in the methods of the document can be produced by a lysine- producing organism lacking diaminopimelate decarboxylase.
  • the 2,6 DAP is derived from any fermentable carbon source, including sugars, glycerol, fatty acids, syngas, methane or methanol.
  • the present document features a bioderived nylon-7, nylon-7, x nylon-x,7, and polyester. It also includes a Nylon-7 produced by a process that includes polymerizing 7 AHA, where the 7 AHA is derived from PAS or AAS.
  • nylon-7, 7 produced by a process that includes polymerizing PA and 1,7 DHA, where the PA is derived from PCoA; PACP; or 2 HDA and the 1,7 DHA is derived from 7 AHA.
  • the document also embodies nylon-7 produced by a process that includes polymerizing 7 AHA made by any of the methods described above that lead to the production of 7 AHA.
  • Another aspect of the invention is a nylon 7,7 produced by a process comprising polymerizing 1,7 DHA and PA, where the 1,7 DHA is made by any of the methods described above that lead to the production of 1,7 DHA and the PA is made by any of the methods described above that lead to the production of PA.
  • the present document also provides a substantially pure culture of host cells, a substantial number of which comprise and express one or more exogenous nucleic acids encoding one or more enzymes involved in the biosynthesis of one or more polymer monomers selected from 7 AHA; PA; 1,7 DAH; ENTL; and 7 HHA.
  • These enzymes include fatty-acyl-CoA reductases, acyl-[acp] reductases, thioester hydrolases, ammonia lyases, enoate reductases, amino acid aminotransferases, CoA transferases, ,acid-thiol ligases, hydro-lyases, carbonyl reductases, carboxylic acid reductases, ⁇ -transaminases, a-keto acid decarboxylases, aldehyde dehydrogenases, deaminating dehydrogenases, alcohol dehydrogenases, aldehyde oxidases, alcohol oxidases, amidohydrolases, decarboxylating acetolactate synthases and enzymes catalyzing a-keto acid chain elongation such as AksA, AksD, AksE and AksF.
  • a "substantial number" is at least 10% (e.g., at least: 20%; 30%; 40%; 50%; 60%; 70%; 75%; 80%; 85%; 90%; 95%; 97%; 98%; 99%; or even 100%) of the cells in the culture.
  • the cells of the culture can be prokaryotic cells, such as, without limitation, the genus Escherichia such as the species Escherichia coli; the genus Clostridia such as the species Clostridium ljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; the genus Corynebacteria such as the species Cory neb acterium glutamicum; the genus Cupriavidus such as the species Cupriavidus necator or Cupriavidus metallidurans; the genus Pseudomonas such as the species Pseudomonas fluorescens, Pseudomonas putida or Pseudomonas oleavorans; the genus Delftia such as the species Delftia acidovorans; the genus Bacillus such as the species Bacillus subtillis; the genus Lactobacillus
  • the cells of the culture can be eukaryotic cells, e.g., fungal cells such as yeast cells.
  • the yeast cells can be of a yeast or fungus such as, without limitation, the genus Aspergillus such as the species Aspergillus niger; he genus Saccharomyces such as the species Saccharomyces cerevisiae; the genus Candida such as thye species C. tropicalis, C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. maltosa, C. parapsilosis, and C.
  • the zeylenoides the genus Pichia such as the species Pichia pastoris; the genus Yarrowia such as the species Yarrowia lipolytica; the genus Issatchenkia such as the species Issathenkia orientalis; the genus Debaryomyces such as the species Debaryomyces hansenii; the genus Arxula such as the species Arxula adenoinivorans; the genus Kluyveromyces such as the species Kluyveromyces lactis; the genus Exophiala, the genus Mucor, the genus Trichoderma, the genus Cladosporium, the genus Phanerochaete, the genus Cladophialophora, the genus Paecilomyces, the genus Scedosporium, or the genus Ophiostoma.
  • the ammonia lyases can include an ammonia lyase in EC 4.3.1 , e.g., EC 4.3.1.1 ; EC 4.3.1.2; EC 4.3.1.3; EC 4.3.1.9; EC 4.3.1.12; EC 4.3.1.13; EC 4.3.1.14, EC 4.3.1.23 or EC 4.3.1.24.
  • the enoate reductases can include an enoate reductase in EC 1.3.1, e.g EC 1.3.1.8; EC 1.3.1.9; EC 1.3.1.10, EC 1.3.1.31 ; EC 1.3.1.38; EC 1.3.1.39; EC 1.3.1.44 or pimeloyl-CoA dehydrogenase in EC 1.3.1.62 such as PimC/PimD or ThnJ/ThnK.
  • the enoate reductase may also be in the EC 1.3, such as EC 1.3.8.1 or EC 1.3.99.3; EC 1.3.99.B 10 or Syntrophus aciditrophicus 2,3-didehydropimelyl-CoA reductase and homologs thereof.
  • the carboxylic acid reductases include a carboxylic acid reductase in EC 1.2.99, e.g. EC 1.2.99.6.
  • the carboxylic acid reductase may also belong to the NAD-dependent non-acylating aldehyde dehydrogenases, such as ThnG in Sphingomonas and Cupriavidus spp..
  • the ⁇ -transaminases can include a transaminase in EC 2.6.1, e.g., EC 2.6.1.18; EC 2.6.1.19; EC 2.6.1.1 1 ; EC 2.6.1.13; EC 2.6.1.39EC 2.6.1.48or EC 2.6.1.62.
  • the thioesterases can include a thioesterase in EC 3.1.2, e.g., such as the gene product of YciA, tesB or Acotl 3; EC 3.1.2.2; EC 3.1.2.18; EC 3.1.2.19; or EC 3.1.2.20.
  • Thioesterases acting on acyl-[acp] thioesters include EC 3.1.2.14, such as FatA, FatB; and EC 3.1.2.21.
  • the acid-thiol ligases or CoA synthtases can include an acid-thiol ligase in EC 6.2.1 , e.g., EC 6.2.1.3; EC 6.2.1.5; EC 6.2.1.14; or EC 6.2.1.23.
  • the CoA transferases can include a CoA transferase in EC 2.8.3, e.g., EC 2.8.3.12; EC 2.8.3.13 or EC 2.8.3.14, or or the gene product of ThnH that catalyses the reversible transfer of CoA to pimelic acid.
  • the acyl-[acp] -thioesterase include a thioesterase in EC 3.1.2, e.g. 3.1.2.14 and EC 3.1.2.21.
  • the acyl-[acp] synthetase can include an acid-thiol ligase in EC 6.2.1, e.g. EC 6.2.1.14, such as BioW; and EC 6.2.1.20.
  • the aminoacid aminotransferases can include an aminotransferase in EC 2.6.1, e.g., EC 2.6.1.39; EC 2.6.1.21 ; EC 2.6.1.42; and EC 2.6.1.67.
  • the deaminating dehydrogenase can include dehydrogenases from EC 1.4.1, e.g. 1.4.1.2; EC 1.4.1.3; EC 1.4.1.4; and EC 1.4.1.16.
  • the carbonyl reductases can include reductases in EC. l .1.1.184 EC 1.1.1.79, EC 1.1.1.B3; EC 1.1.1.B4 or 2-hydroxyglutaryl dehydogenases/alpha ketogluterate reductases from EC 1.1.99.2. or EC 1.1.99.6
  • the a-keto acid decarboxylase can include a-keto acid decarboxylases in EC 4.1.1, e.g. 4.1.1.1 ; 4.1.1.7; 4.1.1.72; or an acetolactate synthase in the class EC 2.2.1.6.
  • the a-amino acid decarboxylase can include EC 4.1.1.1 1; EC 4.1.1.15; EC 4.1.1.16; EC 4.1.1.18; EC 4.1.1.20, EC 4.1.1.45; and EC 4.1.1.86.
  • the fatty-acyl-CoA reductases can include a fatty-acyl-CoA reductase in EC 1.2.1, e.g EC 1.2.1.3; EC 1.2.1.10; EC 1.2.1.22; EC 1.2.1.50; and EC 1.2.1.76..
  • the acyl-[acp]- reductase can include acyl-acyl-carrier-protein reductase from e.g. EC 1.2.1.80.
  • the aldehyde dehydrogenases can include an aldehyde dehydrogenase in EC 1.2.1, e.g., EC 1.2.1.3; EC 1.2.1.4 or EC 1.2.1.63.
  • the aldehyde dehydrogenase/ carboxylic acid reductase may also be in EC 1.2.99, e.g., EC 1.2.99.6.
  • the carboxylic acid reductase may also belong to the NAD-dependent non-acylating aldehyde dehydrogenases, such as ThnG in Sphingomonas and Cupriavidus spp..
  • the aldehyde dehydrogenase/ carboxylic acid reductase may also belong to the NAD-dependent non-acylating aldehyde dehydrogenases, such as ThnG in Sphingomonas and Cupriavidus spp..
  • the aldehyde oxidase can be in EC 1.2.3, e.g. 1.2.3.1.
  • the amidohydrolase can be an amidohydrolase from EC 3.5.2, e.g. 3.5.2.11.
  • the hydro-lyases include a hydro-lyase in EC 4.2.1, e.g., EC 4.2.1.2; EC 4.2.1.59; 4.2.1.61; 4.2.1.17 or 4.2.1.18.
  • the dehydratase may also be 2-hydroxyglutaryl-CoA dehydratase described in clostrida and fusobacteria to which an EC number had not yet been assigned, that is expressed with its activator (HgdCAB) or a 2-hydroxyacyl_CoA dehydratase of anaerobic bacteria..
  • the enzymes catalyzing a-keto acid chain elongation can include one or more enzymes of the set including AksA, AksD, AksE and AksF enzymes.
  • the AksA can include an AksA enzyme in EC 2.3.3, e.g., EC 2.3.3.13 or 2.3.3.14.
  • the AksD can include an AksD enzyme in EC 4.2.1, e.g.,EC 4.2.1.36.
  • the AksF can include an AksF enzyme in EC 1.1.1, e.g., EC 1.1.1.87.
  • the alcohol dehydrogenases can include, e.g., EC. 1.1.1.1 or EC 1.1.1.2 or EC 1.1.1.21.
  • alcohol dehydrogenases can include, for example, adhA from Zymomonas mobiiis, adhB from Zymomonas mobilis, butanol dehydrogenase from Clostridium acetobutylicum, Saccharomyces ADHIV, and ADH6 from 5 * . cerevisiae.
  • the alcohol oxidase can be in class EC 1.1.3.13.
  • Another aspect of the present document is an isolated host cell comprising and expressing one or more exogenous nucleic acids encoding one or exogenous nucleic acids involved in the biosynthesis of one or more polymer monomers selected from 7 AHA; PA; 1,7 DAH; ENTL; and 7 HHA.
  • the enzymes include ammonia lyases, enoate reductases, carboxylic acid reductases,ro-transaminases, thioesterases, acid-thiol ligases or CoA synthtases, CoA transferases, acyl-[acp]thioesterases, acyl-[acp] synthetases, amino acid aminotransferases, deaminating dehydrogenases, carbonyl reductases, a-keto acid decarboxylases, acetolactate synthases, a-amino acid decarboxylases, fatty-acyl-CoA reductases, acyl-[acp]-reductases, aldehyde dehydrogenases, aldehyde oxidases, amidohydrolases, hydro-lyases, , and enzymes catalyzing a-keto acid chain elongation.
  • the cell and enzymes can be any of
  • FIG. 1 is a schematic showing the biotransformation of pimelic acid semialdehyde, to various difunctional C7 alkanes useful for nylon 7, nylon 7,7 and nylon 7,x production, as well as polyesters.
  • FIG. 2 is a schematic of the formation of pimelic acid semialdehyde from pimeloyl- CoA, pimeloyl-[acp], or a-ketosuberate, all of which are intermediates in naturally occurring pathways as indicated, and from tetralin degradation.
  • FIG. 3 is a schematic of the series of reactions in a-ketoacid chain elongation.
  • Three basic reactions are involved in chain elongation: a condensation between an a-ketodiacid and acetyl-CoA to form a citrate homolog, isomerization of the citrate homolog to an isocitrate homolog, and oxidative decarboxylation of the isocitrate homolog to an a-ketodiacid containing an additional methylene group [Howell98: Howell DM, Harich K, Xu H, White RH (1998).
  • FIG. 4 is a schematic of the formation of pimeloyl-CoA through condensation of three malonyl-CoAs.
  • FIG. 5 is a schematic of the formation of pimelic acid by oxidative cleavage of the [acp] thioesters of long chain fatty acids by Biol from Bacillus subtilis.
  • Pimelic acid, or its CoA or [acp] thioesters are precursors in 7-keto-8-aminopelargonate biosynthesis in biotin biosynthesis pathway II.
  • 7-keto-8-aminopelargonate is the key intermediate for biotin biosynthesis.
  • Degradation of pimeloyl-CoA in the host organism can be prevented by deletion or inactivation of BioF, the gene encoding 7-keto-8-aminopelargonic acid synthetase (E.C. 2.3.1.47).
  • FIG. 6 is a schematic of the formation of pimeloyl-[acp] or its methyl ester in 7-keto- 8-aminopelargonate biosynthesis in biotin biosynthesis pathway I.
  • Pimelic acid or pimelic acid semialdehyde can be prepared from these precursors by the action of aaaS (acyl-ACP synthetase), FatA (fatty acyl-ACP thioesterase A), FatB (fatty acyl-ACP thioesterase B), or acyl-[acp] reductase as shown in bold text.
  • aaaS acyl-ACP synthetase
  • FatA fatty acyl-ACP thioesterase A
  • FatB fatty acyl-ACP thioesterase B
  • acyl-[acp] reductase as shown in bold text.
  • FIG. 7 is a scheme showing formation of pimeloyl-CoA from benzoate or cyclohexane carboxylate degradation.
  • FIG. 8 is a scheme showing the formation of pimelic acid semialdehyde in the tetralin degradation pathway in Sphingomonas and Corynebacterium spp.
  • FIG. 9 is a scheme showing formation of pimeloyl-CoA in the cyclohexane carboxylate pathway from crotonate by methanogens. See Mouttaki et ah, Applied and Environmental Microbiology, pages 930-938 (2001).
  • FIG. 1 OA is a schematic showing examples of engineered pathways to pimelic acid semialdehyde and 7-aminoheptanoic acid from D,L-diaminopimelate as a starting point via 2- aminoheptanedioic acid.
  • a-keto-pimelate derived from 2-aminoheptanedioic acid can undergo one round of chain elongation to a-keto-suberate, which can either be decarboxylated to form pimelic acid semialdehyde, or converted to a-aminosuberate, which will lead directly to 7-aminoheptanoic acid upon decarboxylation
  • a-keto-pimelate also can be derived from a-keto-adipate, which in turn can be derived from a-keto-glutarate, through two successive rounds of a-keto-acid chain elongation.
  • FIG 10B is a schematic diagram showing the conversion of either a-hydroxypimelate or its CoA ester to 2-heptenedioic acid or its corresponding CoA ester, a-hydroxypimeloyl-CoA, respectively, by a hydro-lyase.
  • 2-heptenedioic acid or its corresponding CoA ester can be reduced to Pimelic acid or pimeoyl-CoA, respectively, by an enoate reductases that acts on the free acid or the CoA activated thioester.
  • the activation of a-hydroxypimelate or 2- heptenedioic acid may not be to the CoA thioester, but instead to the [acp] thioester by a acyl-[acp]- synthetase.
  • the hydro-lyase may catalyse the elimination of water from a-hydroxypimeloyl-[acp], and the enoate reductases may reduce the double bond of 2-heptenedioic acid-[acp].
  • Pimeloyl-CoA or pimeloyl-[acp] may subsequently be hydrolysed by a transferase or acid-thiol ligase, for recycling of CoA or [acp], or by a thioesterase to produce pimelic acid.
  • FIG. 1 1A shows an SDS-PAGE analysis on soluble and insoluble fractions from expression of the Citrobacter amalonaticus ammonia lyase protein.
  • FIG. 1 1B shows an SDS-PAGE analysis on soluble and insoluble fractions from expression of the Clostridium tetanomorphum (15) and Aspergillus oryzae (16) ammonia lyase proteins.
  • FIG. 12 shows an SDS-PAGE analysis on soluble and insoluble fractions from expression of the Nocardia sp. carboxylic acid reductase gene.
  • FIG. 13 shows the change in absorbance over time due to reduction of pimelic acid, adipic acid and benzoic acid to pimelic acid semialdehyde, adipic acid semialdehyde and benzaldehyde by Nocardia CAR reductase activity.
  • Benzoic acid was used as positive control and reaction mixtures with biocatalyst harbouring the empty vector as negative control.
  • FIG. 14 shows an SDS-PAGE analysis on soluble fraction and insoluble fraction from expression of the Haemophilus influenzae YciA thioesterase protein (18)
  • FIG. 15 shows an SDS-PAGE analysis of the aminotransferase IlvE-Omega Vf fusion
  • FIG. 16 shows Results of the biotransformation of sodium pyruvate and 7- aminoheptanoic acid with the measured concentrations of 7-aminoheptanoic acid substrate and L-alanine product.
  • FIG. 17 shows a Summary of the HPLC results from the L- and D-2-aminosuberic acid enantiomer concentrations in the six biotransformations after 48 and 96 hours.
  • FIG. 18 shows an SDS-PAGE analysis on soluble and insoluble fractions from the bead lysis of expression of the E.coli GadA (129), E.coli LysA (130), E.coli GadA ilvE (131) and E.coli LysA ilvE (132) proteins.
  • FIG. 19 shows the formation of 7-aminoheptanoic acid from the decarboxylation of 2- aminosuberate.
  • FIG. 20 shows protein alignment using the ClustalW algorithm showing significant homology between MJ0277 and comD (SEQ ID 41)/comE (SEQ ID 42) proteins
  • FIG. 21 shows homology of MJ0277 to LACLA acetolactate synthase from Lactococcus lactis (SEQ ID 43).
  • FIG. 22 shows an analysis of the functional domains in LACLA acetolactate synthase, comD/comE from Methanocella paludicola and the hypothetical acetolactate synthase MJ0277 showed that all of them have a similar structure containing a thiamine phosphate binding site.
  • FIG. 23 depicts a map of pI G2022 vector, containing the IlvE-Omega Vf fusion gene.
  • FIG. 24 depicts a map of pING2030 vector containing the IlvE-Ad optimized omega fusion gene.
  • FIG 25 sets forth the SEQ. ID 1-40
  • FIG. 26 depicts a bar graph of the formation of pimelic acid from pimeloyl-CoA by the acid thiol ligase (EC 6.2.1.5) of Thermococcus kodakaraensis (41/42), the CoA transferase (EC 2.8.3.12) of Acidominococcus fermentans (43/44) and the acid-thiol ligase (EC 6.2.1.14) from Pseudomonas mendocina (45).
  • FIG 27 depicts the formation of adipic acid from trans-2-hexenedioic (A) and the formation of adipoyl-CoA from trans-2,3-didehydroadipoyl-CoA by selected enoate reductases.
  • this disclosure relates to methods and materials for biosynthetically producing di- or trifunctional C7 alkanes that are useful as intermediates in the production of nylon-7, nylon-7,x and nylon-x,7, as well as the production of polyesters.
  • one or more e.g., two or more; three or more; four or more; five or more; six or more; seven or more; eight or more; nine or more; ten or more; eleven or more; twelve or more; thirteen or more; fourteen or more; fifteen or more; sixteen or more; seventeen or more; eighteen or more; nineteen or more; twenty or more; or even more
  • isolated enzymes e.g., an ammonia lyase, enoate reductase, aminotransferase, CoA transferase, thioestesterase, acid -thiol ligase, hydro-lyase, carbonyl reductase, thioesterase, carboxylic acid reductase, fatty-acyl CoA reductase, ⁇ -transaminase, a-keto acid decarboxylase, enzymes catalyzing a-keto acid chain elongation, acyl-ACP synthe.
  • a recombinant host that includes one or more (e.g., two or more; three or more; four or more; five or more; six or more; seven or more; eight or more; nine or more; ten or more; eleven or more; twelve or more; thirteen or more; fourteen or more; fifteen or more; sixteen or more; seventeen or more; eighteen or more; nineteen or more; twenty or more; or even more) exogenous nucleic acids encoding one or more (e.g., two or more; three or more; four or more; five or more; six or more; seven or more; eight or more; nine or more; ten or more; eleven or more; twelve or more; thirteen or more; fourteen or more; fifteen or more; sixteen or more; seventeen or more; eighteen or more; nineteen or more; twenty or more; or even more) enzymes (e.g., one or more (as above) of an ammonia lyase, enoate reductase, aminotransfer) enzymes (e.g.
  • nylon-7 is a polyamide produced by polymerization of the monomer 7-aminoheptanoic acid or by ring-opening condensation enantholactam. Nylon-7 also is known as “polyenanthamide” or “polyheptanamide.”
  • nylon-x,7 refers to the family of polyamides that are produced by the polymerization of heptane- 1,7-dioic acid (also known as pimelic acid or pimelate) with a diamine.
  • the integer x indicates the number of carbon atoms in the diamine with which the heptane- 1,7-dioic acid is reacted. In some embodiments, the integer x is an integer greater than 3, e.g., greater than 5, greater than 7, or greater than 9.
  • nylon-5,7 is produced by reacting heptane- 1,7-dioic acid and 1,5-pentanediamine.
  • Nylon 7,7 is produced by reacting heptane- 1,7-dioic acid and 1,7 diaminoheptane.
  • nylon-7,x refers to the family of polyamides that are produced by the polymerization reaction of heptamethylenediamine with a dicarboxylic acid.
  • the integer x indicates the number of carbon atoms in the dicarboxylic acid with which the heptamethylenediamine is reacted.
  • nylon-7,5 is produced by the reaction of heptamethylenediamine (also known as 1,7 -diaminoheptane or 1,7-heptanediamine) and pentane-l,5-dioic acid.
  • the integer x is an integer greater than 3, e.g., greater than 5, greater than 7 or greater than 9..
  • polyester refers to a category of polymers which contains an ester functional group in their main chain.
  • Polyesters can be homopolymers, for example polycaprolactone, that is produced by ring-opening condensation of caprolactone.
  • Copolymer polyesters are formed by the esterification condensation of polyfunctional alcohols and acids.
  • adipic acid and ethylene glycol are used to produce polyethylene adipate.
  • Pimelic acid can be used in the place of adipic acid in such copolymers.
  • 7- hydroxyheptanoic acid can be used to produce polyenantholactone.
  • enzyme refers to a protein catalyst capable of catalyzing a reaction.
  • the term does not mean only an isolated enzyme, but also includes a host cell expressing that enzyme. Accordingly, the conversion of A to B by enzyme C should also be construed to encompass the conversion of A to B by a host cell expressing enzyme C.
  • difunctional C7 alkane refers to straight or branched, saturated or unsaturated, hydrocarbon having seven carbon atoms and two functional groups.
  • the functional groups may be positioned at any point along the hydrocarbon chain. In some embodiments, the functional groups are located at the terminal ends of the hydrocarbon chain (e.g., one functional group at each terminus).
  • the term is also intended to encompass cyclic compounds such as enantholactam.
  • Exemplary difunctional C7 alkanes include, but are not limited to, pimeloyl-CoA, pimeloyl-[acp], heptane- 1,7-dioic acid (pimelic acid or pimelate), 7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine, 1,7-heptanediol, 7-aminoheptanol, 7-aminoheptanal, enantholactam, pimelic acid semialdehyde (also known as 7-oxoheptanoate), 2-heptenediacid, and 2-heptene diacid-CoA.
  • trifunctional C7 alkane refers to straight or branched, saturated or unsaturated, hydrocarbon having seven carbon atoms and three functional groups.
  • the functional groups may be positioned at any point along the hydrocarbon chain. In some embodiments, two of the three functional groups are located at the terminal ends of the hydrocarbon (e.g., one functional group at each terminus).
  • Exemplary trifunctional C7 alkanes include, but are not limited to, 2-oxopimelate (a-ketopimelate), 2-aminopimelate (a- aminopimelate), 2-hydroxypimelate (a-hydroxypimelate), and 2-hydroxypimelate-CoA (a- hydroxypimelate-CoA).
  • the term "functional group” refers to an amine group, a carboxylic acid group, an aldehyde group, an alcohol group, a co-enzyme A group or a keto group.
  • amine group refers to the radical -NH2.
  • carboxylic acid group refers to the radical -COOH.
  • aldehyde group refers to the radical -C(0)H.
  • alcohol group refers to the radical -OH.
  • keto group refers to the radical C(O).
  • co-enzyme A group refers to the organic cofactor, or prosthetic group (nonprotein portion of an enzyme), whose presence is required for the activity of many enzymes (the apoenzyme) to form an active enzyme system.
  • Coenzyme A functions in certain condensing enzymes, acts in acetyl or other acyl group transfer and in fatty acid synthesis and oxidation, pyruvate oxidation and in other acetylation.
  • acyl carrier protein refers to a protein (which typically is active in fatty acid synthesis) that picks up acetyl and malonyl groups from acetyl coenzyme A and malonyl coenzyme A and links them by condensation to form ⁇ -keto acid acyl carrier protein, releasing carbon dioxide and the sulfhydryl form of the acyl carrier protein.
  • the functional groups on a di- or trifunctional C7 alkane can be the same or different.
  • both functional groups can be amine groups, both functional groups can be carboxylic acid groups, or both functional groups can be alcohol groups.
  • one of the functional groups on a difunctional C7 alkane is an amine group and the other is a carboxylic acid group. In some embodiments, one of the functional groups on a difunctional C7 alkane is an alcohol group and the other is a carboxylic acid group. In some embodiments, one of the functional groups on a difunctional C7 alkane is an alcohol group and the other is an amine group.
  • heterologous is used herein to refer to any nucleic acid or polypeptide that is not derived from a cell of the same species as the host cell. Accordingly, as used herein, “homologous" nucleic acids or proteins are those that occur in, or are produced by, a cell of the same species as the host cell.
  • exogenous refers to any nucleic acid that does not occur in (and cannot be obtained from) that particular cell as found in nature.
  • a non-naturally-occurring nucleic acid is considered to be exogenous to a host cell once introduced into the host cell. It is important to note that non-naturally-occurring nucleic acids can contain nucleic acid subsequences or fragments of nucleic acid sequences that are found in nature provided that the nucleic acid as a whole does not exist in nature.
  • a nucleic acid molecule containing a genomic DNA sequence within an expression vector is non-naturally-occurring nucleic acid, and thus is exogenous to a host cell once introduced into the host cell, since that nucleic acid molecule as a whole (genomic DNA plus vector DNA) does not exist in nature.
  • any vector, autonomously replicating plasmid, or virus e.g., retrovirus, adenovirus, or herpes virus
  • retrovirus e.g., adenovirus, or herpes virus
  • genomic DNA fragments produced by PCR or restriction endonuclease treatment as well as cDNAs are considered to be non-naturally-occurring nucleic acid since they exist as separate molecules not found in nature. It also follows that any nucleic acid containing a promoter sequence and polypeptide-encoding sequence (e.g., cDNA or genomic DNA) in an arrangement not found in nature is non-naturally-occurring nucleic acid.
  • a nucleic acid that is naturally-occurring can be exogenous to a particular cell. For example, an entire chromosome isolated from a cell of yeast x is an exogenous nucleic acid with respect to a cell of yeast y once that chromosome is introduced into a cell of yeast y.
  • exogenous nucleic acids can be “homologous” or “heterologous” nucleic acids.
  • endogenous as used herein with reference to nucleic acids or genes (or proteins encoded by the nucleic acids or genes) and a particular cell refers to any nucleic acid or gene that does occur in (and can be obtained from) that particular cell as found in nature.
  • the term "recombinant host” refer to a host, the genome of which has been augmented by at least exogenous nucleic acid sequence.
  • nucleic acid sequences include, but are not limited to, nucleic acids that are not naturally present in the host (i.e,. a heterologous nucleic acid), DNA sequences that are not normally transcribed into RNA or translated into a protein ("expressed"), and other nucleic acid sequences which one desires to introduce into the non-recombinant host (e.g., a regulatory region to alter expression of a particular nucleic acid sequence).
  • the genome of a recombinant host described herein is augmented through the stable introduction of one or more exogenous nucleic acids.
  • the exogenous nucleic aicd is not originally resident in the host that is the recipient of the DNA (i.e., it is a heterologous nucleic acid), but it is within the scope of the invention to isolate a nucleic acid from a given host, and to subsequently introduce one or more additional copies of that nucleic acid into the same host, e.g., to enhance production of an encoded product or alter the expression pattern of a nucleic acid.
  • the exogenous nucleic acid will modify or even replace an endogenous gene or DNA sequence by, e.g., homologous recombination or site-directed mutagenesis. Modification or replacement of an endogenous gene can result in a deficiency in a particular encoded product, e.g, an enzyme.
  • Suitable recombinant hosts are described below. Recombinant hosts can also contain nucleic acids that are not incorporated into the genome of the host cell but exist (and preferably replicate) episomally in the cell.
  • a "promoter” refers to a DNA sequence that enables a gene to be transcribed.
  • the promoter is recognized by RNA polymerase, which then initiates transcription.
  • a promoter contains a DNA sequence that is either bound directly by, or is involved in the recruitment, of RNA polymerase.
  • a promoter sequence can also include "enhancer regions,” which are one or more regions of DNA that can be bound with proteins (namely, the trans-acting factors, much like a set of transcription factors) to enhance transcription levels of genes (hence the name) in a gene-cluster.
  • An enhancer while typically at the 5' end of a coding region, can also be separate from a promoter sequence and can be, e.g., within an intronic region of a gene or 3 ' to the coding region of the gene.
  • operably linked means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest.
  • feedstocks can be used to produce di- or trifunctional alkanes.
  • a recombinant host cell, or a lysate prepared from a cell is contacted with a renewable feedstock to produce a di- or trifunctional C7 alkane, such as 7-aminoheptanoic acid.
  • renewable feedstocks which may be used as a carbon source include, but are not limited to, cellulosic feedstocks, plant-derived carbohydrates and fatty acids.
  • the feedstock with which the recombinant host cell is contacted is glucose, sucrose, xylose, fatty acids or glycerol.
  • the recombinant host cell can be modified for growth on synthesis gas, also known as SynGas, as a source of carbon.
  • synthesis gas also known as SynGas
  • the recombinant host cells of the invention are engineered to provide an efficient metabolic pathway for utilization of SynGas, or other gaseous carbon sources, to produce di- or trifunctional C7 alkanes.
  • recombinant hosts described herein, or a lysate prepared from a recombinant host cell can be contacted with an aromatic hydrocarbon feedstock.
  • aromatic hydrocarbon feedstocks rather than renewable feedstocks, provides a way to convert such petroleum derived materials to compounds that are more benign to the environment, reducing environmental impact.
  • suitable aromatic hydrocarbon feedstocks include, but are not limited to, toluene, benzene, benzoic acid and shikimate.
  • embodiments of the present invention relate to methods for producing di- or trifunctional C7 alkanes in the presence of one or more isolated enzymes in the presence of a recombinant host cell expressing that/those enzyme(s), or in the presence of a cell lysate (or a partially purified lysate) of cell (e.g., recombinant cell) that expresses the enzyme(s).
  • the production of the di- or trifunctional C7 alkanes starts with pimelate or pimeloyl-CoA or pimeloyl-[acp] and proceeds through a common intermediate, namely, pimelic acid semialdehyde.
  • the invention relates to a method of producing the difunctional C7 alkane 7-aminoheptanoic acid by using an enzyme that catalyzes the semialdehyde amination of pimelic acid semialdehyde to 7-aminoheptanoic acid. See FIG. 1.
  • a semialdehyde aminotransferase such as a ⁇ - transanimase can be used.
  • Exemplary ⁇ -transaminases include, but are not limited to transaminases classified under EC 2.6.1 such as EC 2.6.1.18; EC 2.6.1.19; EC 2.6.1.1; EC 2.6.1.13;; EC 2.6.1.48; and EC 2.6.1.62.
  • the aminotransferase is ⁇ - alanine aminotransferase classified in EC 2.6.1.18 from V. fluvialis, B. weihenstephanensis, P. aureginosa, B. subtilis, or P. syringae. See WO201 1/031 147, which is incorporated herein in its entirety.
  • 7-aminoheptanal can be produced using an enzyme that catalyzes the aldehyde dehydrogenation of 7-aminoheptanoic acid to 7-aminoheptanal. See, FIG. 1.
  • An aldehyde dehydrogenases classified under EC 1.2.1, such as EC 1.2.1.4 or EC 1.2.1.63, can be used to produce 7-aminoheptanal from 7-aminoheptanoic acid.
  • the aldehyde dehydrogenase/ carboxylic acid reductase may also be in EC 1.2.99, e.g., EC 1.2.99.6.
  • the carboxylic acid reductase may also belong to the NAD-dependent non-acylating aldehyde dehydrogenases, such as ThnG in Sphingomonas and Cupriavidus spp..
  • heptamethylenediamine also known as 1,7-diaminoheptane
  • Suitable aminotransferases include, but are not limited to, aminotransferases in EC 2.6.1 such as EC 2.6.1.18; EC 2.6.1.19; EC 2.6.1.1 1; EC 2.6.1.13; EC 2.6.1.39EC 2.6.1.48; and EC 2.6.1.62.
  • the aminotransferase is ⁇ - alanine aminotransferase classified in EC 2.6.1.18 from V. fluvialis, B. weihenstephanensis, P. aureginosa, B. subtilis, or P. syringae. See WO201 1/031 147, which is incorporated herein in its entirety.
  • enantholactam can be produced from 7-aminoheptanoic acid using an enzyme that catalyzes the amide hydrolysis of 7-aminoheptanoic acid. See, FIG. 1.
  • Suitable amidohydrolases include amidotransferases classified under EC 3.5.2 such as EC 3.5.2.12 or EC 3.5.2.1 lean be used.
  • 7-hydroxyheptanoate is produced from pimelic acid semialdehyde or 7-aminoheptanol is produced from 7-aminoheptanal using an enzyme that catalyzes the reduction of an aldehyde. See FIG. 1.
  • an alcohol dehydrogenase classified under EC 1.1.1 such as EC 1.1.1.2 or EC 1.1.1.2 lean be used.
  • 1,7 heptanediol can be produced from 7-hydroxyheptanoate using an aldehyde dehydrogenase and an alcohol dehydrogenase.
  • a suitable aldehyde dehydrogenase can be classified under EC 1.2.1 (e.g., EC 1.2.1.4 or EC 1.2.1.63 or EC 1.1.1.21). See, FIG. 1.
  • a suitable alcohol dehydrogenase can be classified under E.C. 1.1.1. such as EC 1.1.1.1 or EC 1.1.1.2.
  • Suitable aldehyde dehydrogenases include EC 1.2.1.3 : EC 1.2.1.4; and EC 1.2.1.63.
  • the aldehyde dehydrogenase/ carboxylic acid reductase may also be in EC 1.2.99, e.g., EC 1.2.99.6.
  • the aldehyde dehydrogenase/ carboxylic acid reductase may also belong to the NAD-dependent non-acylating aldehyde dehydrogenases, such as ThnG in Sphingomonas and Cupriavidus spp..
  • aldehyde oxidase may be used, such as an aldehyde oxidase from EC 1.2.3.1.
  • the conversion to the toxic compound can be performed in vitro using isolated enzymes or a lysate from the recombinant host.
  • the toxic compound is produced by the host and then subsequently converted (using isolated enzymes, a recombinant host expressing an enzyme, or chemically converted) to enantholactam.
  • Pimelate and/or pimeloyl-CoA can be obtained via a number of different ways including: (i) a-ketosuberate produced from the a-keto acid chain elongation pathway, or (ii) the biotin biosynthesis pathway I; (iii) biotin biosynthesis pathway II; (iv) the condensation of three malonyl-CoA molecules into a single pimeloyl-CoA molecule; (v) the benzoate degradation pathway; (vi) cyclohexane carboxylate pathway, (vi) D,L-diaminopimelate pathway; and (vii) a biosynthetic pathway where the starting carbon source is crotonate. See FIG. 2.
  • pimelate can be produced from pimeloyl-CoA using an enzyme that catalyzes the hydrolysis of a thioester.
  • enzymes that can hydrolyze a thioester include, but are not limited to, thioesterases, acid-thiol ligases, and CoA transferases.
  • Suitable thioesterases include thioesterases classified in EC 3.1.2 such as such as the gene product of YciA, tesB or Acotl3; EC 3.1.2.2; EC 3.1.2.18; EC 3.1.2.19; or EC 3.1.2.20.
  • Thioesterases acting on acyl-[acp] thioesters include EC 3.1.2.14, such as FatA, FatB; and EC 3.1.2.21.
  • one of the following enzymes can be used: a thioester hydrolases/acyl-CoA thioesterase classified in EC 3.1.2 such as an ADP-dependent medium-chain-acyl-CoA hydrolase in EC 3.1.2.19 and 3.1.2.18, an acyl- CoA hydrolase in EC 3.1.2.20; or the gene product of YciA, tesB or Acotl3; oleoyl-[acyl-carrier- protein] hydrolases in EC 3.1.2.14. .
  • Suitable acid-thiol ligases include acid-thiol ligases classified in EC 6.2.1 such as EC 6.2.1.3; EC 6.2.1.14; and EC 6.2.1.23.
  • Suitable CoA transferases include CoA transferases classified under EC 2.8.3 such as EC 2.8.3.6, EC 2.8.3.8, EC 2.8.3.12; EC 2.8.3.13 or EC 2.8.3.14 , or the gene product of ThnH that catalyses the reversible transfer of CoA to pimelic acid.
  • Heptane- 1, 7 -dioic acid can be obtained from pimeloyl-[acp] by the use of enzymes acting on [acp] -thioesters, such as acyl-[acp]thioesterases in EC 3.1.2.14, such as fatA and fatB or EC 3.1.2.21.
  • enzymes acting on [acp] -thioesters such as acyl-[acp]thioesterases in EC 3.1.2.14, such as fatA and fatB or EC 3.1.2.21.
  • acid-thiol ligases acting on [acp] thioesters can be used, such as EC 6.2.1.14 and EC 6.2.1.20.
  • pimelic acid can be obtained form pimeloyl-[acp]methyl ester by hydrolysis of the ester bond by AaasaS (acyl-ACP synthetase), followed by removal of the methyl group by a lipase/esterase.
  • AaasaS acyl-ACP synthetase
  • Pimelate can be transformed to pimelic acid semialdehyde using an enzyme that catalyzes the reduction of a carboxylic acid.
  • Suitable reductases include, but are not limited to, carboxylic acid reductases classified in EC 1.2.99;
  • the carboxylic acid reductase may also belong to the NAD-dependent non-acylating aldehyde dehydrogenases, such as ThnG in Sphingomonas and Cupriavidus spp..or fatty acyl-CoA reductases classified in EC 1.2.1 such as EC 1.2.1.3; EC 1.2.1.10; EC 1.2.1.22; EC 1.2.1.50; ECl.2.1.57; and EC 1.2.1.76.
  • a fatty-acyl-CoA reductase can directly convert pimeloyl-CoA or pimeloyl- [acp] to pimelic acid semialdehyde.
  • Exemplary carbonyl reductases in EC 1.2.99 include, but are not limited to EC 1.2.99.6 from Norcardia sp. (Aimin et ah, Appl. Environ. Microbiol. 70: 1874-1881 (2004), incorporated by reference) or S. griseus (Suzuki et al, J. Antibiot. (Tokyo) 60: 380-387 (2007) incorporated by reference).
  • Exemplary fatty-acyl-CoA reductases in EC 1.2.1 include, but are not limited to EC 1.2.1.75 from, e.g., C. aurantiacus (Hugler, M, et al, J. Bacteriology 184: 2404-2410 (2002), incorporated by reference), M. sedula (Kockelkorn, D. and Fuchs, G., J. Bacteriology 191: 6352-6362 (2009), incorporated by reference), or S. tokodai (Alber, B. et al, J. Bacteriology 188: 8551-8559 (2006), incorporated by reference); and 1.2.1.50 from, e.g., Acinetobacter sp., A.
  • platyrhynchos Ishige, T., et al, Appl. Envtl Microbiology 68: 1192-1195 (2002), incorporated by reference
  • A. thaliana Doan, T.T., et al, Journal Plant Physiology 166: 787-796 (2009) and Hooks, M.A., et al, Plant J. 20: 1-13 (1999) 3 both of which are incorporated by reference
  • Homo sapiens McAndrew, R.P. et al, J. Biol Chem. 283: 9435-9443 (2008), incorporated by reference
  • M. Musculis P. leiognathi, P. phosphoreum, P. sativum, or 5 * .
  • acyl-[acp]reductases in EC 1.2.1 include EC 1.2.1.80, from e.g. Synecoccus elongates (Shirmer, A. et al, (2010). Microbila biosynthesis of alkanes. Science, 329 (5991 : 559-562)
  • synthase/AksA alpha-isopropylmalate synthase
  • EC 4.2.1.114 homoaconitase or AksD/AksE, which catalyzes the dehydration reactions of (R)-homocitrate, dihomocitrate and trihomocitrate, as well as the hydration reactions of cis-homoaconitate, cis-(homo) 2 aconitate and cis- (homo) 3 aconitate
  • EC 1.1.1.87 homoisocitrate dehydrogenase or AksF (multifunctional 3-isopropylmalate dehydrogenase/D-malate dehydrogenase ⁇ which catalyzes the NAD(P)+ dependent oxidative decarboxylation of homoisocitrate, threo-iso(homo) 2
  • Exemplary AksA enzymes include, but are not limited to, those in EC 2.3.3 such as EC 2.3.13 or 2.3.3.14.
  • Exemplary AksD enzymes include those in EC 4.2.1 such as EC 4.2.1.36.
  • Exemplary AksF enzymes include, but are not limited to enzymes in EC 1.1.1 such as EC 1.1.1.87.
  • the chain elongation enzyme is AksA MTH1630, AksD MTH1631, AksE MTH0829 or AksF MTH1388. See, e.g., WO2010/104391, which is incorporated by reference herein.
  • malonyl-CoA can be used as the source of pimeloyl-CoA as certain organisms are able to condense three malonyl-CoA molecules into a single pimeloyl- CoA molecule. See FIG. 4. See also Lin, S. and Cronan, J.E., Molecular Biosystems 7: 181 1- 21 (201 1), which is incorporated by reference herein. Accordingly, a recombinant host can be produced in which pimeloyl-CoA is produced from malonyl-CoA. In some embodiments, the host cell is an organism other than Achromobacter. In some embodiments, the pimeloyl-CoA may be subsequently converted to pimelic acid or other difunctional C7 alkanes.
  • a recombinant host cell produces pimelic acid and/or pimeloyl- [acp] or pimeloyl-CoA from acetyl-CoA derived from glycerol and/or fatty acids. See, e.g., Lin, S., Nature Chem. Biol. 6: 682-688 (2010); and Cronan, J.E. and Lin, S., Current Opinion in Chem. Biology 15: 1-7 (201 1), both of which are incorporated by reference herein.
  • FIG. 5 shows formation of pimeloyl-[acp] through oxidative cleavage of long chain fatty acid-[acp] thioesters by Biol in Bacillus subtilis that is converted to pimelic acid or pimeloyl-CoA which are precursors in biotin biosynthesis II (gram positive bacteria).
  • FIG. 6 shows formation of pimelyl-[acp] or its methyl esters in biotin biosynthesis I (gram negative bacteria).
  • FIG. 6 also shows pathways for the conversion of pimeloyl-[acp] methyl ester into pimelate monomethyl ester.
  • the pimelate monomethyl ester is subsequently converted to pimelic acid (heptane- 1,7-dioic acid) in the presence of, e.g., a lipase in EC 3.1.1.
  • FIG. 6 also shows the conversion of pimeloyl-[acp] methyl ester into pimeloyl-[acp] in the presence of an esterase (e.g., an enzyme in EC 3.1.1, such as EC 3.1.1.85, BioH).
  • an esterase e.g., an enzyme in EC 3.1.1, such as EC 3.1.1.85, BioH.
  • the pimeloyl-[acp] is subsequently converted into pimelic acid (heptane- 1,7-dioic acid) in the presence of a acyl- [acp]-thioesterase, e.g., an enzyme in EC 3.1.2.14, such as FatA or FatB.
  • a acyl- [acp]-thioesterase e.g., an enzyme in EC 3.1.2.14, such as FatA or FatB.
  • the invention contemplates a recombinant host cell capable of producing pimelic acid (heptane- 1,7-dioic acid) that comprises one or all of the enzymes shown in FIG. 6.
  • the recombinant host cell does has a deficiency in 7-keto-8- aminopelargonic acid synthetase, an enzyme capable of converting 6-carboxyhexanoyl-CoA (pimeloyl-CoA) into 7-keto-8-aminopelargonic acid (KAPA), and encoded by the BioF gene in host organisms with a native biotin biosynthesis pathway so that the pimeloyl-CoA cannot be shuttled into biotin biosynthesis.
  • an enzyme capable of converting 6-carboxyhexanoyl-CoA pimeloyl-CoA
  • KAPA 7-keto-8-aminopelargonic acid
  • Pimeloyl-[acp] methyl ester can also be produced as a result of the metabolism of long-chain acyl-[acp] and/or free fatty acids by Biol (for example Biol from Bacillus subtilis).
  • Pimelic acid and/or pimeloyl-CoA can also be obtained from eukaryotic biotin biosynthesis pathways. See, e.g., oje, S., Phytochemistry 68: 1904-1921 (2007); and Charles R. Hall, The Contribution of Horizontal Gene Transfer to the Evolution of Fungi (May 10, 2007) (unpublished Ph.D. thesis, Duke University) (on file with Duke University Libraries), both of which are incorporated by reference herein.
  • acetyl-CoA is biotransformed to acetoacetyl CoA using an enzyme classified under EC 2.3.1.9; acetoacetyl-CoA is biotransformed into (S)-3-hydroxybutanoyl-CoA using an enzyme classified under E.C.
  • (S)-3-hydroxybutanoyl-CoA is biotransfored to crotonyl-CoA using an enzyme classified under EC 4.2.1.17; crotonyl-CoA is biotransformed into glutaconyl-l-CoA using an enzyme classified under EC 4.1.1.70; glutaconyl-l-CoA is biotransformed to glutaryl-CoA using an enzyme classified under EC 1.3.99.7; glutaryl-CoA is biotranformed into 3-ketopimelyl-CoA using an enzyme classified under EC 2.3.1.43; 3- ketopimelyl-CoA is biotransfomred into 3-hydroxypimelyl-CoA using an enzyme classified under EC 1.1.1.4259; 3-hydroxypimelyl-CoA is biotransformed into 2,3-didehydro-pimeloyl- CoA using an enzyme classified under EC 4.2.1.-, and 2,3-didehydro-pimeloyl-CoA is biotransformed into pi
  • the invention provides a recombinant host cell comprising the enzymes for converting acetyl CoA into heptane- 1,7-dioic acid.
  • the recombinant host cell has a deficiency in 7-keto-8-aminopelargonic acid synthetase, an enzyme capable of converting 6-carboxyhexanoyl-CoA (pimeloyl-CoA) into 7-keto-8-aminopelargonic acid (KAPA), and encoded by the BioF gene in the host, if the host organisms has a native biotin biosynthesis pathway, so that the pimeloyl-CoA or pimeloyl- [acp] cannot be shuttled into biotin biosynthesis.
  • pimeloyl-CoA 6-carboxyhexanoyl-CoA
  • KAPA 7-keto-8-aminopelargonic acid
  • Biol is overexpressed to increase the pimeloyl-[acp] formed via oxidative cleavage of fatty acid- [acp] esters.
  • [acp]-transferases/synthetases activating long chain fatty acids to their corresponding [acp]-thioesters for cleravage by Biol is overexpressed in the host cell.
  • the embodiments of the invention also provide a method of producing heptane- 1,7- dioic acid comprising the use of such recombinant host cells.
  • pimeloyl-CoA and/or pimelic acid contemplated herein is the benzoate degradation pathway shown in FIG. 7. See, e.g., Bernsteinb, J.R., et al, Metabolic Eng'g 10: 131-140 (2008); Harwood, C.S., et al, FEMS Microbiology Reviews 22: 439-458 (1999); and Harrison, F.H. and Harwood, C.S., Microbiology 151: 727-736 (2005), all of which are incorporated by reference herein. A number of transformations are illustrated in FIG.
  • cyclohexa-l,5-dienecarbonyl CoA is biotransformed to 6-hydroxycyclohex-l-ene-l- carboxyl-CoA using an enzyme classified under EC 4.2.1.100; 6-hydroxycyclohex-l-ene-l- carboxyl-CoA is biotransformed to 6-ketoxycyclohex-l-ene-l-carboxyl-CoA using an enzyme classified under EC 1.1.1-; 6-ketoxycyclohex-l-ene-l-carboxyl-CoA is biotransformed to 3-hydroxypimelyl-CoA using an enzyme classified under EC 3.7.1.-; 3- hydroxypimelyl-CoA is biotransformed to 6-carboxylhex-2-enoyl-CoA using an enzyme classified under EC 4.2.1.-; 3-hydroxypimelyl-CoA is biotransformed to pomelyol-CoA using an enzyme classified under EC 1.3.1.62.
  • the pimeloyl-CoA is converted into pimelic acid (heptane- 1,7-dioic acid).
  • cyclohexa-l,5-dienecarbonyl CoA is biotransformed to cyclohex-l-ene-l-carboxyl-CoA
  • cyclohex- 1 -ene- 1 -carboxyl-CoA is biotransformed to 2-hydroxycyclohexane-l-carboxyl CoA using an enzyme classified under EC 4.2.1.-
  • 2-hydroxycyclohexane-l-carboxyl CoA is biotransformed to 2- ketocyclohexane-l-carboxyl-CoA using an enzyme classified under E.C.
  • 2- ketocyclohexanecarboxyl-CoA is biotransformed to pimeloyl-CoA using an enzyme classified under EC 3.1.2.-_(e.g., a -ketocyclohexanecarboxyl-CoA hydrolase Rp-badl, an enzyme classified under EC 3.1.2.-).
  • the recombinant host cell capable of producing heptane-1,7- dioic acid includes one or more of the enzymes listed in FIG. 7 or in this section.
  • D,L diaminopimelate also known as 2,6- diaminopimelate.
  • D,L-diaminopimelate is an intermediate in the production of lysine.
  • an endogenous lysine pathway can be modified to preferentially produce pimelate by creating recombinant host cells that have a deficiency in diaminopimilate decarboxylase., See, e.g., FIG. 10.
  • the D,L diaminopimelate can be biotransformed into an unsaturated monoaminopimelic acid by an enzyme that catalyzes the reductive deamination of D,L diaminopimelate to 6-amino-2-heptenedioic acid.
  • enzymes that catalyze reductive deamination of D,L diaminopimilate include ammonia lyases classified in EC 4.3.1 such as EC 4.3.1.1; EC 4.3.1.2; EC 4.3.1.3; EC 4.3.1.9; EC 4.3.1.12; EC 4.3.1.13; EC 4.3.1.14; EC 4.3.1.23; and EC 4.3.1.24.
  • the ammonia lyase is a methyl aspartate ammonia lyase classified under EC 4.3.1.2 from C. amalonaticu , C. tetanomorphum or A. oryzae. See, e.g., Botting, Biochemistry 27: 2953-2955 (1988); and Kato, Appl. Microbiol. Biotechnol. 50: 468-474 (1998), the entireties of which are incorporated by reference herein.
  • 6-amino-2-heptenedioic acid can be biotransformed into 2-amino-2-heptenedioic acid using an enzyme that catalyzes the enoate reduction of 6-amino-2-heptenedioic acid to 2-amino-2-heptanedioic acid (also known as 2-aminopimelic acid or a-aminopimelate).
  • an enzyme that catalyzes the enoate reduction of 6-amino-2-heptenedioic acid to 2-amino-2-heptanedioic acid (also known as 2-aminopimelic acid or a-aminopimelate).
  • Examples of enzymes that catalyze the enoate reduction of 6-amino-2-heptenedioic acid include enoate reductases classified in EC 1.3.1 such as EC 1.3.1.8; EC 1.3.1.9; EC 1.3.1.10, EC 1.3.1.31 ; EC 1.3.1.38; EC 1.3.1.39; EC 1.3.1.44 or pimeloyl-CoA dehydrogenase in EC 1.3.1.62 such as PimC/PimD or ThnJ/ThnK.
  • the enoate reductase may also be in the EC 1.3, such as EC 1.3.8.1 or EC 1.3.99.3; EC 1.3.99.B10 or Syntrophus aciditrophicus 2,3- didehydropimelyl-CoA reductase and homologs thereof...
  • the enoate reductase is YqjM or OPR1/3 in EC 1.3.1.31 from B. subtilis or L. esculentum, respectively. See, e.g., Stueckler, Org. Lett. 9: 5409-5411 (2007); Kitzing, J. Biol. Chem. 280: 27904- 27913 (2005); Hall, Angew. Chem. Int. Ed. 46: 3934-3937 (2007); and Breitmaschine, Proc. Natl. Acad. Sci USA 103: 14337-14342 (2006), the entireties of which are incorporated by reference herein.
  • the 2-aminopimelic acid can be converted to the corresponding 2-heptenedioic acid (also known as 2,3-didehydropimelic acid) by an enzyme that catalyzes the reductive deamination of 2-aminopimelic acid.
  • enzymes that catalyze reductive deamination of 2-aminopimelic acid include ammonia lyases classified in EC 4.3.1 such as EC 4.3.1.1; EC 4.3.1.2; EC 4.3.1.3; EC 4.3.1.9; EC 4.3.1.12; EC 4.3.1.13; EC 4.3.1.14; EC 4.3.1.23; or EC 4.3.1.24 as described above.
  • the 2-heptene dioic acid can be transformed to 2-heptene dioic acid-CoA using a CoA transferase (e.g,. a CoA transferase classified in EC2.8.3 such EC 2.8.3.12; EC 2.8.3.13 or EC 2.8.3.14 or or the gene product of ThnH that catalyses the reversible transfer of CoA to pimelic acid) and an acid thiol ligase (e.g., an acid-thiol ligases classified n EC 6.2.1 such as EC 6.2.1.3; EC 6.2.1.5; EC 6.2.1.14; or EC 6.2.1.23).
  • a CoA transferase e.g,. a CoA transferase classified in EC2.8.3 such EC 2.8.3.12; EC 2.8.3.13 or EC 2.8.3.14 or or the gene product of ThnH that catalyses the reversible transfer of CoA to pimel
  • 2-heptene dioic acid-CoA is then converted to pimeloyl-CoA by an enzyme that catalyzes the enoate reduction of 2-heptene dioic acid such as an enoate reductase enzyme classified in in EC 1.3.1.
  • Pimeloyl-CoA can be converted to pimelate or pimelic acid semialdehyde as described above.
  • a keto pimelate also can undergo one round of chain elongation to a-keto-suberate using chain elongation enzymes, such as those present in methanogenic bacteria (e.g., Methanobacterium autotrophicum).
  • chain elongation enzymes include, but are not limited to AksA, AksD, and AksE or F as discussed above.
  • Exemplary AksA enzymes include, but are not limited to, those in EC 2.3.3.
  • Exemplary enzymes in EC 2.3.3 include, but are not limited to EC 2.3.13 or 2.3.3.14.
  • Exemplary AksD enzymes include those in EC 4.2.1.
  • An exemplary enzyme in EC 4.2.1 includes, but is not limited to EC 4.2.1.36.
  • Exemplary AksF enzymes include, but are not limited to enzymes in EC 1.1.1.
  • An exemplary enzyme in EC 1.1.1 includes, but is not limited to EC 1.1.1.87.
  • the fourth set of enzymes comprises a decarboxylase.
  • Exemplary decarboxylases include those in EC 4.1.1.
  • Exemplary decarboxylases in EC 4.1.1 include, but are not limited to EC 4.1.1.11 ; EC 4.1.1.15; EC 4.1.1.17; EC 4.1.1.18; EC 4.1.1.19; EC 4.1.1.20 and EC 4.1.1.86.
  • acetyl-CoA can be fed into the tricarboxylic acid (TCA) cycle, in which acetyl-CoA is transformed into succinic acid (butan-l,4-dioic acid).
  • TCA tricarboxylic acid
  • the succinic acid is then diverted from the TCA cycle into a pathway for synthesizing pimelic acid starting with the biotransformation of 2-oxoglutarate into 2-hydroxy-l,2,4-butanetricarboxylic acid (homocitrate) (e.g., by an enzyme in EC 2.3.3.14); biotransformation of homocitrate to homo cis aconitase and homo cis aconitase to homoisocitrate using an enzyme classified under EC 4.2.1.36; biotransformation of homoisocitrate to oxaloglutarate using an enzyme classified under EC 1.1.1.87; biotransformation of oxaloglutarate to 2-oxoadipate using an enzyme classified under EC 1.1.1.87, and biotransformation of 2-oxoadipate to glutaryl-CoA using an enzyme classified under EC 1.2.4.2.
  • Glutaryl-CoA can be converted to pomeloyl-CoA as discussed above. The homocitrate is
  • the a-keto-suberate can either be decarboxylated by a a-keto acid decarboxylase to form pimelic acid semialdehyde, or converted to a-aminosuberate, using an amino acid aminotransferase which will lead directly to 7-aminoheptanoic acid upon decarboxylation by an a-amino acid decarboxylase.
  • the pimelic acid semialdehyde can be converted to 7-aminoheptanoic acid using a ⁇ -aminotransferase.
  • Exemplary a-keto acid decarboxylases include those in EC 4.1.1 such as EC 4.1.1.1; EC 4.1.1.7; and EC 4.1.1.72 or an acetolactate synthase in the class EC 2.2.1.6.
  • Exemplary a-amino acid decarboxylase include those in EC 4.1.1.1 1; EC 4.1.1.15; EC 4.1.1.17; EC 4.1.1.18; EC 4.1.1.19; EC 4.1.1.20; EC 4.1.1.45 and EC 4.1.1.86.
  • the decarboxylase comprises ketoisovalerate decarboxylase kivD in EC 4.1.1.1 from L. lactis; benzoylformate decarboxylase mdIC A460I or BFD in EC4.1.1.7 from P. putida; pyruvate decaroxylase isozymes 1 and 2 Pdcl in EC 4.1.1.1 from S.
  • exemplary amino acid aminotransferases include those in EC 2.6.1, such as EC 2.6.1.21; EC 2.6.1.39; EC 2.6.1.42; and EC 2.6.1.67.
  • Exemplary ⁇ -aminotransferases include those in EC 2.6.1, such as EC 2.6.1.1 1; EC 2.6.1.13; EC 2.6.1.18; EC 2.6.1.19; EC 2.6.1.48; and EC 2.6.1.62.
  • a keto pimelate can be contacted with one or more enzymes that catalyze the ketone reduction to a hydroxypimelate (e.g., a carbonyl reductase such as EC 1.1.1.184), which in turn can be converted to a -hydroxypimelate CoA using an enzyme that catalyzes the transfer of CoA (e.g., a CoA transferase described above).
  • a - hydroxypimelate CoA can be converted to 2-heptene dioic acid CoA with an enzyme that catalyzes the dehydration of the a-hydroxypimelate-CoA (e.g., a hydro-lyase classified under EC 4.2.1.
  • the 2-heptene dioic acid CoA can be converted to pimelic CoA using an enzyme that catalyzes the enoate reduction of 2-heptene dioic acid CoA. Suitable enoate reductases are described above.
  • the invention provides a recombinant host cell comprising one or more enzymes necessary to convert D,L-diaminopimelate preferentially to pimelate.
  • a source of pimeloyl-CoA, and ultimately a source of pimelate is a biosynthetic pathway where the starting carbon source is crotonate. That biosynthetic pathway is illustrated in FIG. 9. See, e.g., Mouttaki, FL, et ah, Applied Envtl. Microbiology 73: 930-938 (2001). The crotonate is broken down to acetyl-CoA. It has been reported that two thirds of the acetyl-CoA produced in the biosynthetic pathway shown in FIG. 9 is transformed into acetate.
  • the invention provides a recombinant host cell that is deficient in the enzymes that would convert acetyl-CoA into acetate (e.g., a phosphate acetyltransferase and acetate kinase such that the acetyl-CoA is instead converted to acetoacetyl-CoA and subsequently to glutaconyl-CoA in a number of metabolic steps as shown in FIG. 9.
  • the glutaconyl-CoA is subsequently converted to glutaryl-CoA.
  • the glutaryl-CoA is chain-extended to form 3-oxopimelyl-CoA. Following a reduction, dehydration, and a second reduction, pimelyl-CoA is obtained.
  • the invention provides a recombinant host cell in which the genes encoding the enzymes that would convert pimeloyl-CoA into cyclohex-1- ene-l-carboxyl-CoA and ultimately into cyclohexane carboxylate, are are "knocked out", such that the metabolic pathway ends at pimeloyl-CoA.
  • pimelic acid semialdehyde is produced using the tetralin (benzocyclohexane) degradation pathway from Sphingomonas and Corynebacterium spp. 7- oxoheptanoic acid is produced as an intermediate is the tetralin degradation pathway in Sphingomonas macrogolitabid. Lopez-Sanchez, A., et al, Appl. Environ. Microbiol. 76: 110- 118 (2010). Tetralin is metabolized by cleavage of both aromatic rings yielding pyruvate and pimelic acid semialdehyde. Tetralin, an organic solvent, is a complex starting material that is derived from naphthalene.
  • Naphthalene is currently produced from coal tar, but biosynthetic pathways for naphthalene synthesis are known to occur in termites, fungi and some types of plants. Chen, J. et al, Nature 392: 558-559 (1998); Daisy, B.H., et al, Microbiology 148: 3737-3741 (2002); and Azuma, H., et al, Phytochemistry 42: 999-1004 (1996).
  • Pimelic acid semialdehyde can be converted to 7-amino heptanoic acid or 7-hydroxyheptanoic acid as discussed above.
  • Some embodiments of the invention also provides a method of producing hexamethylenediamine from 2-oxopimelate comprising using recombinant host cells.
  • 2- oxopimelate is a known intermediate in the Coenzyme B biosynthesis pathway in methanogenic Archae.
  • the invention provides a recombinant host cell comprising: an enzyme capable of converting 2-oxopimelate into 2-aminopimelate (e.g., an aminotransferase enzyme in EC 2.6.1.67), an enzyme capable of converting 2-aminopimelate to 2-amino-7-oxoheptanoate (e.g., a reductase enzyme in EC 1.4.1), an enzyme capable of converting 2-amino-7- oxoheptanoate to 2,7-diaminoheptanoate (e.g., a 1 -aminotransferase enzyme in EC 2.6.1), and an enzyme capable of converting 2,7-diaminoheptanoate to hexamethylenediamine (e.g., a decarboxylase enzyme in EC 4.1.1).
  • an enzyme capable of converting 2-oxopimelate into 2-aminopimelate e.g., an aminotransferase enzyme in EC 2.6.1.
  • the recombinant host cell capable of producing hexamethylenediamine comprises any or all of these enzymes.
  • Embodiments of the invention also provide a method of producing hexamethylenediamine comprising the use of these recombinant host cells.
  • the invention provides a method for producing hexamethylenediamine from renewable feedstocks such as sugars, fatty acids, glycerol, and SynGas.
  • the non-naturally occurring host cell capable of producing hexamethylenediamine comprises any or all of the enzymes required to convert renewable feedstocks to hexamethylenediamine.
  • This disclosure features recombinant host cells that recombinantly express one or more (e.g., two or more; three or more; four or more; five or more; six or more; seven or more; eight or more; nine or more; ten or more; eleven or more; twelve or more; thirteen or more; fourteen or more; fifteen or more; sixteen or more; seventeen or more; eighteen or more; nineteen or more; twenty or more; or even more) of the enzymes used for producing a compound in one of the pathways described herein and methods of using such host cells to produce di- and trifunctional C7 alkanes.
  • one or more e.g., two or more; three or more; four or more; five or more; six or more; seven or more; eight or more; nine or more; ten or more; eleven or more; twelve or more; thirteen or more; fourteen or more; fifteen or more; sixteen or more; seventeen or more; eighteen or more; nineteen or more; twenty or more; or even more
  • a host cell can include one or more (as above) exogenous nucleic acids encoding one or more (as above) of the following: an enzyme that catalyzes the reductive deamination of D,L diaminopimelate or alpha- aminopimielate; an enzyme that catalyzes the enoate reduction of 2-heptenedioic acid to pimelic acid; an enzyme that catalyzes the carboxylic acid reduction of pimelic acid to pimelic acid semialdehyde, an enzyme that catalyzes the semialdehyde amination of pimelic acid semialdehyde to 7-aminoheptanoic acid, an enzyme that catalyzes the amido hydrolysis of 7-aminoheptanoic acid to enantholactam, an enzyme that catalyzes the aldehyde dehydrogenation of 7-aminoheptanoic acid to 7-aminoheptanal; an enzyme that cata
  • the host cells that are used typically possess a number of properties: they may be easily genetically modified, are tolerant of the conditions used in the method of the invention, and grow to cells densities which are industrially useful. .
  • the host cell may be a single celled microorganism, or may be a cell of a cell line.
  • the host cell may be of a wild type genotype.
  • the enzyme that is used to catalyze one or more steps in the method of the invention is naturally present in the host cell and is expressed at a level that has industrial use in the methods of the invention.
  • the host cell has been genetically modified to express the enzyme at a level that has industrial use in the methods of the embodiments of invention.
  • the enzyme may be sourced from the cell in which it is expressed.
  • the enzyme is sourced from a different strain or species of cell.
  • the host cell is a prokaryote. In another alternative it is a eukaryote. Typically single celled microorganisms are used.
  • prokaryotic cell includes gram positive and gram negative bacteria.
  • suitable gram negative bacteria include 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 Lac
  • Eukary otic host cells include those from yeast and other fungi, as well as, for example, insect, mouse, rat, primate, or human cells.
  • suitable eukaryotic host cells include yeasts and fungi from the genus Aspergillus such as Aspergillus niger; from the genus Saccharomyces such as Saccharomyces cerevisiae; from the genus Candida such as C. tropicalis, C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. maltosa, C. parapsilosis, and C.
  • Pichia such as Pichia pastoris
  • Yarrowia such as Yarrowia lipolytica
  • Issatchenkia such as Issathenkia orientalis
  • Debaryomyces such as Debaryomyces hansenii
  • Arxula such as Arxula adenoinivorans
  • Kluyveromyces such as Kluyveromyces lactis or from the genera Exophiala, Mucor, Trichoderma, Cladosporium, Phanerochaete, Cladophialophora, Paecilomyces, Scedosporium, and Ophiostoma.
  • the host cells may be provided in a variety of different forms.
  • the cells may be resting cells. That is to say that the cells are grown up in cultures and removed from the culture medium before being employed as the biocatalyst.
  • the cells may be used directly following growth, or they may be stored before use. Typical methods of storage include freezing.
  • the cells are lyophilized prior to use.
  • the cells are growing cells. That is to say that the cells perform their biocatalytic action while being cultured. If the substrate for a particular biocatalytic reaction cannot cross the cell membrane to be transformed by host cells, then in one alternative, a crude lysate may be used.
  • a crude lysate is the initial suspension of cellular components produced following lysis of the cells.
  • Lysis of the cells may be performed by any means, including chemical or mechanical. Further methods of lysis will be evident to the skilled person in light of their general knowledge and the teachings herein.
  • a clarified lysate may be used.
  • a clarified lysate may be prepared by centrifuging the crude lysate to pellet unlysed cells and other cellular debris.
  • substantially pure cultures of recombinant host cells are provided.
  • a "substantially pure culture" of a recombinant cell is a culture of that cell in which less than about 40% (i.e., less than about : 35%; 30%; 25%; 20%; 15%; 10%; 5%; 2%; 1%; 0.5%; 0.25%; 0.1%; 0.01%; 0.001%; 0.0001%; or even less) of the total number of viable cells in the culture are viable cells other than the recombinant cells, e.g., bacterial, fungal (including yeast), mycoplasmal, or protozoan cells.
  • Such a culture of recombinant cells includes the cells and a growth, storage, or transport medium.
  • Media can be liquid, semi-solid (e.g., gelatinous media), or frozen.
  • the culture includes the cells growing in the liquid or in/on the semi-solid medium or being stored or transported in a storage or transport medium, including a frozen storage or transport medium.
  • the cultures are in a culture vessel or storage vessel or substrate (e.g., a culture dish, flask, or tube or a storage vial or tube).
  • the recombinant cells of this disclosure can be harvested from a fermentation process by conventional methods such as filtration or centrifugation and formulated into a dry pellet or dry powder formulation while maintaining high activity.
  • Processes for production of a dry powder whole recombinant cell composition exhibiting one or more of the activities disclosed herein can include spray-drying, freeze-drying, fluidised bed drying, vacuum drum drying, or agglomeration and the like.
  • the compositions can be a shelf-stable, dry biocatalyst composition suitable for the biosynthetic methods described herein. Drying methods such as freeze-drying, fluidised bed drying or a method employing extrusion/spheronisation pelleting followed by fluidised bed drying can be particularly useful.
  • Temperatures for these processes may be ⁇ 100°C but typically ⁇ 70°C to maintain high residual activity and stereoselectivity.
  • the dry powder formulation should have a water content of 0 - 10% w/w, typically 2-5% w/w.
  • Stabilising additives such as salts (e.g. KC1), sugars, proteins and the like may be included to improve thermal tolerance or improve the drying characteristics of the recombinant cells during the drying process.
  • partially or wholly purified enzymes may be used instead of or in combination with recombinant hosts or lysates of recombinant or non-recombinant cells.
  • Wholly or partially purified enzymes can be wholly or partially purified lysates of any of the recombinant cells described here or non-recombinant cells.
  • Methods of purification are well known in the art. Partial or complete purification has the advantage that the enzyme of interest is separated from other cellular components which may interfere with the reaction catalyzed by that enzyme (either by also reacting with the substrate or by converting intermediates or the desired product to an unwanted compound). Purification does add further steps to any biocatalyst preparation, however, and therefore may not be suitable in all instances. The determination of the suitability of the use of partially or wholly purified enzymes will be well within the grasp of the skilled person following the teaching herein.
  • one or more of the conversions in the method of the invention will be performed by the biocatalyst under aerobic conditions. In an alternative one or more of the conversions in the method of the invention will be performed under anaerobic conditions. In a further alternative, some steps of the method of the invention will be performed under aerobic conditions and some steps will be performed under anaerobic conditions.
  • the biocatalysts used in the methods of the invention may be unmodified host cells of the species in which the enzyme naturally occurs. Typically, however, it is necessary to modify genetically the host cell to produce a non-naturally occurring (i.e., recombinant or engineered) host cell.
  • the chromosome of the whole cell biocatalyst (i.e., the host cell) has been modified.
  • This modification may be an insertion, a deletion or a substitution of a nucleic acid in the chromosome.
  • Methods of effecting modifications to the chromosome of a cell are well known, e.g., transposon mutagenesis, Cre-Lox mediated recombination, lambda Red and RecET mediated recombination.
  • Stable integration into the chromosome of a nucleic acid is advantageous because it allows the maintenance of the nucleic acid without the need for a selectable marker.
  • Stable integration means that the host cell can be cultured for greater than five generations without loss of the alteration.
  • stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely.
  • the episomal composition of the host cell genome is modified.
  • episome any autonomously replicating element, for example a plasmid (which may be linear or circular), a cosmid, a yeast artificial chromosome (YAC), etc.
  • plasmid which may be linear or circular
  • cosmid a cosmid
  • YAC yeast artificial chromosome
  • Episomal elements frequently place a metabolic load on their host cell, and accordingly, in the absence of any active partitioning mechanism to ensure that at least one copy of the episome is present in each daughter cell following cell division it is necessary to include a selectable marker.
  • the nucleic acids introduced into the cell may comprise one or more (e.g., two or more; three or more; four or more; five or more; six or more; seven or more; eight or more; nine or more; ten or more; eleven or more; twelve or more; thirteen or more; fourteen or more; fifteen or more; sixteen or more; seventeen or more; eighteen or more; nineteen or more; twenty or more; or even more) of a number of elements.
  • one of the elements encodes an enzyme that is used to produce di- or trifunctional C7 alkanes or precursors to such molecules.
  • the nucleic acid encodes a protein which is not an enzyme that functions in the method of the invention.
  • a promoter is operably linked to a nucleic acid.
  • the choice of promoter will depend on the intended application, and can readily be determined by the skilled person. For example, if an enzyme catalyzed reaction may be detrimental to a particular host cell, it may be desirable to use a regulated or inducible promoter, such that gene expression can be turned on or off as necessary. Alternatively, it may be preferred to have expression driven by either a weak or strong constitutive promoter to ensure that the enzyme is expressed at all stages of growth.
  • Exemplary promoters suitable for eukaryotic cell systems include the SV40 early promoter, the cytomegalovirus (CMV) promoter, the mouse mammary tumor virus (MMTV) steroid-inducible promoter, and the Moloney murineleukemia virus (MMLV) promoter.
  • Exemplary yeast promoters include 3-phosphoglycerate kinase promoter, glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) promoter, galactokinase (GAL1) promoter, galactoepimerase promoter, and alcohol dehydrogenase (ADH) promoter.
  • GAP1 galactokinase
  • ADH alcohol dehydrogenase
  • Other suitable promoters in yeast are known to those skilled in the art.
  • Exemplary promoters suitable for bacterial cell systems include but are not limited to: T7, T3, SP6, lac and trp promoters.
  • the introduced nucleic acid also can be operably linked to comprise 5' untranslated region (UTR), 3 ' UTR, enhancer and/or terminator regions, as required.
  • UTR 5' untranslated region
  • enhancer and/or terminator regions as required.
  • a single strain of host cell is used to express more than one (e.g., two or more; three or more; four or more; five or more; six or more; seven or more; eight or more; nine or more; ten or more; eleven or more; twelve or more; thirteen or more; fourteen or more; fifteen or more; sixteen or more; seventeen or more; eighteen or more; nineteen or more; twenty or more; or even more) of the enzymes used in the methods of the invention.
  • the multiple enzymes may be encoded by the same introduced nucleic acid.
  • the enzymes may be encoded on separate introduced nucleic acid fragments.
  • the enzymes may all be expressed from a single promoter (for example by arranging the enzymes in the form of an operon).
  • the enzymes may be expressed from multiple separate promoters.
  • the multiple separate promoters may be induced by the same chemical (for example, each of the multiple enzymes may be expressed from the yeast GAL promoter, thus meaning that each gene is inducible with galactose).
  • Other suitable promoters are known in the art.
  • each of the genes encoding an enzyme used in the method of the invention is under the control of a different promoter. Thus, different enzymes can be introduced individually through the use of different inducing compounds.
  • an intermediate approach is used, wherein a number of enzymes are under the control of the same promoter, and a number of enzymes are under the control of different promoters.
  • This alternative may be particularly advantageous when numerous enzyme pathways have been generated in a host cell and it is desirous to control each member of a pathway in concert with the other pathway members, but to control each pathway separately.
  • each plasmid comprises a different origin and/or a different selectable marker.
  • the expression of multiple enzymes in a single cell is advantageous because, if the co-expressed enzymes act directly after one another in a reaction pathway, it ensures that the product of the upstream enzyme reaction is immediately available to be acted upon by the downstream enzyme. This avoids the need for either (i) purification of the intermediate and the setting up of second reaction vessel to perform the second reaction; or (ii) the need for the product to be exported from the cell comprising the upstream enzyme into the culture medium where it may be acted upon by a cell comprising the downstream enzyme.
  • a cell has been engineered to express a protein under non-natural conditions (for example, when a protein that is native to a species is expressed at levels above the natural level, or in an alternative, when a protein from a different species is expressed in a host cell) in some instances that protein will not be expressed in an active form. Instead it may fold incorrectly, and accumulate as a non-functional "inclusion body" aggregates.
  • the cells used to express the protein may be subjected to genetic modification to further express chaperone proteins which are able either to prevent the mis-folding of the protein, or are able to refold it from the aggregated state.
  • the inclusion of such chaperone proteins is advantageous because it increases the quantity of active protein per cell, and therefore increases the overall efficiency of the method of the invention.
  • the expressed chaperone may be a chaperone protein of the host cell.
  • the chaperone may be from the same species/strain as the protein.
  • Typical chaperone proteins for expression include members of the GroEL/GroES family, and members of the DnaJ/DnaK/GrpE family. Homologs of the archetypal E.
  • coli GroEL/GroES and DnaJ/DnaK/GrpE proteins have been identified in other prokaryotic species (see, for example, ), and eukaryotic homologs are also known (GroEL and GroES correspond to the eukaryotic proteins Hsp60 and HsplO, and DnaJ, DnaK and GrpE correspond to the eukaryotic proteins Hsp70, Hsp40 and Hsp24, respectively). These proteins have been identified in a number of species of yeast (for example, Saccharomyces cerevisiae). The choice of appropriate chaperone proteins for coexpression with an enzyme used in the method of the invention will be evident to the skilled person following the teachings herein.
  • Metabolic engineering is the process of optimizing the parameters in a host cell in order to increase the ability of a cell to produce a compound.
  • the host cells used in the method of the present invention optionally have been engineered to optimize the output of the difunctional C7 alkanes discussed above.
  • Metabolic engineering to increase the ability of a cell to produce a compound is principally performed via two avenues.
  • the first is to optimize the enzymes in the pathway producing the desired product from the starting material.
  • a multi-enzyme pathway resulting in the production of a difunctional C7 alkanes (as shown in the figures and described in the preceding sections)
  • it is possible to determine the concentration of each intermediate in the pathway using techniques known to the skilled person (for example, two dimensional electrophoresis, the use of isotopically labeled precursors, and nuclear magnetic resonance (NMR) spectroscopy), and therefore determine which of the enzyme conversions is the rate limiting step - that is to say which step in the reaction scheme is the slowest.
  • the rate at which this intermediate is reacted should therefore be increased.
  • This can be performed by a number of means. Firstly, the expression level of the limiting enzyme may be increased. Optionally this may be achieved by placing the gene encoding the enzyme under the control of a strong promoter, e.g., the T7 promoter if the enzyme is being expressed in E. coli or the TEF promoter if the enzyme is being expressed in yeast.
  • a strong promoter e.g., the T7 promoter if the enzyme is being expressed in E. coli or the TEF promoter if the enzyme is being expressed in yeast.
  • the second option is to increase the number of copies of the gene encoding the enzyme that are present in cell, for instance by placing the gene on a multicopy plasmid, or by incorporating multiple copies of the gene into the chromosome of the host cell (these copies may be incorporated at the same location in the chromosome or in different locations in the chromosome).
  • the production of difunctional C7 alkanes can also be increased by inactivating or reducing the activity of any enzymes which are capable of diverting the substrate, any of the intermediates, or the product into a metabolic pathway other than that which is the aim of the method. Therefore, the activity of the enzyme may be lowered to increase the yield of the difunctional C7 alkanes.
  • the recombinant host cells disclosed herein can have a deficiency in one or more enzymes (e.g., by deleting the nucleic acid encoding an enzyme of interest or reducing expression of the nucleic acid) that are capable of diverting the starting material of the method of the invention, or any intermediates produced in the reaction pathway producing difunctional C7 alkanes, to a different, unwanted, end product.
  • the enzyme is not deleted, but is instead altered so that it can acts against the substrate, intermediates, or product, at a rate that is less than the rate of the wild type enzyme. In the instance where the enzyme catalyses a reversible reaction, then the enzyme should be altered so that it acts only in the desired direction of reaction.
  • a recombinant host cell can have a deficiency in an enzyme capable of converting pimeloyl-[acp] into 7-keto-8-aminopelargonic acid (KAPA) or converting 6-carboxyhexanoyl-CoA (pimeloyl-CoA) into KAPA (e.g., a deletion or inactivation of BioF, the gene encoding 7-keto-8-aminopelargonic acid synthetase (E.C. 2.3.1.47).
  • KAPA 7-keto-8-aminopelargonic acid
  • pimeloyl-CoA 6-carboxyhexanoyl-CoA
  • a recombinant host cell can have a deficiency in diaminopimilate decarboxylase, which creates a lysine auxotroph.
  • a lysine auxotroph can be particularly useful for deregulating carbon flux to D,L diaminopimelate, the immediate precursor to lysine.
  • a lysine auxotroph would require fed-batch fermentation.
  • host cells are used which are growing (i.e., dividing) at the time the cells perform the conversions in the method of the invention.
  • the cells are cultured under conditions which optimize the production of desired difunctional C7 alkane.
  • Any of the recombinant host cells described herein can be cultured to produce and/or secrete the biosynthetic products of the invention.
  • the term culture is equivalent with fermentor and bioreactor.
  • the carbon source used for growth will be provided in the form of a nutrient broth, for example Luria medium or yeast extract medium.
  • a defined medium that is to say, a medium in which the concentration of each component is known
  • the growth medium comprises glucose as a carbon source.
  • the carbon source used by the host cells in the medium is glucose, sucrose, xylose, fatty acids and glycerol.
  • the enzymes which catalyze the conversions in the method of the invention are present in more than one strain/species of cell, wherein those more than one strain/species of cell are used simultaneously.
  • the strains/species that are chosen must be selected such that one strain does not outcompete the other strain.
  • Such a relationship can be obtained by introducing an artificial symbiosis into the co-culture. That is to say that the two stains/species that are used are each auxotrophic for an essential nutrient, but for different nutrients.
  • the other strain should be engineered to produce an excess of that nutrient so that both strains can survive together in a culture when the growth medium of the culture does not comprise the two essential nutrients. Selection of appropriate auxotrophies will be well within the grasp of the skilled person following the teachings herein.
  • the culture conditions described herein can be scaled up and grown continuously for manufacturing of heptane- 1,7-dioic acid, 7-oxoheptanoic acid, 7-hydroxyheptanoic acid, 7- hydroxyheptanal, 7-aminoheptanoic acid, heptamethylenediamine, 1,7-heptandiol, 7- aminoheptanal, 7-aminoheptanol or enantholactam.
  • Exemplary growth procedures include, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. All of these processes are well known in the art.
  • Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of di- and trifunctional C7 alkanes.
  • the continuous and/or near- continuous production of heptane- 1,7-dioic acid, 7-oxoheptanoic acid, 7-hydroxyheptanoic acid, 7-hydroxyheptanal, 7-aminoheptanoic acid, heptamethylenediamine, 1,7-heptandiol, 7- aminoheptanal, 7-aminoheptanol or enantholactam described previously include culturing a recombinant host cell producing heptane- 1,7-dioic acid, 7-oxoheptanoic acid, 7- hydroxyheptanoic acid, 7-hydroxyheptanal, 7-aminoheptanoic acid, heptamethylenediamine, 1,7-heptandiol, 7-aminoheptan
  • Continuous culture under such conditions can include, for example, 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culture can include 1 week, 2, 3, 4 or 5 or more weeks and up to several months. Alternatively, recombinant cells can be cultured for hours, if suitable for a particular application. It is to be understood that the continuous and/or near-continuous culture conditions also can include all time intervals in between these exemplary periods. It is further understood that the time of culturing the host cell of the invention is for a sufficient period of time to produce a sufficient amount of product for a desired purpose.
  • Fermentation procedures are well known in the art. Briefly, fermentation for the biosynthetic production of heptane- 1,7-dioic acid, 7-oxoheptanoic acid, 7-hydroxyheptanoic acid, 7-hydroxyheptanal, 7-aminoheptanoic acid, heptamethylenediamine, 1,7-heptandiol, 7- aminoheptanal, 7-aminoheptanol or enantholactam described previously can be utilized in, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art.
  • the invention also provides compositions comprising recombinant host cells according to the invention and heptane- 1,7-dioic acid, 7-oxoheptanoic acid, 7- hydroxyheptanoic acid, 7-hydroxyheptanal, 7-aminoheptanoic acid, heptamethylenediamine, 1,7-heptandiol, 7-aminoheptanal, 7-aminoheptanol and enantholactam.
  • the invention also provides a composition comprising a recombinant host cell according to the invention and a feedstock.
  • the feedstock is a renewable feedstock, for example wherein the feedstock is selected from the group consisting of glucose, sucrose, xylose, fatty acids and glycerol.
  • the feedstock is a polyaromatic hydrocarbon, for example benzene, toluene or shikimate.
  • enzyme targets were selected from the ammonia lyase family (EC 4.3.1), including methylaspartate ammonia lyase (MAL; EC 4.3.1.2), based on its broad substrate specificity, presence in a wide range of hosts, ability to be cloned and expressed exogenously, and available crystal structure for rational design protein engineering.
  • the MAL genes from Citrobacter amalonaticus, Clostridium tetanomorphum and Aspergillus oryzae were selected.
  • the MAL enzymes encoded by C. amalonaticus (GeneBank AB005294; fragment 1641- 2882878596..879060NM_NM) and C tetanomorphum (GeneBank S48141; fragment 756- 1997) have significant dissimilarity (57% identity).
  • the Aspergillus oryzae MAL gene (GeneBank XM_001827609) is evolutionarily divergent (44% identity with Citrobacter amalonaticus MAL, 39% identity with Clostridium tetanomorphum MAL), which may show different catalytic properties and/or selectivities.
  • Another unusaual ammonia lyase that is a potential enzyme for the reductive deamination of 2,6-diaminopimelate and 2- aminoheptanedioic acid is the D-glucosaminate ammonia lyase (EC 4.3.1.9) from Pseudomonas fluorescens (accession number BAD69624). This is an unusual ammonia lyase that catalyzes an ⁇ , ⁇ -elimination:
  • the selected MAL gene targets from Citrobacter amalonaticus, Clostridium tetanomorphum, and Aspergillus oryzae were then cloned into the IPTG inducible pET21-a backbone using inABLE technology to generate 14 (pET21-a with the Citrobacter amalonaticus MAL gene), 15 (pET21-a with the Clostridium tetanomorphum MAL gene), and 16 (pET21-a with the Aspergillus oryzae MAL gene).
  • 14 pET21-a with the Citrobacter amalonaticus MAL gene
  • 15 pET21-a with the Clostridium tetanomorphum MAL gene
  • 16 pET21-a with the Aspergillus oryzae MAL gene
  • Potential restriction enzyme e.g., Earl
  • Earl restriction sites located within coding sequences were disrupted, e.g., by incorporation of a silent mutation, as to avoid altering the encoded protein sequence.
  • the resulting DNA was free of Earl sites, and was used the input sequence for software analysis to design corresponding truncated parts flanked by Earl (ordered through DNA 2.0), long and short linker oligonucleotides (ordered through Sigma-Aldrich), and long and short part oligonucleotides (ordered through Sigma-Aldrich).
  • the 5 '-end of the part and linker oligonucleotides were phosphorylated by incubating the primer in the present of T4 kinase under suitable reaction conditions (6 ⁇ oligo, 1 PNK buffer, 1 mM ATP, 10 U T4 kinase, 5 mM DTT, 5% (w/v) PEG8000) at 37°C for 30 minutes, followed by inactivation of the enzyme at 65 °C for 20 minutes.
  • the phosphorylated linker oligos and phosphorylated part oligos were annealed by mixing equimolar amounts of each oligo (50 ⁇ ⁇ at 6 ⁇ ) and then heating the mixture to 65 °C in a thermocycler before gradually lowering the temperature down to 20°C to form partially double-strand part oligos and partially double-strand linker oligos having specific 16 bp overhangs (melting temperature above 75°C).
  • the resulting annealed part oligos (POA) and annealed linker oligos (LOA) were then diluted to a final concentration of 1 ⁇ with TE buffer (Tris-EDTA).
  • the part/linker fragments corresponding to the selected genes were generated by successive cycles of Earl digestion of the cloned truncated parts followed by ligation of their respective annealed part oligos (POA28, POA29 or POA30), their truncated part (TP28, TP29 or TP30), and the annealed linker oligos from the vector backbone (LOA31).
  • Part linker fusions corresponding to the vector backbone were prepared by ligation of its annealed part oligos (POA31), its truncated part (TP31) and the annealed linker oligos from either genes (LOA28, LOA29 or LOA30).
  • LOA31 annealed part oligos
  • TP31 truncated part
  • LOA30 annealed linker oligos from either genes (LOA28, LOA29 or LOA30).
  • Ten and twenty-fold molar excesses of LOA and POA compared to the truncated part were used in the gene and vector reactions respectively to favor ligation between the truncated part and the oligonucleotides during each Earl digestion/ligation cycle.
  • the reactions were performed in a total volume of 50 ⁇ ⁇ and were incubated in a thermocycler (Eppendorf Mastercycler Gradient).
  • Cycles of Earl digestion/ligation were achieved by alternating the temperature between 37°C and 16°C, which correspond to the optimum temperatures for the Earl digestion and for the ligation using the T4 DNA ligase respectively.
  • the samples were loaded on a 0.7% agarose gel, and the part linker was purified from the residual backbone through agarose gel separation and gel extraction (QIAGEN QIAquick Gel Extraction Kit). Expected size fragments were observed after agarose gel electrophoresis for the preparation of part linker fragments. Ligation of the digested fragment with the annealed oligonucleotides displaying compatible 3 bp overhangs resulted in the formation of a non-cleavable part/linker fragment.
  • the gene part/linker fusions were then combined with their respective vector part/linker fusion using a 2-part assembly to construct an E. coli expression vector using the part linkers previously generated.
  • the gene and vector part linker fusions were mixed together in an equimolar amount (0.1 pmol each), which resulted in the expected DNA fragment assembly due to the specific complementary overhangs formed in the annealed part oligonucleotide and the annealed linker oligonucleotide from each part.
  • Reactions were incubated at room temperature for 30 min prior to transformation of high efficiency chemically competent ⁇ E. coli cells using 2 ⁇ of the assembly reaction and 10 ⁇ of the competent cells.
  • Transformed cells were plated on LB-Amp-agar and incubated at 37°C overnight. Five hundred clones were obtained for each assembly. Two random clones were then picked from each assembly and the corresponding vectors were isolated using the QIAGEN QIAprep Miniprep Kit.
  • coli expression vectors were analyzed using Sphl and EcoRY to identify positive clones for the insertion.
  • the restriction products from each sample were analyzed usign agarose gel electrophoresis.
  • the expected band pattern was observed for both clones from each assembly confirming the construction of E. coli expression vectors harbouring the Citrobacter amalonaticus, Clostridium tetanomorphum and Aspergillus oryzae MAL genes.
  • Assembly of the 17 negative control vector (pET21-a control) was performed using the part/linker fusion pET21-a part/pET21-a linker was performed as a negative control, and clones from the generation of the negative control gene-free construct were analyzed using Xmnl and AlwNl.
  • E. coli constructs previously obtained were used to study the expression of the Citrobacter amalonaticus, Clostridium tetanomorphum and Aspergillus oryzae MAL genes in E. coli.
  • the vectors (10 ng DNA from each assembly) were used to transform electro- competent BL21 (DE3) E. coli cells, and transformation samples were plated on LB-Amp- Agar. Transformants were obtained after overnight incubation at 37°C.
  • a single clone was picked from each assembly plate, and was used to inoculate 5 ml of LB-Amp medium as a starting culture. The OD 600 was measured after overnight incubation at 37°C and 250 rpm. Two mL of each starting culture (approximately 4.5xl0 7 - 6.2xl0 7 cells) were used to inoculate 100 mL of LB-Amp, and the cultures were incubated in a 500 mL baffled shake flask at 37°C and 250 rpm until an OD 600 of 0.6 to 0.8 was reached.
  • Protein expression which is controlled by the T7 promoter in pET21-a, was induced by addition of 1 mM IPTG (final concentration) and cultures were further incubated at 37°C and 250 rpm. 1 mL of each sample was taken prior to induction, 4h post-induction, and 24h post- induction. Cell growth was checked by measuring OD 60 o. The remaining culture left after 24h post-induction incubation was transferred to a 50 mL falcon tube, harvested by centrifugation at 5000 rpm for 10 minutes and the cell pellets stored at -20°C.
  • the time-point samples from each assembly were processed for SDS-PAGE analysis.
  • the samples were centrifuged at 13000 rpm for 2 min before the supernatant was removed and the cells were lysed using the Bugbuster protein extraction reagent supplemented with lysozyme (15 mg/mL) and benzonase (3.4 ⁇ / ⁇ ).
  • the lysis reactions were then centrifuged at 13000 rpm for 2 min, and the soluble fraction was transferred to a new tube and the insoluble fraction was re-suspended in water.
  • 16 mL of each starting culture (approximately 4.4xl0 8 cells) were used to inoculate 800 mL of LB-Amp, and the cultures were incubated in a 2 L baffled shake flask at 37°C and 250 rpm until an OD 600 of 0.6 to 0.8 was reached. Protein expression was induced by addition of 1 mM IPTG (final concentration), and cultures were further incubated at 37°C and 250 rpm. After 24h post- induction incubation, the remaining culture was transferred to four 50 mL falcon tubes, harvested by repeated centrifugation at 5000 rpm for 10 minutes (4 rounds of centrifugation per tube; 50 mL of medium per round) and the cell pellets stored at -20°C.
  • Glass bead lysis was used to disrupt the cells for collecting the Citrobacter amalonaticus, Clostridium tetanomorphum and Aspergillus oryzae MAL protein.
  • Cell pellets from the 24 hour post-induction samples were re-suspended in bead lysis buffer consisting of 0.1 mM Tris, 1 mg/ml Pepstatin A and 200 mM PMSF protease inhibitors. Acid washed 212- 300 ⁇ glass beads (0.4 g per 1 mL of lysis buffer) were added to each tube, and lysed in a sonicating water bath in parallel.
  • biotransformations were prepared at either 5g/L or 20g/L whole cell equivalent of the 14, 15 and 16 lyase strains.
  • the enzyme concentration was calculated based on whole cell equivalent which is the pelleted cell mass after centrifugation (wet cell weight) divided by the volume of buffer used to resuspend the cells during lysis.
  • the biotransformations were conducted in 3 ml volumes with lOmM DL-threo-beta methyl aspartate.
  • biotransformations were prepared varying levels of enzyme and substrate concentrations (2 variable, 2 level stat design) to examine 14 and 15 enzyme activity towards 2-aminopimelate.
  • the biotransformations were conducted in 3 ml volumes with varying loadings as indicated below:
  • This method comprises forming a chiral adduct with a Boc-cysteine and ortho-phthaladehyde reagent to form a diastereomeric pair of L- and D-2-aminopimelate derivatives.
  • the diastereomers (representing the enantiomers of 2-aminopimelate) can then be separated by HPLC using a C18 column and a 5mM potassium phosphate buffer pH 7.0 and acetonitrile gradient at 338nm.
  • the peak areas for L- and D-2-aminopimelate signals in each sample were recorded and observed decrease in the L-2-aminopimelate standard is expressed as % relative to the D-2-aminopimelate.
  • enzyme targets were selected from the enoate reductase family (EC 1.3.1), such as EC
  • pimeloyl-CoA dehydrogenase in EC 1.3.1.62 such as PimC/PimD or ThnJ/ThnK.
  • the enoate reductase may also be in the EC 1.3, such as EC 1.3.8.1 or EC 1.3.99.3; EC
  • enoate reductase genes from Bacillus subtilis (YqjM) (GeneBank D84432; fragment 242791-243807) and Solatium lycopersicum (OPR1 and OPR3) (GeneBank NM_001247852, fragment 108-1238 and GeneBank NM_001246944; fragment 94-1284, respectively) were selected because they had been previously cloned and successfully expressed in E. coli, crystal structures of the YqjM and OPR3 enzymes were reported, which enables protein engineering through rational design (The 1.3 A crystal structure of the flavoprotein YqjM reveals a novel classof old yellow enzyme: Kitzing et al. J. Biol. Chem.
  • XenA (EC 1.3.1.31) from Pseudomonas putida
  • TER (EC 1.3.1.44) from Euglena gracilis
  • PECR and MECR from Homo sapiens
  • acdA (EC 1.3.99.-) from Pseudomonas putida
  • ACADS (EC 1.3.99.-) from Bos Taurus and PimC/pimD (EC 1.3.1.62) from Rhodopseudomonas palustris were selected for expression in E. coli and tested for the reduction of 2-heptene-dioic acid and 2-heptenedioic acid-CoA.
  • PECR peroxisomal trans-2-enoyl-CoA reductase
  • Bos taurus acyl-CoA dehydrogenase (ACADS, 44.6 kDa, 412AA) NP 001029573.1
  • Rhodopseudomonas palustris Pimeloyl-CoA dehydrogenase (PimC) Rhodopseudomonas palustris Pimeloyl-CoA dehydrogenase
  • Rhodopseudomonas palustris Pimeloyl-CoA dehydrogenase PimD, NP 949050.1 small subunit, 40.5 kDa, 389AA
  • enr 2-Enoate reductase (Y16137) EC 1.3.1.31 from Clostridium kluyveri: Whole cells of Clostridium kluyveri were found to reduce a broad range of 2-enoates in the presence of molecular hydrogen.
  • the substrates were: tiglic acid, acrylic acid, methacrylic acid, dimethylacrylic acid, (E)- pentenoic acid, (E)-hexenoic acid, sorbic acid, cinnamic acid [19], (E)-2-butenoic acid and (E)-2- methyl-2-butenoic acid, (E)- and (Z)-3-methyl-2-pentenoic acid, p-methoxy-cinnamic acid and p- nitro-cinnamic acid [Buhler, M., Giesel, H., Tischer, W. and Simon, H. (1980) Occurrence and the possible physiological role of 2-enoate reductases. FEBS Lett.
  • enr 2-Enoate reductase (Y09960) EC 1.3.1.31 from C. tyrobutyricum (previously Clostridium sp. La 1): 2-Enoate reductase was found in Clostridium sp. growing on (3 ⁇ 4)-butenoate and purified to homogeneity. It accepted (3 ⁇ 4)-2-methylbutenoate and cinnamate [Elshahed MS, Bhupathiraju VK, Wofford NQ, Nanny MA, Mclnemey MJ.
  • enr 2-Enoate reductase (Y16136) EC 1.3.1.31 from Moorella thermoacetica (previously Clostridium thermoaceticum): An antiserum against enoate reductase from C. tyrobutyricum cross- reacted with a protein of the thermophilic Clostridium thermoaceticum. The gene encoding the reductase was identified and the enzyme was successfully overexpressed in E. coli [Rohdich, 2001].
  • pimeloyl-CoA dehydrogenases (EC 1.3.1.62) should catalyse the reduction of the double bond in 2-heptenedioic acids and the corresponding CoA esters.
  • Pimeloyl-CoA dehydrogenase activity was found in cell extracts of Syntrophus aciditrophicus grown in a co-culture with Desulfovibrio sp. strain Gi l with benzoate or in a pure culture with crotonate and is involved in the benzoate degradation pathway (Elshahed MS, Bhupathiraju VK, Wofford NQ, Nanny MA, Mclnerney MJ.
  • GDH Glutaryl-Coenzyme A Dehydrogenase from Desulfococcus multivorans, a non-decarboxylating glutaconyl-coenzyme A- forming glutaryl-coenzyme A dehydrogenase was characterized in the obligately anaerobic bacteria Desulfococcus multivorans [Wischgoll S, Demmer U, Warkentin E, Gunther R, Boll M, Ermler U. (2010) Structural basis for promoting and preventing decarboxylation in glutaryl- coenzyme A dehydrogenases. Biochemistry. 49: 5350-5357].
  • the enzyme was overexpressed and its crystal structure was determined which makes it a useful target for protein engineering to catalyse reversible oxidation and reduction of the C7 equivalents (2,3-didehydropimeloyl-CoA and pimeloyl-CoA) of its native C5 substrates glutacolnyl-CoA and glutaryl-CoA
  • the residual Earl site was removed using the Quikchange single-site directed mutagenesis kit from Strategene. Forward and reverse primers were designed to disrupt the residual Earl site, and the primers were phosphorylated as described in the section 3.1.1.2 prior to the Quikchange reaction.
  • the Quikchange and the negative control reactions were prepared as follows: [00319]
  • the reactions were run in a thermocycler (95°C 0.5 minutes; 35 cycles of 95°C 0.5 minutes, 55°C 1 minute, 68°C 10 minutes; 68°C 1 minutes, 4°C hold), and then treated with Dpnl (1 ⁇ ) to digest the methylated parental DNA at 37°C for 2 hours.
  • the digestion reactions (3 ⁇ ) were used to transform TOP 10 electrocompetent E.coli cells, and the transformation samples (100 ⁇ ) were plated onto LB-Kan-Agar. About 100 clones were obtained on the Quikchange reaction plate compared to none on the negative control plate after overnight incubation at 37 °C. Ten clones were picked from the plate, the corresponding plasmids were purified, and analyzed by Earl digestion. The absence of a 300 bp band on the agarose gel indicated that the Earl site located in the OPR3 gene was deleted. Earl digestion showed that several clones were positive for the removal of the residual Earl site. DNA sequencing confirmed the clone's sequence.
  • Vector from clone 3 (a clone positive for removal of Earl) was used to transform electrocompetent TOP 10 E.coli cells.
  • QIAGEN Hi-Speed Plasmid Purification Midi Kit was used to prepare a large DNA stock from the culture, and the DNA sample was subsequently concentrated using the QIAGEN PCR purification kit. The final DNA concentration and the volume of the sample was 470.0 ng/ ⁇ in 120 ⁇ (DNA cone. 199.8 nM).
  • the Earl free sequences were then used as input sequences in the part designer software to design the corresponding truncated parts flanked by Earl sites, as discussed in Section 3.1.1.2.
  • the synthesized Bacillus subtilis YqjM (TP25), Solarium lycopersicum OPRl (TP26), and Solanum lycopersicum OPR3 (TP27) truncated parts were inserted into the DNA2.0 pJ201 vector.
  • the pET21-a truncated part vector was fully synthesized with introduction of a fragment including the chloramphenicol resistance gene to produce the vector TP31.
  • part linker fusions were prepared by ligating the 5 '-end of the truncated part with its corresponding part oligo annealed fragment, and the 3 '-end of the truncated part with the linker oligo annealed fragment, as previously described in section 3.1.1.2.
  • Part linker fusions corresponding to the Bacillus subtilis YqjM, Solanum lycopersicum OPRl and Solanum lycopersicum OPR3 genes were prepared by ligation of their respective annealed part oligos, their truncated part, and the annealed linker oligos from the vector backbone.
  • Part linker fusions corresponding to the vector backbone were prepared by ligation of its annealed part oligos, its truncated part, and the annealed linker oligos from either genes.
  • a negative control gene-free assembly was also prepared by ligation of the vector backbone annealed part oligos, its truncated part, and its annealed linker oligos, resulting in self- assembly of the vector backbone.
  • part/linker fusions e.g., Bacillus subtilis YqjM part/pET21-a linker and Solanum lycopersicum OPRl part/pET21-a linker as well as the 3 kb vector backbone; or pET21-a part/Solanum lycopersicum OPR3 linker - 5.3 kb and Solanum lycopersicum OPR3 part/pET21-a linker - 1.2 kb as well as a 1.8 kb band corresponding to the vector backbone.
  • part/linker fusions e.g., Bacillus subtilis YqjM part/pET21-a linker and Solanum lycopersicum OPRl part/pET21-a linker as well as the 3 kb vector backbone
  • the correct size bands were then excised from the gel, and the DNA was gel extracted using the QIAGEN QIAquick Gel Extraction Kit.
  • the DNA concentration ranged from 14.4 ng/ ⁇ to 49.6 ⁇ g/ ⁇ l in a total volume of 30 ⁇ 1 (DNA cone, range 5.4 nM to 35.6 nM).
  • coli pET21-a expression vectors harbouring the Bacillus subtilis YqjM, the Solanum lycopersicum OPR1 and Solanum lycopersicum OPR3 enoate reductase genes.
  • the part junctions between the 3 '-end of the vector backbone and the 5 '-end of the genes as well as between the 3 '-end of the gene and the 5 '-end of the vector backbone were sequenced to confirm the construction.
  • the cloned enoate reductase genes were expressed in E.coli using 1 mM IPTG and 37° induction temperature, as described above in Section 3.1.1.3. Briefly, the OD600 was measured for each culture pre -induction and at various time points after induction. The remaining culture left after 24h post-induction incubation was transferred to a 50 mL falcon tube, harvested by centrifugation at 5000 rpm for 10 minutes, and the protein content of the samples were analyzed using SDS-PAGE.
  • IPTG concentration range of 0.1-1 mM IPTG
  • induction temperature range of 16-37°C
  • E.coli strains expressing the 3 enoate reductases were scaled up to 800 ml cultures and assayed for activity.
  • Enzyme assays for enoyl-CoA reductase activity were based on published methods (Bergler H, et al, (1993).
  • Protein EnvM is the NADH-dependent enoyl-ACP reductase (Fabl) of Escherichia coli. Reactions were performed in phosphate buffer (lOOmM, pH7.5), containing 300 ⁇ NADPH, NADH or FAD depending on the cofactor requirement of the enzyme, and purified enzyme (25-55 ⁇ g/ml). Reactions were started by addition of the substrates, 2,heptane dioic aicd or 2,3-didehydropimloyl-CoA.
  • Reactions were incubated at 30°C for 4 hours, and stopped by the addition of 2M HCL (10% v/v). Products were analysed by LC-MS. Controls were comprised of reaction mixtures with no substrate and reaction mixtures with inactivated (boiled for 5 min) enzymes.
  • acyl-CoA dehydrogenase activity was based on published methods (Le W, Abbas AS, Sprecher H, Vockley J and Schulz H (2000). Long-chain acyl-CoA dehydrogenase is a key enzyme in the mitochondrial ⁇ -oxidation of unsaturated fatty acids. Biochimica et Biophysica Acta. 1485 : 121 -128).
  • Assays were performed in 1ml containing 50 mM potassium phosphate buffer (pH 7.6), 36 ⁇ dichlorophenolindophenol, 0.3 mM N-ethylmaleimide, 1.5 mM phenazine methosulfate, 60 ⁇ acyl-CoA and enzyme (25-55 ⁇ g/ml). Reactions were started by the addition of phenzine methosulfate (Le et al, 2000).
  • Fatty acyl CoA reductases are involved in wax ester synthesis in plants and other higher eukaryotes for cuticle wax formation. In Archaea and bacteria, these enzymes are involved in long- chain alkane biosynthesis, although their physiological function in these organisms is still not always clear.
  • the formation of wax esters in plants begins with the elongation of acyl CoAs to form long- chain fatty acids (LFCA) (or fatty acyl-CoAs), which are then reduced via the decarbonylation pathway or acyl CoA reduction pathways (Cheesbrough and Kolattukudy, 1984).
  • Hydrocarbons are formed via the decarbonylation pathway. This pathway is initiated by a fatty acyl-CoA reductase-catalysed reaction to produce the aldehyde, followed by decarbonylation catalysed by an aldehyde decarbonylase to yield the [Cn-1 ] alkane (Cheesbrough and Kolattukudy, 1984).
  • acyl CoA reduction pathway is also initiated by a reduction of LCFAs to aldehydes, followed by a further reduction of aldehydes by an aldehyde reductase to produce even-chained primary alcohols (Millar et al., 1999) (Kolattukudy, 1971). Similar pathways are thought to exist in prokaryotes (Schirmer et al., 2010).
  • Both pathways are initiated by reduction of long-chain acyl-CoAs to aldehydes catalysed by fatty acyl-CoA reductase (FAR; EC 1.2.1.50) in a reversible reaction.
  • FAR fatty acyl-CoA reductase
  • the aldehydes are reduced further to the alcohols. This can be achieved in either one or two steps, depending on the organism.
  • the two step reduction is initiated using the acyl-CoA reductase- catalysed reaction, which releases the fatty aldehyde for a second reduction catalysed by a fatty aldehyde reductase.
  • fatty acyl CoA reductases can be broadly divided into two types: (1) alcohol-forming acyl-CoA reductases and (2) aldehyde generating acyl-CoA reductases. There are examples of both types in prokaryotes and eukaryotes. Since the target is pimelic semialdehyde, the aldehyde -generating fatty acyl CoA reductases are the most promising candidates for reduction of pimeloyl CoA.
  • the candidates were chosen using the following criteria:- (l)The enzyme must generate aldehyde only or there must be scope to eliminate alcohol formation by protein engineering; (2) The must be evidence of existence of the protein i.e. protein has been identified and purified or isolated in cellular fractions; (3) The enzyme was tested using acyl-CoA or acyl-ACP substrates; (4) The enzyme must not be part of multi-enzyme complex unless activity has been demonstrated independently of the complex. Once candidates were identified, further bioinformatics and literature searches were performed to provide additional data to rank the selections.
  • Table 1 shows the results of database searches carried out against a known aldehyde-forming enzyme (Acrl). Each of the candidates has been purified and assayed in vitro. The candidates were ranked and grouped according to their suitability. The top ranking enzymes (1-3) are considered the best candidates for further study, the intermediate ranked enzymes (4 and 5) are considered to be good candidates but may require further investigation and the lower ranked enzyme (6) would require more effort to obtain enzyme activity data, due to the absence of the protein sequence and the lack of a sequenced genome. [00348]
  • Level of evidence - Enzyme activity means enzyme has been purified either heterologously or homologously, or activity has been measured in crude extracts or other cellular fractions
  • Acinetobacter calcoaceticus Acrl (P94129) (Reiser, S., and Somerville, C. (1997): Isolation of mutants of Acinetobacter calcoaceticus deficient in wax ester synthesis and complementation of one mutation with a gene encoding a fatty acyl coenzyme A reductase.
  • J Bacterid 179, 2969-2975 An aldehyde-generating acyl-CoA reductase has been purified and contains 295 amino acids, with a molecular weight of 32.5 kDa. This enzyme has been expressed in E. coli and cell-free extracts were shown to contain acyl-CoA reductase activity.
  • AAR from Synechococcus elongatus PCC 7942 (YP_400611) (Schirmer, A., Rude, M.A., Li, X., Popova, E., and Del Cardayre, S.B. (2010). Microbial Biosynthesis of Alkanes. Science 329, 559-562): This acyl-ACP reductase is in the cyanobacterium S. elongatus and designated AAR. The enzyme is thought to be involved in alkane biosynthesis. It has been expressed and purified in E. coli MG1655. Both CoA and ACP derivatives are substrates.
  • octadecanal is formed from either oleoyl-CoA and oleoyl-ACP without fatty alcohol formation.
  • oleoyl-ACP was the preferred substrate, with a KM of 8 ⁇ compared to 130 ⁇ for oleoyl-CoA.
  • the enzyme is NADPH- dependent and requires magnesium for activity. No other substrates have been tested.
  • the selectivity for aldehyde formation and the activity with CoA derivatives makes this enzyme a good candidate for testing for pimeloyl-CoA reduction.
  • orthologues of this gene in closely related organisms (Schirmer et al., 2010), although AAR is the best studied.
  • the following patents are associated with the work from (Schirmer et al., 2010): WO 2009/140695 and WO 2009/140696.
  • LuxC is part of a multi-enzyme complex (LuxCDE) consisting of a fatty acyl-CoA reductase, fatty acyl synthetase and fatty acyl thioesterase, and is involved in bioluminescence.
  • LuxCDE multi-enzyme complex
  • the enzyme exhibits acyl-CoA reductase activity with the C14 substrate, tetradecanoyl-CoA (myristoyl-CoA), which is reduced to the corresponding aldehyde, tetradecanal. No other substrates have been tested.
  • tetradecanoyl-CoA myristoyl-CoA
  • the LuxC is part of a multisubunit complex in vivo, the enzyme has been successfully purified from the wild-type organism and has been expressed and partially purified from E. coli. In both cases, LuxC was active independently of the other subunits. This makes LuxC a good candidate to convert pimeloyl-CoA to pimelic acid semialdehyde.
  • FAR from Marinobacter aquaeolei VT8 (YP_959486) (Hofvander, P., Doan, T.T.P., and Hamberg, M. (2011).
  • a prokaryotic acyl-CoA reductase performing reduction of fatty acyl-CoA to fatty alcohol.
  • FEBS Letters 585, 3538-3543 This fatty acyl CoA reductase (designated FAR) was originally identified as a fatty aldehyde reductase (Wahlen, B.D., Oswald, W.S., Seefeldt, L.C., and Barney, B.M. (2009).
  • Aldehyde-generating acyl-CoA reductase from Bottyococcus braunii (AAB35106) (Wang, X., and Kolattukudy, P.E. (1995). Solubilization and purification of aldehyde-generating fatty acyl-CoA reductase from green alga Botryococcus braunii. FEBS Lett 370, 15-18): This enzyme is an aldehyde-generating acyl-CoA reductase from the microalga, Botryococcus braunii.
  • the enzyme has been purified from the wild-type organism and assayed using labeled activated fatty acid, [1-14C]- palmitoyl-CoA.
  • the enzyme was shown to produce the corresponding aldehyde and was NADH- dependent.
  • the substrate range has not been studied and this would need to be addressed.
  • the protein needs to be cloned, expressed and purified in E. coli.
  • Accession number AAB35106 refers to a partial 26 amino acid N-terminal sequence from the purified enzyme.
  • the full protein sequence is not deposited on UNIPROT, BRENDA or NCBI databases. Sequencing of the genome is underway (http://www.jgi.doe.gov/). There might be scope to isolate the gene using primers for the N terminal sequence, but the general lack of information about the enzyme may complicate its exploitation for metabolic engineering.
  • acyl-CoA reductases have been identified that are suitable candidates to be tested for reduction of pimeloyl-CoA to pimelic semialdehyde.
  • the enzyme from Acinetobacter calcoaceticus, Acrl appears to be the best candidate, as it is the best characterized of the enzymes identified even though there is no information on activity with short chain, C-odd substrates. If initial tests turn out to be negative, the enzyme would still be a good target for directed evolution to obtain pimeloyl CoA reduction.
  • AAR from S. elongatus and its orthologues as well as LuxC are also promising candidates. Although these enzymes operate on relatively long chain fatty acyl CoA substrates, they have not been tested with short chain substrates. Again, directed evolution may provide a promising route to obtain the desired substrate selectivity if the initial tests are negative.
  • the two enzymes from M. aquaeolei VT8 may also be promising, although some protein engineering is likely to be required to prevent oxidation of the aldehyde. These enzymes are suitable candidates because they are known to act on the shortest chain fatty acyl CoAs out of all the fatty acyl CoA reductases identified.
  • the gene encoding the enzyme from B. braunii has not yet been identified. This makes it the most difficult of the enzymes to work with, but it could be investigated if other options are not successful. Similar enzymes are also known in higher plants (e.g. Pisum sativum) and provide further options for testing, but, again, are not well characterised.
  • Clostridium kluyveri DSM555 has a CoA dependent succinate semialdehyde dehydrogenase (SucD, EC 1.2.1.76, Accession number AAA92347.1), as well as 2 other aldehyde dehydrogenases (Accession number EDK34221.1) and an acetaldehyde dehydrogenase (EC 1.2.1.10, Accession number EDK331 16.1) that may be used to convert pimloyl-CoA to pimelic acid semialdehyde.
  • Biotransformations to assay for activity of the enzymes with pimeloyl-CoA with purified (Histrap columns) enzymes produced in E. coli were performed under aerobic conditions.
  • a total reaction volume of 250 uL made of DTT (5 mM), MgS04 (5 mM), NADH (1.2 mM), adipoyl-CoA or pimeloyl-coA (1 mM) in tris buffer (50 mM, pH7.5) was prepared in a 96-well plate.
  • the reaction was started by adding 10 to 15 uL of the soluble fraction of the proteins prepared in E. coli. Each reaction was done in triplicate.
  • control buffer was prepared by replacing the enzymatic preparation with Tris (50 mM, pH7.5). This control was prepared in triplicate.
  • control enzyme was prepared using the mixture described above but replacing the hexanoyl-CoA with water for each enzymatic preparation. This control was done in duplicate.
  • Biotransformation reactions with enzyme fractions from C. kluyveri strains were performed in an anaerobic cabinet as follows: The reactions were carried out in Tris buffer (50 mM, pH7.5), DTT (5mM), MgS04 (5 to 10 mM), NADH (1 mM), NADPH (1 mM), adipoyl coenzyme A or pimeloyl coenzyme A (0.5 to 1 mM) in a total volume of 200 uL. The reaction was started by adding 15uL of enzymatic preparation (soluble or insoluble fraction). These reaction were done in triplicate. A "control buffer” was prepared by replacing the enzymatic preparation with Tris (50 mM, pH7.5). This control was prepared in triplicate.
  • a Synergi-2.5u FusionRP column (Phenomenex, Ref: 00B-4423-B0) was used for the analysis.
  • the authentic standards of the C6- and C7-semi-aldehydes were detected in negative mode using ultrapure water supplemented with 0.1% (v/v) of formic acid and acetonitrile supplemented with 5% (v/v) of ultrapure water as running buffers.
  • the method used to detect the semi-aldehydes authentic standards was used to analyse the reactions.
  • C. kluyveri are known to be oxygen sensitive, so the lack of activity observed by the 3 enzymes from C. kluyveri could be due to the assay conditions.
  • C. kluyveri cell fractions produced under anaerobic conditions with succinate as inducer in the medium, and assayed under anaerobic conditions in the native strains, were used to determine activity of SucD (EC 1.2.1.76).
  • Enzyme fractions of both strains of C. kluyveri DSM555 & DSM563 used, produced detectable amounts of pimelic acid semialdehyde from pimeloyl-CoA as analysed by LC-MS.
  • Enzyme targets for the reduction of pimelic acid into pimelic acid semialdehyde were selected from the carboxylic acid reductase family (EC 1.2.99.-, such as EC 1.2.99.6).
  • the Nocardia carboxylic acid reducatse is a monomeric enzyme that displays a large substrate specificity towards aromatic substrates and was successfully cloned and expressed in E. coli (Nocardia sp. Carboxylic acid reductase: Cloning, expression, and characterization of a new aldehyde oxidoreductase family: He et al. Appl. Environ. Microbiol. 2004, 70, 1874-1881).
  • the enzyme was therefore a good candidate for an initial screening to monitor conversion of pimelic acid to pimelic acid semialdehyde.
  • the Nocardia sp. carboxylic acid reductase (CAR) gene (GeneBank AY495697) was expressed in E. coli BL 21 along with Nocardia phosphopantetheine transferase (PPTase) and used as a cell-free extract following cell lysis and centrifugation to remove insoluble fraction.
  • CAR carboxylic acid reductase
  • PPTase Nocardia phosphopantetheine transferase
  • the activity assay was based on spectrophotometric detection of CAR activity towards sodium benzoate as positive control and sodium pimelate.
  • the conditions for the assay were taken from Applied and Environmental Microbiology 2004, 70, p i 874- 1881.
  • Each assay comprised of final concentrations of 50mM substrate ( ⁇ ), lOOmM magnesium chloride ( ⁇ ), 50mM tris-hydrochloric acid, ImM ethylenediammetetraacetic acid, ImM dithiothreitol added together from one stock solution (200 ⁇ 1), l OmM adenosine tri-phosphate ( ⁇ ), 1.5mM nicotinamide-dinucleotide phosphate hydrate (reduced) ( ⁇ ⁇ ) and the biocatalyst, which was charged as cell free extract to give a final concentration of 4.3g/L whole cell equivalents.
  • the reactions were carried out at ambient temperature ( ⁇ 20degC) in a 2mL cuvette at pH 7.5.
  • Each assay was charged first with magnesium chloride ( ⁇ ⁇ ), 50mM tris-hydrochloric acid, ImM ethylenediammetetraacetic acid, ImM dithiothreitol (200 ⁇ 1), lOmM adenosine tri-phosphate ( ⁇ ) and 1.5mM nicotinamide-dinucleotide phosphate hydrate (reduced) ( ⁇ ) and absorbance measurements recorded at thirty second (30s) intervals from time zero (0s) to two minutes (120s). Biocatalyst was then charged and absorbance recorded at thirty second (30s) intervals from time zero (0s) to two minutes (120s). Lastly the substrate was charged and absorbance recorded at thirty second (30s) intervals from time zero (0s) to three minutes (180s).
  • CAR from Nocardia catalysed the reduction of pimelic acid to pimelic acid semialdehyde with >100% of the activity for benzoic acid.
  • Other suitable enzymes for the reduction of pimelic acid from EC 1.2.99.6 include for example that of Streptomyces griseus (Suzuki et al, 2007.
  • GriC and GriD constitute a carboxylic acid reductase involved in grixazone biosynthesis in Streptomyces griseus. J Antibiot (Tokyo). Jun;60(6):380-7).
  • ThnG a non-acylating aldehyde dehydrogenase in Sphingomonas and Cupriavidus species, converts pimelic acid semialdehyde to pimelic acid during tetralin degradation (See, Fig. 8) and may be reversible.
  • Enzymes that are able to catalyse the hydrolysis or transfer of CoASH or acyl-[acyl-carrier protein] thioesters include thioester hydrolases from EC 3.2.1.-, such as EC 3.1.2.2, EC 3.1.2.14EC 3.1.2.18; 3.1.2.19, 3.1.2.20 EC 3.1.2.21, acid-thiol ligases from EC 6.2.1.-, such as EC 6.2.1.3, EC 6.2.1.5; EC 6.2.1.14, EC 6.2.1.20; EC 6.2.1.23 and CoA transferases from EC 2.8.3.-, such as EC 2.8.3.12 and EC 2.8.3.13 or or the gene product of ThnH that catalyses the reversible transfer of CoA to pimelic acid (Aroa Lopez-Sanchez, Belen Floriano, Eloisa Andujar, Maria Jose Hernaez, and Eduardo Santero, 2010.
  • thioesterases present the best candidates for the hydrolysis of pimeloyl-CoA due to the large number of characterized enzymes and their broad substrate specificity in terms of chain length (C-3 to C-26) and in terms of functional groups (Dicarboxylyl-CoA substrate).
  • Acid thiol ligases catalyse the formation of carbon-sulfur bonds with concomitant hydrolysis of adenosine triphosphate (ATP).
  • ATP adenosine triphosphate
  • the CoA ligases have not been tested rigorously or systematically for reversibility. No crystal structures are available, so the active site structure is not known. Therefore, it is not clear whether AMP, pyrophosphate or magnesium ions would be needed for the reverse reaction, and in vitro assays should be done in the presence and absence of combinations of these cofactors. Of course, the ultimate goal is to obtain the reverse reaction in whole cells, and this might be more challenging. The later reactions in the pathway will, of course, pull the equilibrium in the thiolytic direction.
  • Pimelic acid CoA synthase (6-carboxyhexanoate-CoA ligase) was discovered in Bacillus sphaericus using cell free extracts. It is involved in the biotin biosynthesis pathway. The enzyme activity was determined indirectly by a reaction coupled with 7-keto-8-aminopelargonic acid synthetase and microbiological assay using Saccharomyces cerevisiae[l].T e gene encoding pimelic acid CoA synthase (bioW) was identified together with 7-keto-8-aminopelargonic acid synthetase (bioF) by complementation studies of E. coli mutants with the library carrying sequences from the B.
  • bioW pimelic acid CoA synthase
  • bioF 7-keto-8-aminopelargonic acid synthetase
  • the enzyme was active in the presence of disodium pimelate, ATP, CoASH and MgCl 2 .
  • the products of the reaction were pimeloyl-CoA and AMP, no ADP was detected.
  • the enzyme was specific only for ATP and pimelate.
  • Other nucleotides such as GTP, AMP
  • alternative substrates such as glutarate, adipate, suberate
  • PauA GenBank: AJ012480.1 from Pseudomonas mendocina 35 (EC 6.2.1.14)
  • Catabolic pimeloyl-CoA ligase, pauA is involved in degradation of dicarboxylic acids (C6-C10) in Pseudomonas mendocina 35 [Binieda A, Fuhrmann M, Lehner B, Rey-Berthod C, Frutiger-Hughes S, Hughes G, Shaw NM. (1999) Purification, characterization, DNA sequence and cloning of a pimeloyl-CoA synthetase from Pseudomonas mendocina 35.Biochem J. 340:793-801].
  • the enzyme activates dicarboxylic acids which are subsequently metabolized via the beta-oxidation pathway.
  • the enzyme was purified to homogeneity and the partial amino acid sequence was determined.
  • PauA showed a broader substrate range than bioW, and accepts pimelate (100% relative activity), adipate (72%) and azelate (18%).
  • the enzyme was between one and two orders of magnitude more active than bioW involved in the biotin biosynthesis pathways.
  • the gene pauA was identified and the ligase was successfully overexpressed in E. coli.
  • the ligase pauA from Pseudomonas mendocina was selected as an example from this class because of its substrate range and specific activity (preferred substrate is pimelic acid, Binieda et al., 1999. Purification, characterization, DNA sequence and cloning of a pimeloyol-CoA synthetase from Pseudomonas mendocina 35. Biochem. J. 340: 793-801).
  • Rhodopseudomonas palustris pimA which likewise has been expressed in E.coli and shown to have ligase activity with a broad range of substrates (C7-C14 dicarboxylic acids), although pimelic acid was not the preferred substrate (Harrison and Harwoo, 2005.
  • the pimFABCDE operon from Rhodopseudomonas palustris mediates dicarboxylic acid degradation and participates in anaerobic benzoate degradation. Microbiol. 151 :727-736). For this reason the pauA gene from Pseudomonas mendocina seems to be a better choice and was chosen for exemplification.
  • the succinyl-CoA synthetase from the hyperthermophilic Archaea Thermococcus kodakaraensis (SCS») is a heteromeric enzyme ( ⁇ 2 ⁇ 2) encoded by TK1880 (a-subunit) and TK0943 ( ⁇ -subunit).
  • SCS catalyzes the reversible conversion of succinyl-CoA to succinate and CoA concomitant with substrate level phosphorylation of ADP/GDP.
  • SCS is relatively specific, displaying relevant levels of activity for only a few acids.
  • the enzyme was purified about 34-fold with 79% recovery [Izumi Y, Morita H, Tani Y, Ogata K. (1974) The pimeloyl-CoA synthetase responsible for the first step in biotin biosynthesis by microorganisms. Agr.Biol. Chem. 38, 2257-2262].
  • the reaction required pimelate, ATP, CoASH and MgCl 2 .
  • the enzyme was specific towards pimelate only, and other dicarboxylic acids were not accepted. Interestingly, ATP could be replaced with ADP, suggesting that pyrophosphate cleavage is not essential.
  • the amino acid and nucleotide sequences are not known. However three complete genomes of Bacillus megaterium have been published since 1974. Therefore, a BLAST search for bioW homologs was undertaken. Unfortunately, this did not show any matches.
  • the activity was found in the soluble fraction of pea protein extracts [7].
  • the enzyme required ATP/Mg 2+ and CoASH.
  • the molecular weight of the ligase was determined by western blot technique using polyclonal antibodies against the ligase from Lysinibacillus sphaericus.
  • the enzyme accepted pimelic acid as well as azelaic acid, but not nonanoic acid.
  • GTP could be used instead of ATP, but no reaction was observed for ADP.
  • the gene encoding the soluble pimeloyl-CoA synthetase was not identified.
  • the enzyme was located in the peroxisomal protein fraction [GerblingH,AxiotisS,Douce R. (1994) A new acyl-CoA synthetase, located in higher plant cytosol. J Plant Physiol. 143 : 561-564.]. Pimelic acid was activated to pimeloyl-CoA and subsequently degraded to acetyl-CoA and malonyl- CoA by the beta-oxidation pathway. The substrate range of this enzyme was not tested. It showed a higher pH-optimum than soluble acyl-CoA synthetase from the same organism.
  • Dicarboxy lie acids (C5-C16) were converted into their CoA esters by a dicarboxylyl-CoA synthetase from the microsomal protein fraction from a rat liver [Vamecq J, de Hoffmann E, Van Hoof F. (1985)The microsomal dicarboxylyl-CoA synthetase. Biochem J. 230:683-693.].
  • the enzyme uses ATP, but not GTP. The reaction is inhibited by its products, and AMP is more inhibitory than pyrophosphate. The enzyme showed the highest activity for the C12 substrate. In some assays of dicarboxylyl-CoA synthetase, the plateau of CoA consumption was followed by CoA release.
  • the activity was found in environmental cultures of denitrifying bacteria from activated sludge enriched to grow on sodium nitrate and pimelate [Gallus C, Schink B.(1994) Anaerobic degradation of pimelate by newly isolated denitrifying b&cteh&.Microbiology. 140: 409-416.].
  • the enzyme was active in the presence of ATP and Mg 2+ .
  • the product of the reaction was AMP rather than ADP.
  • the enzyme did not accept benzoic acid.
  • CoA transferases are less attractive candidates than thioesterases, due to either a narrow substrate specificity or a lack of gene/protein characterization such as for the succinate- hydroxymethylglutarate CoA transferase (EC 2.8.3.13, despite showing activity towards the C-6 derivative of pimeloyl-CoA, adipyl-CoA- (Substrate specificity of a dicarboxyl-CoA:dicarboxylic acid coenzyme A transferase from rat liver mitochondria: Deana et al. Biochem Int. 1992, 26, 767- 773).
  • pimeloyl-CoA transferase Only one succinyl-CoA: pimeloyl-CoA transferase has been reported but the gene responsible for activity is not available and the organism reported was not identified (Gallus and Schink (1994) Microbiol. 10: 409-416 Anaerobic degradation of pimelate by newly isolated denitrifying bacteria). Some enzymes from this family may still be useful, and the following ernzymes were identified as potential candidates:
  • Glutaconate coenzyme A-transferase (Get) from Acidaminococcus fermentans is an enzyme catalysing the formation of (R)-2-hydroxyglutaryl-CoA from acetyl-CoA and the free acid. Get was characterized as a heterooctamer ( ⁇ 4 ⁇ 4 ) composed of two different subunits (GctA, 35725 Da and GctB, 29168 Da). Both subunits: GctA, and GctB have been co-expressed in E.coli and shown to be functional as glutaconate CoA-transferase (EC 2.8.3.12).
  • Get is also active on unsaturated and saturated as well as hydroxylated substrates (2-Hydroxyglutarate), but is not tested against C 7 coenzyme derivatives (Mack et al., 1994. Location of the two genes encoding glutaconate coenzyme A- transferase at the beginning of the hydroxyglutarate operon in Acidaminococcus fermentans.). Eur. J. Biochem, 226:41-51).
  • This enzyme was successfully expressed in E. coli and used for enzymatic biosynthesis of glutaconyl-CoA, 3-methylglutaconyl-CoA, butynedioyl-CoA, 2-hydroxyadpioyl-CoA, oxalocrotonyl-CoA and muconyl-CoA (Parthasarthy A, Pierik A, Kahnt J, Zelder O, Buckel W. (2011) Substrate specificity of 2-hydroxyglutaryl-CoA dehydratase from Clostiridium symbiosum: Toward a bio-based production of adipic acid. Biochemistry. 50: 3540-35500).
  • CoA transferases include the CoA transferase from Rattus norvegious (Succinate-hydroxymethylglutarate CoA-transferase EC 2.8.3.13), since it accepts adipyl-CoA as substrate, with higher affinity than that observed for succinyl-CoA, malonyl-CoA, glutaryl-CoA (Substrate specificity of a dicarboxyl-CoA:dicarboxylic acid coenzyme A transferase from rat liver mitochondria (Deana Biochem Int. 1992, 26, 767-773).
  • the acetyl-CoA:5- hydroxypentanoate CoA-transferase from Clostridium aminovalericum (EC 2.8.3.14) has not been tested for activity towards C7 substrates, but does show activity towards C3-C5 substrates and is specific for saturated substrates (Eikmanns and Buckel, 1990. Properties of 5 -hydroxy valerate CoA- transferase from Clostridium aminovalericum. Biol. Chem. 371 : 1077-1082). The gene(s) responsible for this activity were not listed.
  • the enzyme from Pseudomonas mendocina was entirely insoluble and only trace amounts could be refolded, thus only the enzymes from Acidaminococcus and Thermococcus were used in activity assays with their native substrates. Activity for both enzymes was confirmed using colorimetric assays monitoring the formation of CoA esters of their natural substrates, (E)-glutaconate and succinate, by measuring absorbance at 520 nm and 405 nm, respectively.
  • Biotransaformation reactions to assay the disappearance of Adipoyl-CoA and Pimeloyl- CoA and formation of adipic acid or pimelic acid was performed in 1 ml reactions by adding 30 ul protein solution to 300 ul reaction mixture that contained (per ml) in the reaction mixtures as described below:
  • Thioestrases are hydrolytic enzymes that catalyze the hydrolysis of CoA or [acp] thioesters: O o o o
  • Thioesterases are ubiquitous in all organisms acting on CoA or [acp] activated carboxylic acids and belong to EC 3.1.2, including EC 3.1.2.2; EC 3.1.2.28; EC 3.1.2.19; EC 3.1.2.20 for CoA ester hysdorlases, while [acp] thioester hydrolases are found in EC 3.1.2.14 and EC 3.1.2.21. Only two thioesterases (ACOT8 and ACOT4) have been tested for activity towards dicarboxylyl-mono- CoA esters such as succinyl-CoA.
  • ACOT8 peroxisomal acyl-CoA thioesterase 8 (formerly PTE-2) from Mus musculus
  • ACOT8 is a homologue of human PET1 thioesterase.
  • the enzyme was overexpressed as a His-Tag fusion protein in E. coli and purified to homogeneity using HisTrap column chromatography [Westin MA, Hunt MC, Alexson SE. (2005) The identification of a succinyl-CoA thioesterase suggests a novel pathway for succinate production in peroxisomes. J Biol Chem. 280: 38125-38132].
  • the activity was inhibited at substrate concentrations > 5-10 ⁇ for long-chain acyl-CoAs, but addition of BSA prevented inhibition.
  • the substrate range of the thioesterase was tested using saturated and unsaturated straight- chain acyl-CoAs (C2-C20).
  • ACOT8 was also tested for activity towards dicarboxylyl-mono-CoA derivatives and preferentially hydrolyzed the longer-chain substrates (activity increase with C chain length from C3 to C12) [Hunt MC, Solaas K, Kase BF, Alexson SE. (2002) Characterization of an acyl-coA thioesterase that functions as a major regulator of peroxisomal lipid metabolism. J Biol Chem. 277: 1128-1138].
  • ACOT3 (formerly PTE- la) from Mus musculus
  • ACOT3 was expressed in E. coli and purified to homogeneity [19].
  • the enzyme showed activity for monocarboxylic acid esters from C8 to C26, with the highest activity towards longer chain substrates (CI 6).
  • the enzyme did not accept bile acid esters. There was no inhibition with free CoASH. Dicarboxylic acid esters were not tested.
  • ACOT5 (formerly PTE-lc) from Mus musculus
  • ACOT5 was expressed in E. coli and purified to homogeneity [Westin MA, Alexson SE, Hunt MC. (2004) Molecular cloning and characterization of two mouse peroxisome proliferator-activated receptor alpha (PPARalpha)-regulated peroxisomal acyl-CoA thioesterases. J Biol Chem. 279: 21841- 21848].
  • the enzyme showed activity for monocarboxylic acid esters from C6 to C20, with the highest activity towards medium chain substrates (CIO). The enzyme did not accept bile acid esters. There was no inhibition with free CoASH. Dicarboxylic acid esters were not tested.
  • ACOT4 peroxisomal acyl-CoA thioesterase 4 (formerly PTE- lb) from Mus musculus
  • ACOT4 was expressed in E. coli and purified to homogeneity [18].
  • the enzyme showed a narrow substrate range, accepting succinyl-CoA and glutaryl-CoA.
  • the reaction was not inhibited by CoASH at concentrations up to 500 ⁇ .
  • ACOT8, ACOT5 and ACOT3 are thus excellent candidates for the hydrolysis of dicarboxylyl-CoAs and other acyl-CoAs that are involved in the pimelic acid biosynthesis pathway. 3.1.5.3.2 Cloning, expression and biotransformations with selected thioesterases
  • Both genes were selected as wild-type sequences for the construction of the corresponding E. coli expression vectors and used to transform E. coli BL21 (DE3).
  • Expression of Haemophilus influenzae YciA thioesterase was achieved in E. coli, ( Figure 14h) but not of the Mus musculus Acot8 thioesterase.
  • a protein band at the expected size (17 kDa) was clearly detected after IPTG induction in the soluble fraction (Lanes 2 to 3) and in the insoluble fraction (Lanes 6 and 7). No corresponding protein was observed in the negative control experiment (Lanes 4 and 8) suggesting that the expected 17 kDa Haemophilus influenzae YciA thioesterase protein was successfully expressed in E.
  • Coenzyme A sodium salt (150mg, 195 ⁇ 1) was suspended in a mixture of acetone (22.5mL) and water (225 ⁇ ). The suspension was cooled to OdegC on ice and adjusted to pH 8 with 200mM sodium bicarbonate solution. Acid chloride (586 ⁇ 1, 3eq) was added slowly to the suspension with stirring whilst maintaining the reaction pH at 8 by further addition of sodium bicarbonate solution. The reaction mixture was allowed to stir at 0°C for a further lh.
  • reaction mixture was concentrated in vacuo and the white residue was resuspended in water (20mL), adjusted to pH 3 with 5M phosphoric acid and extracted with diethyl ether (3 x 20mL). The remaining aqueous layer was then purified by solid phase extraction (SPE) using a Strata X 33 ⁇ polymeric reverse phase lg/20mL cartridge supplied by Phenomenex.
  • SPE solid phase extraction
  • the SPE cartridge was primed with methanol and equilibrated with 1 OmM sodium phosphate (pH4.2) buffer prior to use.
  • the aqueous layer containing the crude acyl-CoA product was loaded onto the cartridge.
  • l OmM sodium phosphate (pH4.2) buffer washes (3 x 5mL) were carried out, followed by a stepwise gradient using 5, 10 and 20% MeOH/buffer elutions (3 x 5mL). Each fraction was collected and analysed by HPLC. The fractions containing product only were combined and concentrated in vacuo. The isolated products were used directly in the biotransformations without further purification. 1H NMR or LCMS were used to characterise the products.
  • Biotransformations were carried out in a final volume of 2mL.
  • the biotransformations comprised of ImM substrate, 200mM KC1 and 50mM potassium Hepes (pH 7.5).
  • Negative controls comprised of cells harbouring the empty vector
  • the biocatalyst was charged as a cell free extract to give a final concentration of ⁇ 15g/L whole cell equivalents.
  • the reactions were carried out at ambient temperature (20°C) in 15mL falcon tubes with stirring.
  • Each biotransformation was sampled after lh, 4h and 24h and analysed by HPLC (Agilent 1 100) on a Phenomenex Luna C18 250 x 4.6mm, 5 micron column..
  • each sample Prior to injection, each sample was heated to 100°C for 5 mins to stop the reaction, spun at 14000 rpm for 2 minutes and filtered through a 0.2 micron syringe filter.
  • the filtrates were analysed by HPLC at 260 nm for the disappearance of pimeloyl-CoA (Rt 12.39 mins) and the formation of CoA (Rt 7.98 minutes), both which absorbs strongly at this wavelength, while pimlic acid was not detectable by UV at 260 nm. Pimelic acid formation was confirmed by LC-MS.
  • a low level of background hydrolysis was observed in the negative control, which can be ascribed to low levels of endogenous thioesterase activity in the E. coli harbouring the empty vector.
  • Complete hydrolysis of pimeloyl-CoA to pimelic acid and acetyl-CoA was observed after 1 hour of reaction.
  • Transaminases or aminotransferases are enzymes that catalyse the reaction between an amino acid and a keto acid. This reaction involves removing the amino group from the amino donor, leaving behind an a-keto acid, and transferring it to the acceptor a-keto acid and converting it into an amino acid. They are important enzymes in the synthesis of amino acids and are ubiquitous throughout nature. Many of these enzymes contain pyridoxal-phosphate (PLP) as cofactor. These enzymes belong to a large diverse family covering a wide range of cellular functions, using a wide range of substrates. 3.1.6.1 Selection of transaminases
  • a number of candidates, identified by literature searching, BLAST searching and protein database searching are suitable enzymes for te conversion of pimelic acid semialdehyde to 7- aminoheptanoic acid.
  • the enzyme has a relatively wide substrate range, including 6-aminohexanoate and 7- aminoheptanoate, using 2-oxoglutatarate as the amino acceptor, and forming the corresponding semialdehydes (Yonaha, K., K. Suzuki, and S. Toyama, 4-Aminobutyrate: 2-oxoglutarate aminotransferase of Streptomyces griseus: purification and properties. Eur J Biochem, 1985. 146(1): p. 101-6). Although this is very promising, the reversibility of these reactions has not been tested.
  • S. griseus IFO 3102 S. griseus subsp.griseus
  • the cofactor is pyridoxal 5'-phosphate.
  • a protein is deposited on the Pubmed database (Table 1). The gene encoding this (SGR_1829), annotated as a putative 4-aminotransferase, is present in the genome sequence of S. griseus IFO 13350, which is closely related to IFO 3102.
  • this protein is a close homologue of the enzyme described and studied (Yonaha, K., K. Suzuki, and S. Toyama, 4-Aminobutyrate:2-oxoglutarate aminotransferase of Streptomyces griseus: purification and properties. Eur J Biochem, 1985. 146(1): p. 101 -6). In light of the substrate range data available, this protein is a very good candidate for testing as a suitable aminotransferase enzyme.
  • AAAAT alpha-aminoadipate aminotransferases
  • PDB ID: 2ZP7 It is a very well studied enzyme and its crystal structure is available (PDB ID: 2ZP7), which offers mechanistic insights into substrate specificity ( Ouchi, T., et al., Dual roles of a conserved pair, Arg23 and Ser20, in recognition of multiple substrates in alpha-aminoadipate aminotransferase from Thermus thermophilus. Biochem Biophys Res Commun, 2009. 388(1): p. 21 -7; Tomita, T., et al., Mechanism for multiple-substrates recognition of alpha-aminoadipate aminotransferase from Thermus thermophilus. Proteins, 2009. 75(2): p. 348-59).
  • This enzyme is similar to the enzyme from Thermus thermophilus described previously. It is annotated as a kynurenine/aminoadipate aminotransferase, although there is some evidence to suggest that two distinct proteins carry out these reactions (Mawal, M.R. and D.R. Deshmukh, Alpha- aminoadipate and kynurenine aminotransferase activities from rat kidney. Evidence for separate identity. J Biol Chem, 1991. 266(4): p. 2573-5). The protein has been purified from rat kidney and expressed in human embryonic kidney fibroblast cells and was found to be around 100 kDa in size. The enzyme can accept 2-oxoadipate as a substrate.
  • Diamino aminotransferases from EC 2.6.1.48 such as the 5-aminovalerate transaminases are also suitable catalysts for the preparation of 7-aminoheptanoic aicd form pimelic acid semialdehyde.
  • a further 5 genes (Bacillus weihenstephanensis KBAB4, Pseudomonas aeruginosa (gi9951072), Bacillus subtilis (gil6078032), Pseudomonas syringae class III, Rhodobacter sphaeroides class III) were selected (DSM patent (Preparation of 6-aminocaproic acid from 5-formyl valeric acid: DSM - US201 1-0171699 Al patent).
  • the three biotransformations contained 50mM sodium pyruvate and lOmM 7-aminoheptanoic acid. The reactions were held in a 30°C orbital incubator and aliquots removed periodically from the biotransformations for analysis. Sample ID sodium pyruvate/mM 7-aminoheptanoic acid/mM Enzyme loading v/v% rc0212-40-1 50 10 0.33 rc0212-40-2 50 10 0.66 rc0212-40-3 50 10 1.67
  • L-2-aminosuberic acid 2-ketosuberic acid [00426] An aminotransferase conversion of L-2-aminosuberic acid to 2-ketosuberic acid provides an opportunity to modulate biotransformations.
  • ING66 biotransformations (rc0212-46-1 and -2) showed significantly higher levels of L-2-aminosuberic acid degradation compared to the pING2022 and pING2030 aminotransferases.
  • ING 66 branched chain aminoacid aminotransferase is already known to exhibit excellent selectivity for L-2-aminoglutarate (L-glutamic acid) which is a shorter (C5) homologue of L-2-aminosuberic acid so this preference in activity for ING 66 is not unexpected.
  • Bacillus weihenstephanensis ⁇ -amino acid transaminase (Accession number JAl 14119.1) was expressed and purified using standard techniques. Reactions were performed with both adipic acid semialdehyde and pimelic acid semialdehyde and product formation (6-aminocaproic acid and 7-aminoheptanoic acid) was confirmed by HPLC (Agilent 1200, GraceSmart RP C18 column).
  • ester aldehyde 3a/b (lg, 6.9mmol, leq) was suspended in 50mM sodium phosphate buffer (pH 8) (20mL). Immobilised lipase resin (lOOmg) was charged and the suspension was heated to 50degC and stirred for 45 mins. After this time, GC-MS indicated the ester aldehyde starting material 3a/b had been consumed. The suspension was cooled to RT, the lipase beads were filtered and the resultant filtrate was concentrated in vacuo to afford the crude C6/C7 semi aldehydes 4a/4b in phosphate buffer. The products were characterised by ! H NMR.
  • ester aldehyde 3a/b (lg, 6.9mmol, l eq) was suspended in 50mM sodium phosphate buffer (pH 8) (20mL). Immobilised lipase resin (lOOmg) was charged and the suspension was heated to 50degC and stirred for 45 mins. After this time, GC-MS indicated the ester aldehyde starting material 3a/b had been consumed. The suspension was cooled to RT, the lipase beads were filtered and the resultant filtrate was concentrated in vacuo to afford the crude C6/C7 semi aldehydes 4a/4b in phosphate buffer. The products were characterised by ! H NMR.
  • WCE whole cell equivalents
  • V.f. ⁇ amino acid transaminase biotransformation protocol [00440] 6 biotransformations were then carried out each in a final volume of 5mL. Each biotransformation comprised of 5mM semi aldehyde, lOOmM L-monosodium glutamate and the biocatalyst, which was charged as cell free extract to give a final concentration of 15g/L whole cell equivalents. The reactions were carried out at 30degC in 15mL falcon tubes with stirring at pH 7. Each biotransformation was sampled periodically and analysed by HPLC. Prior to injection, each sample was heated to lOOdegC for 5 mins, spun at 14000rpm for 2 mins and filtered through a 0.2 micron syringe filter.
  • V.f. ⁇ -amino acid transaminase has activity towards 6-oxo hexanoic acid and 7-oxo heptanoic acid. Formation of the corresponding 6-amino hexanoic acid and 7-amino heptanoic acid products were measured by HPLC. Identification of the product peaks were determined by correlating the retention times of their respective external reference standards.
  • biotransformations were then carried out in a final volume of lmL.
  • Each biotransformation comprised of lOmM semi aldehyde, l OOmM L-monosodium glutamate and a range of purified enzyme aliquot volumes (100, 10 and ⁇ ⁇ ). This was done to evaluate transaminase activity at different protein concentrations.
  • Each biotransformation was carried out at pH 7 and placed in a shaking incubator at 30degC (250rpm). Sampling was done periodically and analysed by
  • 6-oxo-hexanoic acid E.coli (BL21 DE3) ⁇ ⁇ , Negative control using host cells with empty vector. Subjected to the same purification process as the over- expressed strain.
  • Derivatisation was done in the autosampler of the HPLC using the following injector program: 2 ⁇ 1, sample, 6 ⁇ derivatisation mixture, 6 ⁇ potassium borate buffer pHIO was mixed in the injector loop and held for 4 mins prior to injection.
  • D- and L-specific amino acid aminotransferases for the production of D- and L-2-aminosuberate substituted for D- or L-specific decarboxylases to produce 7-aminoheptanoic acid
  • IlvE L-AAAT from E.coli (WT gene: X02413.1 (fragment 301..1230); WT protein: 110425 OA) was selected as the L-selective aminotransferase and Bacillus sphaericus D-AAAT (WT gene: U26732.1 (fragment 427..1278) WT protein: P54693.1) as an example of a D-selective alpha amino acid aminotransferase to demonstrate the conversion of a-ketosuberate to a-aminosuberate.
  • the product was extracted with methyl tert butyl ether (3 x 250mL), dried over sodium sulfate, filtered and concentrated in vacuo to afford the crude diethyl ester (2) as a colourless oil in 70% isolated yield.
  • the diethyl ester was purified by high vacuum distillation prior to the next stage.
  • biotransformations were carried out each in a final volume of 5mL. Each biotransformation comprised of lOmM substrate and 50mM L-monosodium glutamate. The biocatalyst was charged as whole cells to give obtain a final concentration of 1 OOg/L. The reactions were carried out at 30degC in 15mL falcon tubes with stirring. Each biotransformation was sampled periodically and analysed by HPLC. Prior to injection, each sample was heated to lOOdegC for 5 mins, spun at 14000rpm for 2 mins and filtered through a 0.2 micron syringe filter.
  • biotransformations were carried out in a final volume of 5mL. Each biotransformation comprised of lOmM substrate, 50mM D-Alanine and 0.4mM pyridoxal phosphate. The biocatalyst was charged as a cell free extract to obtain a final concentration of 21 g/L whole cell equivalents. The reactions were carried out at 30degC in 15mL falcon tubes in a shaking incubator. Each biotransformation was sampled periodically and analysed by HPLC. Prior to injection, each sample was heated to lOOdegC for 5 mins, spun at 14000rpm for 2 mins and filtered through a 0.2 micron syringe filter.
  • biotransformation results were determined by correlating HPLC retention times of the external reference standards to the observed product peaks formed in each biotransformation.
  • a non-chiral derivatisation HPLC method was employed to enable detection of the corresponding derivatised product adducts at 338nm on a reverse phase CI 8 column.
  • IlvE is well- known to be enantiospecific forming only L-amino acids from keto-acids so an achiral analytical method was used for simplicity.
  • the details for the non-chiral derivatisation protocol are outlined in the Appendices section.
  • D-2-amino glutamic acid positive control
  • D-2-amino-suberic acid and D-2-amino-pimelic acid products were generated from 2-oxo glutarate, 2-oxo suberate and 2-oxo pimelate respectively.
  • biotransformation results were determined by correlating HPLC retention times of external reference standards to the observed product peaks formed in each biotransformation.
  • HPLC was also employed to demonstrate the enantioselectivity of the enzyme.
  • a pre-column denvatisation protocol for each sample in the presence of a chiral derivatisation reagent, it was possible to separate the corresponding diastereomeric adducts on a reverse phase CI 8 column at 338nm wavelength.
  • the details for the chiral derivatisation protocol are outlined in the Appendices section.
  • Derivatisation was done in the autosampler of the HPLC using the following injector program: 2 ⁇ sample, 6 ⁇ . derivatisation mixture, 6 ⁇ ⁇ potassium borate buffer pHIO was mixed in the injector loop and held for 4 mins prior to injection.
  • Derivatisation was done in the autosampler of the HPLC using the following injector program: 2 ⁇ 1, sample, 6 ⁇ L derivatisation mixture, 6 ⁇ potassium borate buffer pHIO was mixed in the injector loop and held for 4 mins prior to injection.
  • Suitable catalysts were selected from the a-amino acid decarboxylases as candidates for the decarboxylation of a-amino suberic acid, in particular the aspartate 4-decarboxylase (EC 4.1.1.1 1), the glutamate decarboxylase (EC 4.1.1.15), the lysine decarboxylase (EC 4.1.1.18), the diaminopimelate decarboxylase (EC 4.1.1.20), and the diaminobutyrate decarboxylase (EC 4.1.1.86).
  • the aspartate 4-decarboxylase EC 4.1.1.1 1
  • the glutamate decarboxylase EC 4.1.1.15
  • the lysine decarboxylase EC 4.1.1.18
  • the diaminopimelate decarboxylase EC 4.1.1.20
  • diaminobutyrate decarboxylase EC 4.1.1.86
  • the Escherichia coli GadA glutamate decarboxylase and Escherichia coli LysA diaminopimelate decarboxylase were initially selected from those groups of proteins.
  • Other suitable enzymes include LysA from Methanocaldococcus jannaschii (AAB99100) (EC 4.1.1.20) (Ray, S.S., et ah, Cocrystal structures of diaminopimelate decarboxylase: mechanism, evolution, and inhibition of an antibiotic resistance accessory factor. Structure, 2002. 10(1 1): p. 1499-508), and the lysine decarboxylases present in E.
  • CadA (AAA23536), which is inducible under anaerobic conditions and by lysine, and has a very high activity and Ldc (accession D87518) that is constitutively expressed but has lower activity for lysine (Kikuchi, Y., et al, Characterization of a second lysine decarboxylase isolated from Escherichia coli. J Bacteriol, 1997. 179(14): p. 4486-9).
  • Biotransformations were performed to assay activity for 2-aminosuberate as well as 2- aminoheptanoic acid.
  • Each biotransformation comprised of l OmM substrate, l Oug/mL pyridoxal phosphate and ImM ⁇ -mercaptoethanol, adjusted to pH 7 with 1M sodium hydroxide.
  • the biocatalyst was charged as cell free extract to obtain a final concentration of 20g/L whole cell equivalents.
  • the reactions were carried out at 37degC in 15mL falcon tubes with stirring.
  • Each biotransformation was sampled periodically and analysed by HPLC. Prior to injection, each sample was heated to 100°C for 5 mins, spun at 14000rpm for 2 mins and filtered through a 0.2 micron syringe filter.
  • Derivatisation was done in the autosampler of the HPLC using the following injector program: 2 ⁇ sample, 6 ⁇ derivatisation mixture, 6 ⁇ potassium borate buffer (pHIO) was mixed in the injector loop and held for 4 mins prior to injection.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
EP12816586.7A 2011-12-21 2012-12-21 Bioconversion process for producing nylon-7, nylon-7,7 and polyesters Withdrawn EP2794897A2 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US201161578272P 2011-12-21 2011-12-21
US201161578265P 2011-12-21 2011-12-21
US201161578289P 2011-12-21 2011-12-21
PCT/US2012/044984 WO2013003744A2 (en) 2011-06-30 2012-06-29 Bioconversion process for producing nylon-7, nylon-7,7 and polyesters
PCT/US2012/071472 WO2013096898A2 (en) 2011-12-21 2012-12-21 Bioconversion process for producing nylon-7, nylon-7,7 and polyesters

Publications (1)

Publication Number Publication Date
EP2794897A2 true EP2794897A2 (en) 2014-10-29

Family

ID=48669717

Family Applications (1)

Application Number Title Priority Date Filing Date
EP12816586.7A Withdrawn EP2794897A2 (en) 2011-12-21 2012-12-21 Bioconversion process for producing nylon-7, nylon-7,7 and polyesters

Country Status (5)

Country Link
EP (1) EP2794897A2 (https=)
JP (2) JP2015500663A (https=)
CN (1) CN104126012A (https=)
BR (1) BR112014015077A2 (https=)
WO (1) WO2013096898A2 (https=)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108998430A (zh) * 2018-06-29 2018-12-14 浙江工业大学 一种界面自组装羰基还原酶及在(r)-3-羟基-3-苯基丙酸乙酯合成中的应用

Families Citing this family (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2720997A1 (en) 2011-06-17 2014-04-23 Invista Technologies S.à.r.l. Use of hydrolases to increase monomer content in waste stream
EP2726627A2 (en) * 2011-06-30 2014-05-07 Invista Technologies S.à.r.l. Bioconversion process for producing nylon-7, nylon-7,7 and polyesters
US9102960B2 (en) 2011-12-16 2015-08-11 Invista North America S.á.r.l. Methods of producing 6-carbon chemicals via CoA-dependent carbon chain elongation associated with carbon storage
US9102958B2 (en) 2011-12-16 2015-08-11 Invista North America S.á.r.l. Methods of producing 6-carbon chemicals via CoA-dependent carbon chain elongation associated with carbon storage
US9790525B2 (en) 2012-12-14 2017-10-17 Invista North America S.A.R.L. Methods of producing 7-carbon chemicals via CoA-dependent carbon chain elongation associated with carbon storage
EP2938734A2 (en) * 2012-12-31 2015-11-04 Invista Technologies S.A R.L. Methods of producing 7-carbon chemicals via aromatic compounds
WO2014105793A1 (en) * 2012-12-31 2014-07-03 Invista North America S.A.R.L. Methods of producing 7-carbon chemicals from long chain fatty acids via oxidative cleavage
CN105408487A (zh) * 2012-12-31 2016-03-16 英威达技术有限责任公司 通过甲酯保护的碳链延伸生产7碳化学物的方法
EP2938736A2 (en) * 2012-12-31 2015-11-04 Invista Technologies S.A R.L. Methods of producing 7-carbon chemicals via c1 carbon chain elongation associated with coenzyme b synthesis
CN105073214A (zh) 2012-12-31 2015-11-18 英威达技术有限责任公司 通过甲酯保护的碳链延伸生产6碳化学物的方法
EP2938731A2 (en) * 2012-12-31 2015-11-04 Invista Technologies S.A R.L. Methods of producing 7-carbon chemicals via carbon chain elongation associated with cyclohexane carboxylate synthesis
WO2014105797A2 (en) * 2012-12-31 2014-07-03 Invista North America S.A.R.L. Methods of producing 7-carbon chemicals via pyruvate and succinate semialdehyde aldol condensation
US9745607B2 (en) 2014-05-15 2017-08-29 Invista North America S.A.R.L. Methods of producing 6-carbon chemicals using 2,6-diaminopimelate as precursor to 2-aminopimelate
US20170152531A1 (en) 2014-06-16 2017-06-01 North America S.A.R.L. Process for producing glutarate and glutaric acid methyl ester
CN106795535A (zh) 2014-06-16 2017-05-31 英威达技术有限责任公司 用于生物合成化合物的方法，试剂和细胞
US9988654B2 (en) 2014-06-16 2018-06-05 Invista North America S.A.R.L. Methods, reagents and cells for biosynthesizing compounds
US9957535B2 (en) 2014-06-16 2018-05-01 Invista North America S.A.R.L. Methods, reagents and cells for biosynthesizing compounds
EP3224366A1 (en) * 2014-11-26 2017-10-04 Invista Technologies S.A.R.L. Methods and materials for producing 7-carbon chemicals via a c9 route
CN116790698B (zh) * 2023-06-21 2024-06-18 南京大学 基于氧化脱羧酶的硫醛合成方法及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009121066A1 (en) * 2008-03-28 2009-10-01 The Regents Of The University Of California Producing dicarboxylic acids using polyketide synthases
WO2011031147A1 (en) * 2009-09-11 2011-03-17 Dsm Ip Assets B.V. Preparation of a compound comprising an amine group from an alpha-keto acid

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3719561A (en) * 1966-08-26 1973-03-06 Kyowa Hakko Kogyo Kk Process for producing diaminopimelic acid
FR2921364B1 (fr) * 2007-09-20 2009-11-06 Arkema France Procede de coproduction de 7-oxoheptanoate de methyle et d'acide undecylenique a partir d'acide ricinoleique
US8192976B2 (en) * 2008-12-12 2012-06-05 Celexion, Llc Biological synthesis of difunctional alkanes from carbohydrate feedstocks
WO2010104391A2 (en) * 2009-03-11 2010-09-16 Dsm Ip Assets B.V. Preparation of adipic acid
EA201200454A1 (ru) * 2009-09-11 2013-01-30 ДСМ АйПи АССЕТС Б.В. Получение альфа-кетопимелиновой кислоты
WO2012031910A2 (en) * 2010-09-10 2012-03-15 Dsm Ip Assets B.V. Method for preparing alpha-ketopimelic acid by c1-elongation
EP2726627A2 (en) * 2011-06-30 2014-05-07 Invista Technologies S.à.r.l. Bioconversion process for producing nylon-7, nylon-7,7 and polyesters

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009121066A1 (en) * 2008-03-28 2009-10-01 The Regents Of The University Of California Producing dicarboxylic acids using polyketide synthases
WO2011031147A1 (en) * 2009-09-11 2011-03-17 Dsm Ip Assets B.V. Preparation of a compound comprising an amine group from an alpha-keto acid

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CANTU D C ET AL: "Thioesterases: A new perspective based on their primary and tertiary structures", PROTEIN SCIENCE, WILEY, US, vol. 19, no. 7, 1 July 2010 (2010-07-01), pages 1281 - 1295, XP002722509, ISSN: 0961-8368, [retrieved on 20100517], DOI: 10.1002/PRO.417 *
JING DU ET AL: "Engineering microbial factories for synthesis of value-added products", JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY, vol. 38, no. 8, 28 April 2011 (2011-04-28), pages 873 - 890, XP055107701, ISSN: 1367-5435, DOI: 10.1007/s10295-011-0970-3 *
PER HOFVANDER ET AL: "A prokaryotic acyl-CoA reductase performing reduction of fatty acyl-CoA to fatty alcohol", FEBS LETTERS, ELSEVIER, AMSTERDAM, NL, vol. 585, no. 22, 10 October 2011 (2011-10-10), pages 3538 - 3543, XP028107918, ISSN: 0014-5793, [retrieved on 20111019], DOI: 10.1016/J.FEBSLET.2011.10.016 *
ROBERT M. WILLIS ET AL: "Characterization of a Fatty Acyl-CoA Reductase from Marinobacter aquaeolei VT8: A Bacterial Enzyme Catalyzing the Reduction of Fatty Acyl-CoA to Fatty Alcohol", BIOCHEMISTRY, vol. 50, no. 48, 6 December 2011 (2011-12-06), pages 10550 - 10558, XP055064364, ISSN: 0006-2960, DOI: 10.1021/bi2008646 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108998430A (zh) * 2018-06-29 2018-12-14 浙江工业大学 一种界面自组装羰基还原酶及在(r)-3-羟基-3-苯基丙酸乙酯合成中的应用
CN108998430B (zh) * 2018-06-29 2020-11-13 浙江工业大学 一种界面自组装羰基还原酶及在(r)-3-羟基-3-苯基丙酸乙酯合成中的应用

Also Published As

Publication number Publication date
JP2017131229A (ja) 2017-08-03
CN104126012A (zh) 2014-10-29
WO2013096898A3 (en) 2014-03-13
BR112014015077A2 (pt) 2020-10-27
WO2013096898A2 (en) 2013-06-27
JP2015500663A (ja) 2015-01-08

Similar Documents

Publication Publication Date Title
US10577634B2 (en) Bioconversion process for producing nylon-7, nylon-7,7 and polyesters
EP2794897A2 (en) Bioconversion process for producing nylon-7, nylon-7,7 and polyesters
JP7751361B2 (ja) アジペート、ヘキサメチレンジアミン、及び６－アミノカプロン酸の生合成のための微生物及び方法
US20230022583A1 (en) Adipate (ester or thioester) synthesis
US10415063B2 (en) Semi-synthetic terephthalic acid via microorganisms that produce muconic acid
CN107384846B (zh) 生产1,4-丁二醇的微生物和相关方法
EP2855688A1 (en) Biosynthetic pathways, recombinant cells, and methods
CA2985279A1 (en) Process for the biological production of methacrylic acid and derivatives thereof
WO2020219863A1 (en) Engineered microorganisms and methods for improved aldehyde dehydrogenase activity
EP3164495A1 (en) Microorganisms for producing 4c-5c compounds with unsaturation and methods related thereto
WO2022210708A1 (ja) 組換え微生物及びｃ６化合物の製造方法
WO2017190056A1 (en) Conversion of 1-carbon compounds to products
US10774349B2 (en) Alpha omega bifunctional fatty acids
San et al. Alpha omega bifunctional fatty acids
AU2015203845B2 (en) Adipate ester or thioester synthesis

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20140610

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAX Request for extension of the european patent (deleted)
PUAG Search results despatched under rule 164(2) epc together with communication from examining division

Free format text: ORIGINAL CODE: 0009017

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20161205

B565 Issuance of search results under rule 164(2) epc

Effective date: 20161205

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: INVISTA TEXTILES (U.K.) LIMITED

P01 Opt-out of the competence of the unified patent court (upc) registered

Effective date: 20230528

RAP3 Party data changed (applicant data changed or rights of an application transferred)

Owner name: INVISTA TEXTILES (U.K.) LIMITED

RAP3 Party data changed (applicant data changed or rights of an application transferred)

Owner name: INVISTA TEXTILES (U.K.) LIMITED

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20250701