LINOLEIC ACID

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional Application Se . No. 62/289,877, filed February 1, 2016, which is incorporated herein by reference in its entirety.

SEQU ENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 31 , 2017, is named 12444 J)626-00304 _SL.txt and is 1 76,780 bytes in size.

TECHNICAL FIELD

This invention provides methods for biosynthesizing 7-carbon monomers. For example, the present invention provides methods for making two 9-carbon monomers from an 18-carbon intermediate and enzymatically converting the two 9-carbon monomers to 7-carbon monomers. For example, the present invention provides methods for making non-3 -enal and 9-oxononanoate using a polypeptide having the activity of a hydroperoxide lyase and enzymatically converting non-3 -enal and 9-oxononanoate to pimeloyl-CoA or a salt thereof using one or more polypeptides having the activity of a dehydrogenase, a CoA ligase, an isomerase, a reductase, a thioesterase, a monooxygenase, a hydratase, and/or a tkiolase, or methods using microorganisms expressing one or more of such polypeptides. This invention also provides methods for converting pimeloyl-CoA or a salt thereof to one or more of pimelic acid, 7- aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine, and 1 ,7- heptanediol, or corresponding salts thereof, using one or more polypeptides having the activity of a thioesterase, a CoA ligase, a CoA transferase, a dehydrogenase, a reductase. an acetyltr ansf erase , a deacylase, and/or a transaminase or methods using recombinant microorganisms expressing one or more such polypeptides. BACKGROUND

Nylons are synthetic polymers composed of polyamides, which are generally synthesized by the condensation polymerization of a diamine with a dicarboxylic acid. Similarly, nylons also may be produced by the condensation polymerization of lactams. Nylon 7 is produced by polymerisation of 7-aminoheptanoic acid, whereas Nylon 7,7 is produced by condensation polymerisation of pimelic acid and heptamethylenediamine. No economically cost competitive petrochemical routes exist to producing the monomers for Nylon 7 and Nylon 7,7.

Given the lack of economically cost competitive petrochemical monomer feedstocks, biotechnology offers an alternative approach via biocatalysis, Biocata!ysis is the use of biological catalysts, such as enzymes, to perform biochemical transformations of, for example, bioderived feedstocks and petrochemical feedstocks, which can both be viable starting materials for the biocatalysis processes.

SUM MARY

Accordingly, against this background, it is clear that there is a need for sustainable methods for producing one or more of pimelic acid, 7 -amino heptanoate, 7- hydroxyheptanoate, heptamethylenediamine, and 1 ,7-heptanediol, or derivatives thereof, wherein the methods are bioeatalyst based. This document is based at least in part on the discovery that it is possible to construct biochemical pathways using, inter alia, a polypeptide having the activity of a hydroperoxide lyase to produce two C9 (9-carbon) aliphatic molecules from a single C I 8 (18-carbon) molecule, and converting the two C9 molecules in one or more enzymatic steps to pimelic acid, 7-aminoheptanoic acid, 7- hydroxyheptanoic acid, heptamethylenediamine, or 1 ,7-heptanediol, or corresponding salts thereof, in particular, this document is based at least in part on the discovery that it is possible to construct biochemical pathways using, inter alia, a polypeptide having the activity of a hydroperoxide lyase to produce non-3 -enal and 9-oxononanoate from 9- hydroxyperoxyoctadec- 10, 12-dienoate and converting at least one of non-3 -enal and 9- oxononanoate in one or more enzymatic steps to pimelic acid, 7-aminoheptanoic acid, 7- hydroxyheptanoic acid, heptamethylenediamine or 1 ,7-heptanediol. Pimelic acid and A pimeiate. 7-hydroxyheptanoic acid and 7-hydroxyheptanoate, 9-oxononanoate and 9- oxononanoic acid, and 7-aminoheptanoic and 7-aminoheptanoaie are used interchangeably herein to refer to the compounds in any of their neutrai or ionized forms, including any salt forms thereof, it is understood by those skilled in the art that the specific form will depend on pH.

For compounds containing carboxylic acid groups such as organic monoacids, hydroxyacids, aminoacids, and dicarboxylic acids, these compounds may be formed or converted to their ionic salt form when an acidic proton present in the parent compound either is replaced by a metal ion, e.g. , an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base. Acceptable organic bases include ethanolamine, diethanolaraine, triethanolamine, tromethamine, N-methylglucamine, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. The salt can be isolated as is from the system as the salt or converted to the free acid by reducing the pH to below the pKa through addition of acid or treatvnent with an acidic ion exchange resin.

For compounds containing amine groups such as, but not limited to, organic amines, aminoacids, and diamines, these compounds may be formed or converted to their ionic salt form by addition of an acidic proton to the amine to form the ammonium salt, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cycl operitanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3 -(4- hydroxybenzoy l)benzoic acid, cinnamic acid, mandeiic acid, methanesuLfonic acid, ethanesulfonic acid, 1 ,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4- methylbicyclo-[2.2.2]oct-2-ene- 1 -carboxylic acid, glucoheptonic acid, 4,4'-methylenebis- (3-hydroxy-2-ene-l -carboxylic acid), 3 -pheny lpropionic acid, trimethy!acetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxy-naphthoic acid, salicylic acid, stearic acid, muconic acid, and the like. Acceptable inorganic bases are known in the art and include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. The salt can be isolated as is from the system as a salt or converted to the free amine by raising the pi I to above the p b through addition of base or treatment with a basic ion exchange resin.

For compounds containing both amine groups and carboxylic acid groups such as, but not limited to aminoacids, these compounds may be formed or converted to their ionic salt form by either 1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobroinic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexarioic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maieic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 3 ,2-ethanedisulfonic acid, 2-hydroxyethanesuIfonic acid, benzenesulfonic acid, 2-naphthaienesulforiic acid, 4-methylbicyclo-[2.2.2]oct-2-ene- 1 - carboxylic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2~ene- 1 -carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like; or 2) when an acidic proton present in the parent compound either is replaced by a metal ion, e.g. , an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base. Acceptable organic bases are known in the art and include eihanolamine, diethanolamine, triethanolamine, tromethamine, N- methylglucamine, and the like. Acceptable inorganic bases are known in the art and include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. The salt can be isolated as is from the system or converted to the free acid by reducing the pH to below the p a through addition of acid or treatment with an acidic ion exchange resin,

it has been discovered that appropriate non-natural pathways, feedstocks, microorganisms, attenuation strategies to the microorganism's biochemical network, and cultivation strategies may be combined to efficiently produce pimeloyl-CoA as a C7 (7- carbon) building block, or convert pimeloyl-CoA to other C7 building blocks such as pirnelic acid, 7-aminohepianoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine, or 1 ,7-heptanediol.

In some embodiments, a terminal earboxy! group can be enzymatically formed using a polypeptide having the activity of a thioesterase, a CoA transferase, a Co A ligase, an aldehyde dehydrogenase, a succinate-semialdehyde dehydrogenase, a 5- oxopentanoaie dehydrogenase, a 6-oxohexanoate dehydrogenase, or a 7-oxoheptanoate dehydrogenase. See FIG. 3.

in some embodiments, a terminal amine group can be enzymatically formed using a polypeptide having the activity of a co-lransaminase or a deacylase. See FIG. 4 and FIG. 5. The polypeptide having the activity of a ω -transaminase can have at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 7-12. Furthermore, the polypeptide having the activity of a ω-transaminase can have at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 7-12 and be capable of transferring at least one amine group separated from a carbonyl group by at least one methylene insertion.

In some embodiments, a terminal hydroxyl group can be enzymatically formed using a polypeptide having the activity of an alcohol dehydrogenase. See FIG. 4 and FIG.

In one aspect, this document features a method of producing non-3 -enai and 9- oxononanoate from 9-hydroxyperox octadec- 10,12-dienoate using a polypeptide having the activity of a hydroperoxide lyase classified under EC 4.2.99.-.

In any of the methods, non-3 -enal and 9-oxononanoate can be enzymatically produced from 9-hydroxyperoxyoctadec- 10,12-dienoate, which itself can be enzymatically produced from octadecanoyl-CoA using one or more polypeptides having the activity of a delta9-desatitrase, a de!tal 2-desaturase, a thioesterase, and/or a 9- lipoxygenase. The polypeptide having the activity of a delta9-desaiurase can be classified under EC 1.14.19.1, such as, for example, the gene product of Le-FADl from Lentin la edodes (UniProtKB Accession No. Q76CI9), the gene product of SCD1 from Mesocricetus auratus (UniProtKB Accession No. A7LCI9), an acyl-CoA -delta9-3a~ desa urase from Dendrolimus punctatus (UniProtKB Accession No. B7SB75), the gene product of scdl from Rattiis norvegicus (UniProtKB Accession No. P07308), the gene product of PF3D7 0511200 from Plasmodium falciparum (UniProtKB Accession No. Q810W9), or the gene product of desBl from Bombus lucorum (UniProtKB Accession No. A5CKE1 }.

The polypeptide having the activity of a delta 12-desatwase can be classified under EC 1.14.19.6, such as, for example, the gene product of D12Des from Acheta domesticus (UniProtKB Accession No. B7SB91), the gene product of FAD2 from Gossypium hirsutum (UniProtKB Accession No. Q8W2B9), the gene product of CFad6 from Chlorella vulgaris (UniProtKB Accession No. D3U658), a delta 12 fatty acid desaturase from Triadica sebifera (UniProtKB Accession No. A5J295), the gene product of Pc-fad2 from Phanerochaeie chrysosporium (UniProtKB Accession No. D4Q8H2), the gene product of Cs-fad2 from Ceriporiopsis subvermispora (UniProtKB Accession No. D4Q8S6), or the gene product of AN 1037.2 from Emericella nidulans (UniProtKB Accession No. Q5BEJ3).

The polypeptide having the activity of a thioesterase can be classified under EC 3.1.2.-, such as, for example, the gene product of BT_2075 from Bacteroides thetaiotaomicron (strain ATCC 29148 / DSM 2079 / NCTC 10582 / E50 / VPT-5482) (GenBank Accession No. AA077182.1 , SEQ ID NO: 20), the gene product of lp 708 from Lactobacillus plantarum (strain ATCC BAA-793 / NCIMB 8826 / WCFS1) (GenBank Accession No. CCC78182.1 , SEQ ID NO: 22), the gene product oi CPF 2954 from Clostridium perfringens (strain ATCC 13124 / DSM 756 / JCM 1290 / NCIMB 6125 / NCTC 8237 / Type A) (GenBank Accession No. ABG82470.1, SEQ ID NO: 18), or the gene product of HMPREF0077_1317 from Anaerococcus telradius ATCC 35098 (GenBank Accession No. EEI82564.1 , SEQ ID NO: 23).

The polypeptide having the activity of a 9-lipoxygenase can be classified, for example, under EC 1.13.1 1.58,EC L 13. i l .60, EC 1.13.1 1.61 , or EC 1.13.1 1.62, such as, for example, an allene oxide synthase-lipoxygenase protein from Plexaura homomalla (UniProtKB Accession No. 016025), a Psi-producing oxygenase A from Emericella nidulans (UniProtKB Accession No. Q6RET3), a. 5,8-linoleate dial synthase from Aspergillus fumigatus (UniProtKB Accession No. C1KH66), or a linoleate dial synthase from Gaeumannomyces graminis (UniProtKB Accession No. Q9UUS2),

The method includes enzymaticaily converting non-3-enal to azelaic acid via two alternative enzymatic pathways. The method also includes enzymaticaily converting 9- oxononanoate to azelaic acid.

In one aspect, the method includes converting non-3-enal to azelaic acid using one or more polypeptides having the enzymatic activities of an aldehyde dehydrogenase, a CoA ligase, a dodecenoyl-CoA isomerase, a irans-2~enoyl~CoA reductase, a thioesterase, a monooxygenase, an alcohol dehydrogenase, a succinate semi ldehyde dehydrogenase, a 5-oxopentanoate dehydrogenase, a 6-oxohex noate dehydrogenase, and/or a 7-oxoheptanoate dehydrogenase.

The polypeptide having the activity of an aldehyde dehydrogenase can be classified under EC 1.2.1.-, for example, EC 1.2.1.3, EC 1 .2.1.4, EC 1.2.1.5, or EC 1.2.1.48, such as, for example, the gene product of Bt-aldh from GeobaciU thermoleovorans B23 (UniProtKB Accession No. Q9FAB 1), the gene product of dhaS from Bacillus subtilis (UniProtKB Accession No. 034660), the gene product of ALD5 from Saccharomyces cerevisiae (UniProtKB Accession No. A6ZR27), the gene product of ALDH2C4 from Arabidopsis thaliana (UniProtKB Accession No. Q56YU0), the gene product of aldh.7 from Rhodococcus ruber (UniProtKB Accession No. Q840S9), the gene product of alkll from Pseudomonas oleovorans (UniProtKB Accession No. PI 2693). the gene product of aldl from cinetobacter sp. M-l (UniProtKB Accession No. Q9FDS 1 ), or the gene product of acoD from Ralstonia eutropha (UniProtKB Accession No. P46368).

The polypeptide having the activity of a CoA ligase can be classified under EC 6.2.1.-, such as, for example, the gene product of acs6 from Brassica napus (UniProtKB Accession No. Q9FNT6), the gene product of PCS60 from Saccharomyces cerevisiae (UniProtKB Accession No. P38137), the gene product of olkK from Pseudomonas oleovorans (UniProtKB Accession No. Q00594), the gene product of ACSM5 from Homo sapiens (UniProtKB Accession No. Q6NIJN0), or the gene product of alkK from Aeropyrum pernix (UniProtKB Accession No. Q9YF45).

The polypeptide having the activity of a dodecenoyl-CoA isomerase can be classified under EC 5.3.3.8, such as, for example, the gene product of ECU from Saccharomyces cerevisiae (SEQ ID NO: 17 and SEQ ID NO: 19).

The polypeptide having the activity of a trans~2~e.noyl.-CoA reductase can be classified under EC 1.3.1.38, EC 1 .3.1.44, or EC 1.3.1.8. The polypeptide having the activity of a thioesterase can be classified under EC 3.1.2.-. The polypeptide having the activity of a moriooxygena.se can be classified under EC 1.14.14, 1 , EC 1.14.14.3, EC 1 ,14.15.1 , or EC 1.14.15.3. The polypeptide having the activity of an alcohol dehydrogenase can be classified under EC 1.1.1.-, such as, for example, a 4- hydroxybutanoate dehydrogenase classified under EC 1 , 1.1.61 , such as, for example, the gene product of gabD from Escherichia coli (Bartsch et al. , J. Bacteriol, 1990, 172(12), 7035), or a 6-hydroxyhexanoate dehydrogenase classified under EC 1.1 , 1.258, such as, for example, the gene product of clrnD from Acinetohacter sp. NCIMB9871 (Donoghue et al, Eur. J. Biochern, 1975, 60: 1 -7). The poiypeptide having the activity of a succinate semialdehyde dehydrogenase can be classified under EC 1.2, 1.16, EC 1.2, 1.24, or EC 1 ,2, 1.79. The polypeptide having the activity of a 5-oxopentanoate dehydrogenase can be classified under EC 1 .2.1.20, such as that encoded by cpnE from Comamonas sp. The poiypeptide having the activity of a 6-oxohexanoate dehydrogenase can be classified under EC 1.2.1.63, such as that encoded by chnE from Acinetohacter sp., and the polypeptide having the activity of a 7-oxoheptanoate dehydrogenase can be classified under EC 1.2.1.- (e.g. , the gene product of thnG from Sphingomonas maerogolitahida).

In an alternative aspect, the method includes converting non-3 -enal to azelaic acid using one or more polypeptides having the enzymatic activities of an enal isomerase, an enoate reductase, an aldehyde dehydrogenase, a monooxygenase, an alcohol dehydrogenase, a succinate semialdehyde dehydrogenase, a 5-oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, and/or a 7-oxoheptanoate dehydrogenase. The polypeptide having the activity of an enal isomerase can be classified under EC 5.3.3.-, such as, for example, the gene product of ECU from Saccharomyces cerevisiae (SEQ ID NO: 17 and SEQ ID NO: 19); Geisbrecht et al J. Biol. Chem, 1998 273 (50) 33184-33191.) The polypeptide having the activity of an enoate reductase can be classified under EC 1.3.1.31. The polypeptide having the activity of an aldehyde dehydrogenase can be classified under EC 1.2.1.-, for example, EC 1.2.1.3, EC 1.2.1.4, EC 1.2.1.5, or EC 1.2.1.48. The polypeptide having the activity of a monooxygenase can be classified under EC 1.14.14.1, EC 1.14.14.3, EC 1.14.15.1, or EC 1.14.15.3. The polypeptide having the activity of an alcohol dehydrogenase can be classified under EC 1.1 .1.-, for example, a 4 '-hydroxy butanoate dehydrogenase classified under EC 1.1.1.61 , such as, for example, the gene product of gabD from Escherichia coli (Bartsch et al , J, Bacterial. , 1990, 172(12), 7035), or a 6-hydroxyhexanoate dehydrogenase classified under EC 1.1.1.258, such as, for example, the gene product of chnD from Acinetobacter sp. NCIMB9871 (Donoghue et al, Eur. J. Biochem, 1975, 60: 1-7).

The polypeptide having the activity of a succinate semialdehyde dehydrogenase can be classified under EC 1.2.1.16, EC 1.2.1.24, or EC 1.2.1.79, such as, for example, the gene product of ALDH5F1 from Arabidopsis thaliana (UniProtKB Accession No. Q9SAK4), the gene product of araE from Azospirillum brasilense (UniProtKB Accession No. Q3JUP4), the gene product of Ssadh from Drosophila melanogaster (UniProtKB Accession No. Q9VBP6), the gene product of ALDH5AI from Gorilla gorilla (UniProtKB Accession No. Q6A2H1), the gene product of ALDH5A1 from Hylobates tar (UniProtKB Accession No. Q3MSM3), the gene product of ssadh from Lucilia cuprina (UniProtKB Accession No. B0JFD4), the gene product of ALDH5A 1 from Pan paniscus (UniProtKB Accession No. Q3MSM4), the gene product of ALDH5A 1 from Pan troglodytes (UniProtKB Accession No. Q6A2H0), the gene product of ALDH5A1 from Pongo abelii (UniProtKB Accession No. Q6A2H2), the gene product of ALDH5A1 from Pongo pygmaeus (UniProtKB Accession No. Q6A2H2), or the gene product of gapN-1 from Sulfolobus solfataricus (UniProtKB Accession No. Q97XS9).

The polypeptide having the activity of a 5-oxopentanoate dehydrogenase can be classified under EC 1.2.1 .20, such as that encoded by cpnE from Comamonas sp. The polypeptide having the activity of a 6-oxohexanoate dehydrogenase can be classified under EC 1 ,2.1.63, such as that encoded by chnE from Acinetobacter sp., and the polypeptide having the activity of a 7-oxoheptanoale dehydrogenase can be classified under EC 1.2.1.- (e.g. , the gene product of thnG from Sphingomonas macrogolitabida).

In a further aspect, the method includes converting 9-oxononanoate to azelaic acid using a polypeptide classified under EC 1.2.1 .-, such as EC 1.2.1 .3, EC 1.2.1.16, EC 1.2.1.20, EC 1 .2.1.24, EC 1.2. 1.63, or EC 1.2.1.79. The polypeptides classified under EC 1.2.1.3 have the activity of an aldehyde dehydrogenase. The polypeptides classified under EC 1.2.1.16, EC 1.2.1 .24, or EC 1.2, 1 .79 have the activity of a succinate semialdehyde dehydrogenase, such as, for example, the gene product of ALDH5FI from Arabidopsis thaliana (Uni ProtKB Accession No. Q9SAK4), the gene product of araE from Azospirillum brasilense (UniProtKB Accession No. Q1 JUP4), the gene product of Ssadh from Drosophila melanogasier (UniProtKB Accession No. Q9VBP6), the gene product of ALDH5A1 from Gorilla gorilla (Uni ProtKB Accession No. Q6A2H 1 ), the gene product of ALDH5A1 from Hylobates lar (UniProtKB Accession No. Q3MSM3), the gene product of ssadh from Lucilia cuprina (UniProtKB Accession No. B0JFD4), the gene product of ALDH5A1 from Pan paniscus (UniProtKB Accession No. Q3MSM4), the gene product of ALDH5A1 from Pan troglodytes (UniProtKB Accession No. Q6A2H0), the gene product of ALDH5A1 from Pongo abelii (UniProtKB Accession No. Q6A2H2), the gene product of ALDH5A1 from Pongo pygmaeus (UniProtKB Accession No. Q6A2H2), or the gene product of gapN-1 from Sulfo!obus solfataricus (UniProtKB Accession No, Q97XS9). The polypeptides classified under EC 1.2.1.20 have the activity of a 5-oxopentanoate dehydrogenase, such as that encoded by cpnE from Comamonas sp. The polypeptides classified under EC 1.2.1 ,63 have the activity of a 6-oxohexanoate dehydrogenase, such as that encoded by chnE from Acinetobacter sp. Further polypeptides classified under EC 1.2.1.- have the activity of a 7-oxoheptanoate dehydrogenase (e.g. , the gene product of thnG from Sphingomonas macrogolitabida). in the above-described enzymatic pathways, both non-3 -enal and 9-oxononanoate are converted to azelaic acid. Azelaic acid is then converted to pimeloyl-CoA using one or more polypeptides having the enzymatic activities of a Co A ligase, an acyl-CoA dehydrogenase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, a 3- oxoacyl-ACP reductase, and/or a β-ketothiolase.

The polypeptide having the activity of a CoA ligase can be classified under EC 6.2.1 ,-, such as, for example, the gene product of acs6 from Brassica napits (UniProtKB Accession No. Q9FNT6), the gene product of PCS60 from Saccharomyc.es cerevisiae (UniProtKB Accession No. P38137), the gene product of alkK. from Pseudomonas oleovorans (UniProtKB Accession No. QQ0594), the gene product of ACSM5 from Homo sapiens (UniProtKB Accession No. Q6 UN0), or the gene product of alkK from Aeropyrum pernix (UniProtKB Accession No. Q9YF45). The polypeptide having the activity of an acyl-CoA dehydrogenase can he classified under EC 1.3.8.-, such as EC 1.3.8.6, EC 1.3.8.7, or EC 1.3.8.8. The polypeptide having the activity of an enoy!-CoA hydratase can be classified under EC 4.2.1.17 or EC 4.2.1.1 19. The polypeptide having the activity of a 3-hydroxyacyl-CoA dehydrogenase can be classified under EC 1.1.1.35, EC 1 .1.1 ,36, or EC 1.1.1.157, and the polypeptide having the activity of a 3-oxoacyl-ACP reductase may be classified, for example, under EC 1.1.1 , 100, The polypeptide having the activity of a β-ketothiolase can be classified under EC 2.3.1.16 or EC 2.3.1.174. The polypeptide having the activity of a β-ketothiolase is capable of converting 3-oxo- azeiaoyl-CoA to pimeloyl-CoA.

Any of the methods further can include enzymaticaily converting pimeloyl-CoA to pirnelic acid. 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, or 1,7-heptanediol or their corresponding salts in one or more steps.

For example, pimeloyl-CoA can be enzymaticaily converted to pirnelic acid using one or more polypeptides having the activity of a thioesierase, a CoA ligase, a CoA transferase, an aldehyde dehydrogenase, a 5-oxopenlanoate dehydrogenase, a 6- oxohexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, and or a succinate- semialdehyde dehydrogenase. See FIG. 3.

For example, pimeloyl-CoA can be enzymaticaily converted to 7- aminoheptanoate using one or more polypeptides having the activity of an acetylating aldehyde dehydrogenase and/or a co-transaminase. See FIG. 4. For example, pimeloyl-CoA can be enzymatically converted to pimelate (pimeiic acid) as previously described in FIG. 3, and pimelate can be enzymatically converted to 7-aminoheptanoate using one or more polypeptides having the enzymatic activity of a carhoxyiate reductase and/or a ω-transaminase. See FIG. 4. The polypeptide having the activity of a ω-transaminase can have at least 70% sequence identity to any one of the amino acid sequences set forth in S Q I D NOs: 7 - 12.

For example, pimeloyi-CoA can be enzymatically converted to 7- hydroxyheptanoate using one or more polypeptides having the enzymatic activity of an acetylating aldehyde dehydrogenase and/or an alcohol dehydrogenase. In particular, pimeloyl-CoA can be enzymatically converted to 7-hydroxyheptanoate using one or more polypeptides having the enzymatic activity of an acetylating aldehyde dehydrogenase and one or more polypeptides having the enzymatic activity of a 4-hydroxybutanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, and/or a 6-hydroxyhexanoate dehydrogenase. See FIG. 6.

For example, pimeloyl-CoA can be enzymatically converted to pimelate (pimeiic acid) as previously described in FIG. 3, and pimelate can be enzymatically converted to 7-hydroxyheptanoate using one or more polypeptides having the enzymatic activity of a carhoxyiate reductase and/or an alcohol dehydrogenase. In particular, pimelate can be enzymatically converted to 7-hydroxyheptanoate using one or more polypeptides having the enzymatic activity of a carhoxyiate reductase and one or more polypeptides having the enzymatic activity of a 4-hydroxybutanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, and/or a 6-hydroxyhexanoate dehydrogenase. See FIG, 6.

For example, 7-aminoheptanoate and 7-hydroxyheptanoate can be converted to heptamethylenediamine using one or more polypeptides having the activity of a carhoxyiate reductase, a co-transaminase, an alcohol dehydrogenase, an N- acetyltransferase, and/or a deacylase. See FIG. 5.

The polypeptide having the activity of a carhoxyiate reductase can have at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 3 - 6. The polypeptide having the activity of a ω-transaminase can have at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 7 - 12. For example, pimeloyl-CoA can be converted to pimelate semialdehyde (see FIG. 3, FIG. 4, and FIG. 6), and subsequently pimelate semialdehyde can be converted to heptamethy!enediamine using one or more polypeptides having the activity of a carboxylaie reductase and/or a co- transaminase. See FIG. 5.

The polypeptide having the activity of a carboxylate reductase can have at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 1 - 6. The polypeptide having the activity of a co-transaminase can have at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 7 - 12.

For example, pimeloyl-CoA can be converted to 7-hydroxyheptanoate (see FIG. 6), and subsequently 7-hydroxyheptanoate can he converted to 1 ,7-heptanedioI using polypeptides having the activity of a carboxylate reductase and an alcohol dehydrogenase. See FIG. 7.

The polypeptide having the activity of a carboxylate reductase can have at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 1 - 6,

In any of the methods described herein, pimelic acid can be produced by forming the second terminai functional group in pimeloyl-CoA using a polypeptide having the activity of: (i) a thioesterase classified under EC 3.1.2.-, (ii) a Co A ligase classified under EC 6.2.1.-, such as EC 6.2.1.5 or EC 6.2.1.15, and/or (iii) a CoA transferase classified under EC 2.8.3.-, such as EC 2.8.3.8 or EC 2.8.3.12.

In any of the methods described herein, pimelic acid can be produced by forming the second terminal functional group in pimelate semialdehyde (also known as 7- oxoheptanoate) using a polypeptide having the activity of (i) an aldehyde dehydrogenase classified under EC 1.2.1 ,3, or (ii) a succinate semialdehyde dehydrogenase classified under EC 1.2.1. 16, EC 1.2.1.24, or EC 1 .2.1.79, a 5-oxopenianoate dehydrogenase classified under EC 1.2.1.- , for example. EC 1.2.1.20, such as that encoded by cpriE from Comamonas sp., a 6-oxohexanoate dehydrogenase classified under EC 1.2.1 ,63, such as that encoded by chnE from Acinetobacter sp. , or a 7-oxoheptanoate dehydrogenase classified under EC 1.2.1 ,- (e.g. , the gene product of ihriG from Sphingomonas macrogolitabida ). See FIG. 3.

In any of the methods described herein. 7-aminoheptanoic acid can be produced by forming the second terminal functional group in pimelate semiaidehyde using a polypeptide having the activity of a ω-transaminase classified under EC 2.6.1.-. See FIG. 4.

in any of the methods described herein, 7-hydroxyheptanoic acid can be produced by forming the second terminal functional group in pimelate semiaidehyde using a polypeptide having the activity of an alcohol dehydrogenase classified under EC 1.1.1. -, a 6-hydroxyhexanoate dehydrogenase classified under EC 1.1.1.258, such as, for example, the gene product of chnD from Acineiobacter sp. NCIMB9871 (Donoghue et al., Eur. J. Biochem, 1975, 60: 1-7); a 5-hydroxypentanoate dehydrogenase classified under EC 1.1.1.-, such as, for example, the gene product of cpnD from Comamonas sp. (Iwaki et al, Appl. Emir on. Microbiol , 1999, 65(1 1): 5158 - 5162), or a 4- hydroxybutanoate dehydrogenase classified under EC 1.1.1.61, such as, for example, the gene product of gabD from Escherichia coli (Bartsch et al , J. Bacteriol , 1990, 372(12), 7035). See FIG. 6.

In any of the methods described herein, heptarnethylenediamine can be produced by forming a. second terminal functional group in (i) 7-aminoheptanal using a polypeptide having the activity of a ω-transaminase classified under EC 2.6.1.-, such as, for example, EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 or in (ii) N7-acetyl- 1 ,7-diaminoheptane using a deacylase classified, for example, under EC 3.5.1.-, such as, for example, EC 3.5.1.62 or EC 3.5.1 .82, such as that encoded by dag from Alcaligenes xylosoxydans xylosoxydans (Achromobacier xylosoxidans) (UniProtKB Accession No. P9421 1). See FIG. 5.

in any of the methods described herein, 1 ,7-heptanediol can be produced by forming the second terminal functional group in 7-hydroxyheptanal using a polypeptide having the activity of an alcohol dehydrogenase classified under EC 1.1 .1.- {e.g. , EC 1.1.1.1, EC 1 .1.1.2, EC 1.1.1.21, or EC 1.1.1.184), such as that encoded by YMR318C from Saccharomyces cerevisiae, yqhD from Escherichia coli, or as represented by GenBank Accession No. CAA81612.1 (SEQ ID NO; 21). See FIG. 7.

In some embodiments, the biological feedstock can be or can derive from, monosaccharides, disaccharides, lignocelluiose, hemicelluiose, cellulose, lignin, levulinic acid and formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, plant oils, or municipal waste.

In some embodiments, the non-biological feedstock can be or can derive from natural gas, syngas, CCb/I , methanol, et.ha.noi, benzoate, non-volatile residue (NVR) or a caustic wash waste stream from cycloheptane oxidation processes, or terephtlialic acid / isophthalic acid mixture waste streams.

In some embodiments, the microorganism's tolerance to high concentrations of one or more C7 (7-carbon) building blocks is improved through continuous cultivation in a selective environment.

In some embodiments, the microorganism's biochemical network is attenuated or augmented to (1 ) ensure the intracellular availability of acetyl-CoA or malonyl-CoA, (2) create an NADH or NADPH imbalance that may only be balanced via the formation of one or more C7 building blocks, (3) prevent degradation of central metabolites, central precursors leading to and including C7 building blocks, and/or (4) ensure efficient efflux from the cell.

As used herein, "attenuation" refers to downregulation or inactivation of gene expression.

In some embodiments, a cultivation strategy is used to achieve anaerobic, micro- aerobic, or aerobic cultivation conditions.

in some embodiments, the cultivation strategy includes limiting nutrients, such as limiting nitrogen, phosphate, or oxygen.

In some embodiments, one or more C7 building blocks are produced by a single type of microorganism, e.g. , a recombinant microorganism containing one or more exogenous nucleic acids, using, for example, a fermentation strategy. In some embodiments, one or more C7 building blocks are produced by a single type of microorganism having one or more exogenous nucleic acids which encode polypeptides having the activity of a hydroxperoxide lyase, aldehyde dehydrogenase, a CoA ligase, a dodecenoyl-CoA isomer ase, a trans-2-enoyl-CoA reductase, a thioesterase, a monooxygenase, an alcohol dehydrogenase, a s ccinate-semialdehyde dehydrogenase, a 5-oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, an acyl-CoA dehydrogenase, an enoyl-CoA hydratase, a 3-hydroxyacyl- CoA dehydrogenase, a 3-oxoacyl CoA dehydrogenase, and/or a β-ketothiolase. Said microorganism produces pimeloyl-CoA from 9-hydroxyperoxyoctadec- 10, 12-dienoate. See FIG. 1.

In another aspect, this document features a recombinant microorganism that includes at least one exogenous nucleic acid encoding a polypeptide having the activity of a hydroxperoxide lyase, an e«a/ isome ase, an enoate reductase, an aldehyde dehydrogenase, a monooxygenase, an alcohol dehydrogenase, a succinate-semialdehyde dehydrogenase, a 5-oxopentanoate dehydrogenase, a 6~oxohexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, a o,4 ligase, an acyl-CoA dehydrogenase, an erioyl- Co/4 hydratase, a 3-hydroxyacyl-CoA dehydrogenase, a 3-oxoacyl CoA dehydrogenase, and/or a β-keto hiolase. Said microorganism produces pimeloyl-CoA from 9- hydroxyperoxyoctadec- 10, 12-dienoate. See FIG. 2.

In another aspect, this document features a recombinant microorganism that includes at least one exogenous nucleic acid encoding a polypeptide having the activity of an aldehyde dehydrogenase, a succinate-semialdehyde dehydrogenase, a 5- oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, a Co/i ligase, an acyl-CoA dehydrogenase, an enoyl-CoA hydratase, a J- hydroxyacyl-CoA dehydrogenase, a 3-oxoacyl CoA dehydrogenase, and a β-hetothiolase. Said microorgamsm produces pimeioyi-CoA from 9-oxononanoate. See FIG. 1 and FIG. 2.

A microorganism producing pimeioyi-CoA further can include one or more polypeptides having the activity of: (i) a thioesterase, (ii) a Co.4 £¾se, (iii) a o/i transferase, and/or (iv) an acetylating aldehyde dehydrogenase and one of a succinate- semialdehyde dehydrogenase, a 5-oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, and/or a 7-oxoheptanoate dehydrogenase; the microorganism further producing pimelic acid or a salt thereof. See FIG. 3.

A microorganism producing pimeloyl-CoA further can include one or more polypeptides having the activity of an acetyl ating aldehyde dehydrogenase and/or a ω- transaminase, the microorganism further producing 7-aminoheptanoate or a salt thereof, A microorganism producing pimeloyl-CoA further can include one or more of the exogenous polypeptides as described directly above for producing pimelate, and a microorganism producing pimelate further can include one or more exogenous polypeptides having the activity of: carboxylase reductase and/or a (^-transaminase, the microorganism further producing 7-aminoheptanoate or a salt thereof. See FIG. A.

A microorganism producing pimeloyl-CoA further can include one or more of the following exogenous polypeptides having the activity of: an acetyl ating aldehyde dehydrogenase, an alcohol dehydrogenase, a 4-hydroxybutanoate dehydrogenase, a 5- hydroxypentanoate dehydrogenase, and a 6-hydroxyhexanoate dehydrogenase, the microorganism further producing 7-hydroxyheptanoate or a salt thereof. A microorganism producing pimeloyl-CoA further can include one or more of the exogenous polypeptides as described directly above for producing pimelate, and a microorganism producing pimelate further can include one or more polypeptides having the activity of carboxylate reductase and/or a ahiransamina.se , the microorganis further producing 7-aminoheptanoate or a salt thereof. A microorganism producing pimeloyl- CoA further can include one or more exogenous polypeptides having the activity of: an acetylating aldehyde dehydrogenase, an alcohol dehydrogenase, a 4-hydroxybutanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, and/or a 6-hydroxyhexanoate dehydrogenase, the microorganism further producing 7-hydroxyheptanoate or a salt thereof. A microorganism producing pimeloyl-CoA further ca include one or more of the exogenous polypeptides as described directly above for producing pimelate, and a microorganism producing pimelate further can include one or more exogenous polypeptides having the activity of: a carboxylate reductase and an alcohol dehydrogenase, a 4-hydroxybutanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, and/or a 6-hydroxyhexanoate dehydrogenase, the microorganism further producing 7-hydroxyheptanoate or a salt thereof. See FIG. 6.

A microorganism producing pimeloyl-CoA further can include one or more of the exogenous polypeptides as described above for producing 7-aminoheptanoate or 7- hydroxyheplanoate, and a microorganism producing 7-aminoheptanoate or 7- hydroxyheptanoate can further include one or more of the exogenous polypeptides having the activity of: a carboxylate reductase, a ω-transaminase, an alcohol dehydrogenase, an N-acetyltransferase, and/or a deacylase, said microorganism further producing heptameihylenediamine. A microorganism producing pimelate semialdehyde (see FIG. 3, FIG. 4, and FIG. 6) can further include one or more polypeptides having the activity of a carboxylate reductase and/or a (o~transaminase, said microorganism further producing heptamethylenediamine or a salt thereof. See FIG. 5.

A microorganism producing pimeloyl-CoA further can include one or more of the exogenous polypeptides as described above for producing 7-hydroxyheptanoate, and a microorganism producing 7-hydroxyheptanoate can further include one or more of the exogenous polypeptides having the activity of a carboxylate reductase and/or an alcohol, dehydrogenase, the microorganism further producing 1,7-heptanedioI. See FIG. 7.

Any of the recombinant microorganisms described herein further can include one or more of exogenous polypeptides having the activity of: a delta9-desaturase, a delta 12-desalurase, a thioesterase, and/or a 9-lipoxygenase.

Any of the recombinant microorganisms can be a prokaryote, such as a prokaryote from a genus selected from Escherichia, Clostridia, Corynebacteria, Cupriavidus, Pseudomonas, Delftia, Bacillus, Lactobacillus, Lactococcus, and Rhodococcus. For example, the prokaryote can be selected from Escherichia coli, Clostridium Ijungdahlii, Clostridium autoethanogenum, Clostridium kluyveri, Corynebacterium glutamicum, Cupriavidus necaior, Cupriavidus metalUdurans . Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas oleavorans, Delftia acidovorans. Bacillus subiillis, Lactobacillus delbrueckii, Lactococcus lactis, and Rhodococcus equi. Such prokaryotes also can be sources of genes for constructing recombinant cells described herein that are capable of producing C7 building blocks. Any of the recombinant microorganisms can be a eukaryote such as a eukaryote from a genus selected from Aspergillus, Saccharomyces, Pichia, Y rrowia, IssatchenMa, Debaryomyces, Arxula, and Kluyveromyces. For example, the eukaryote can be selected from Aspergillus niger, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Issathenkia orientalis, Debaryomyces hansenii, Arxula adenoinivorans, and Kluyveromyces lactis. Such eukaryotes also can be sources of genes for constructing recombinant cells described herein that are capable of producing C7 bui lding blocks.

Any of the recombinant microorganisms described herein further can include attenuation of one or more of the following enzymes: a polyhydroxyalkanoate synthase, an acetyl-CoA thioesterase, a phosphotransacetylase forming acetate, an acetate kinase, a lactate dehydrogenase, a menaquinol-fwnarate oxidoreductase, an alcohol dehydrogenase forming ethanol, a triose phosphate isomer ase, a pyruvate decarboxylase, a glucose-6-phosphate isomerase, an NADH-consuniing transhydrogenase, an NADH- specific glutamate dehydrogenase, an NADH NADPH-utilizing glutamaie dehydrogenase, a pimeloyl-CoA dehydrogenase, an acyl-CoA dehydrogenase accepting C7 building blocks and central precursors as substrates, a butyryl-CoA dehydrogenase, or an adipyl-CoA synthetase.

Any of the recombinant microorganisms described herein further can overexpress one or more genes encoding: an acetyl-CoA synthetase, a 6-phosphogluconate dehydrogenase; a transketolase: a puridine nucleotide transhydrogenase; a glyceraldehyde-SP -dehydrogenase; a malic enzyme; a glucose-6-phosphate dehydrogenase: a glucose dehydrogenase; a fructose 1, 6 diphosphatase; a I -alanine dehydrogenase; a L-glutamate dehydrogenase: a formate dehydrogenase; a L-glutamine synthetase; a diamine transporter; a dicarboxylate transporter; and/or a multidrug transporter.

In another aspect, this document features a non-naturally occurring microorganism comprising at least one exogenous nucleic acid encoding at least one polypeptide having the activity of at least one enzyme, at least one substrate, and at least one product, as depicted in any one of FIG. 1 to 7. in another aspect, this document features a nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having carboxylate reductase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having carboxylate reductase activity is selected from: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 1 ; (b) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ I D NO: 2; (c) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 3; (d) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 4, (e) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 5 and (f) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 6.

in another aspect, this document features a nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having ω- transaminase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having ω-transaminase activity is selected from: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 7; (b) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 8; (c) a polypeptide having at least 7G%> sequence identity to the polypeptide of SEQ ID NO: 9; (d) a polypeptide having at least 70¾> sequence identity to the polypeptide of SEQ ID NO: 10: (e) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 11 or SEQ ID NO: 48; and (f) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 12.

In another aspect, this document features a nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having hydroperoxide lyase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having hydroperoxide lyase activity is selected from: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ D NO: 13 and (b) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 14.

In another aspect, this document features a nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having enoate reductase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having enoate reductase activity is selected from: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 15 and (b) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 16.

In another aspect, this document features a nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having isomer ase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having isomer ase activity is a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 17.

In another aspect, this document features a nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having thioesterase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having thioesterase activity is selected from; (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 18, (b) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 20, (c) a polypeptide having at least 70%> sequence identity to the polypeptide of SEQ ID NO: 22, and (d) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 23.

In another aspect, this document features a nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having alcohol dehydrogenase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having alcohol dehydrogenase activity is a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 21. In another aspect, this document features a composition comprising the nucleic acid construct or expression vector of embodiment 103 or 104.

in another aspect, this document features a culture medium comprising the nucleic acid construct or expression vector of embodiment 103 or 104.

In another aspect, this document features a non-naturally occurring biochemical network comprising 9-hydroxyperoxyoctadec- 10, 12-dienoate, an exogenous nucleic acid encoding a polypeptide having the activity of a hydroperoxide lyase classified under EC 4.2.99.-, and non-3-enal and 9-oxononanoate.

In another aspect, this document features a non-naturally occurring biochemical network comprising non-3 -enoyl-Co A, an exogenous nucleic acid encoding a polypeptide having the activity of a dodecenoyl-CoA isomerase classified under EC 5.3.3.8, and non- 2-enoyl-CoA.

In another aspect, this document features a non-naturally occurring biochemical network comprising non-2-enal, an exogenous nucleic acid encoding a polypeptide having the activity of an enoate reductase classified under EC 1 .3, 1.31 , and nonanal.

In another aspect, this document features a non-naturally occurring biochemical network comprising nonanoic acid, an exogenous nucleic acid encoding a polypeptide having the activity of a monooxygenase classified under EC 1.14.14.- or EC 1 .14.15.-, and a 9-hydroxynonanoic acid.

In another aspect, this document features means for producing pirneloyl-CoA, comprising culturing a non-naturally occurring microorganism comprising at least one exogenous nucleic acid encoding a polypeptide having the enzymatic activity of (i) a hydroperoxide lyase, (ii) an acetylating aldehyde dehydrogenase, (iii) a CoA ligase, (iv) a dodecertoyl- Co A isomerase or an enoate reductase, (v) a trans-2-enoyl-CoA reductase, (vi) a thioesterase, (vii) a monooxygenase, (viii) an alcohol dehydrogenase, (ix) an aldehyde dehydrogenase classified under any of EC 1.2.1.3, EC 1.2.1.16, EC 1.2.1 ,20, EC 1 .2.1.24, EC 1.2.1.63, or EC 1.2, 1.79, (x) an acyl-CoA dehydrogenase, (xi) an enoyl- CoA hydrolase, (xii) a 3-hydroxyacyl-CoA dehydrogenas or a 3-oxoacyl ACT reductase, and/or (xiii) a β-kelothiolase, expressed in a sufficient amount in said microorganism to produce pimeloyl-CoA. In another aspect, this document features a bio-derived, bio-based or fermentation-derived product, wherein said product comprises: (i) a composition comprising at least one bio-derived, bio-based, or fermentation-derived compound according to embodiment 112; (ii) a bio-derived, bio-based, or fermentation-derived polymer comprising the bio-derived, bio-based, or fermentation-derived composition or compound of (i), or any combination thereof; (iii) a bio-derived, bio-based, or fermentation-derived resin comprising the bio-derived, bio-based, or fermentation- derived compound or bio-derived, bio-based, or fermentation-derived composition of (i) or any combination thereof or the bio-derived, bio-based, or fermentation-derived polymer of (ii) or any combination thereof: (iv) a molded substance obtained by molding the bio-derived, bio-based, or fermentation-derived polymer of (ii) or the bio-derived, bio-based, or fermentation-derived resin of (iii), or any combination thereof; (v) a bio- derived, bio-based, or fermentation-derived formulation comprising the bio-derived, bio- based, or fermentation-derived composition of (i), bio-derived, bio-based, or fermentation-derived compound of (i), bio-derived, bio-based, or fermentation-derived polymer of (ii), bio-derived, bio-based, or fermentalion-derived resin of (iii), or bio- derived, bio-based, or fermentation-derived molded substance of (iv), or any combination thereof; and (vi) a bio-derived, bio-based, or fermentation-derived semi-solid or a non- semi-solid stream, comprising the bio-derived, bio-based, or fermentation-derived composition of (i), bio-derived, bio-based, or fermentation-derived compound of (i), bio- derived, bio-based, or fermentation-derived polymer of (ii), bio-derived, bio-based, or fermentation-derived resin of (iii), bio-derived, bio-based, or fermentation-derived formulation of (v), or bio-derived, bio-based, or fermentation-derived molded substance of (iv), or any combination thereof.

Embodiments 1-112 recite non-limiting example embodiments of the disclosure. 1. A method of producing non-3 -enai and 9-oxononanoate in a recombinant microorganism, said method comprising enzymatically converting 9- hydroxyperoxyoctadec- 10, 12-dienoate to non-3 -enal and 9-oxononanoate using an exogenous polypeptide having the activity of a hydroperoxide lyase classified under The method of embodiment 1 , wherein said exogenous polypeptide is the gene product of Cucumis sativus (GenBank Accession No. AAF64041.1, SEQ ID NO; 1 3) or a polypeptide having at least 70%, at least 80%, or at least 85% sequence identity with the same or the gene product of Oryza sa!iva (GenBank Accession No. BAG97978J , SEQ ID NO: 14) or a polypeptide having at least 70%, at least 80%, or at least 85% sequence identity with the same.

The method of embodiment 1 , further comprising enzymatically converting non-3 - enal to azelaic acid using a one or more polypeptides, comprising at least one polypeptide having the activity of a dodecenoyl-CoA isomerase classified under EC 5.3.3.8.

The method of embodiment 3, wherein said at least one polypeptide having the activity of a dodecenoyl-CoA isomerase classified under EC 5,3.3.8 enzymatically converts non-3-enoyl-CoA to non-2-enoyl-CoA.

The method of embodiment 1, further comprising enzymatically converting non-3- enai to azelaic acid using a one or more polypeptides, comprising at least one polypeptide having the activity of an enoate reductase classified under EC 1 .3.1.31 . The method of embodiment 5, wherein said at least one polypeptide having the activity of an enoate reductase classified under EC 1 .3.1.31 enzymatically is an enzymatic step in converting non-3 -enal to nonanak

The method of embodiment 3 or embodiment 5, wherein said one or more polypeptides comprises a polypeptide having the activity of a monooxygena.se classified under EC 1.14.14.- or EC 1.14.15.-, such as EC 1 .14.14.1 , EC 1 .14.14.3, EC 1.14.15.1 , or EC 1.14.15.3.

The method of embodiment 7, wherein said polypeptide having the activity of a monooxygenase converts nonanoic acid to 9-hydroxynonanoic acid.

A method of producing azelaic acid in a recombinant microorganism, said method comprising the steps of enzymatically converting 9-h droxyperoxyoctadec- 10,12- dienoate to non-3 -enal and 9-oxononanoate using an exogenous polypeptide having the activit of a hydroperoxide lyase classified under EC 4.2.99.- and enzymatically converting non-3 -enal to azelaic acid using one or more polypeptides, including at least one polypeptide having the activity of a dodecenoyl-CoA isomerase classified under EC 5.3,3.8 or at least one polypeptide having the activity of an enoate reductase classified under EC 1.3.1.3 1.

The method of embodiment 9, wherein said one or more polypeptides further comprises a polypeptide having the activity of a monooxygenase classified under EC 1.14, 14.- or EC 1.14.15.-, wherein said monooxygenase enzymatically converts nonanoic acid to 9-hydroxynonanoic acid.

The method of embodiment 9, wherein said non-3 -enal is converted to azeiaic acid using one or more polypeptides having the enzymatic activities of an enal isomerase, an aldehyde dehydrogenase, a CoA ligase, a dodecenoyl-CoA isomerase, a trans-2-enoyl-CoA reductase, a Ihioesterase, a monooxygenase, and/or an alcohol dehydrogenase.

The method of embodiment 11, wherein said aldehyde dehydrogenase is classified under EC 1.2.1.3, EC 1 .2.1.4, EC 1.2.1.5, or EC 1.2, 1.48.

The method of embodiment 1 1 , wherein said CoA ligase is classified under EC 6.2.1 .-. such as EC 6.2, 1.5 or EC 6.2.1.15.

The method of embodiment 11 , wherein said trans-2-enoyl-CoA reductase is classified under EC 1.3.1.38, EC 1.3.1.44, or EC 1.3.1.8.

The method of embodiment 11, wherein said ihioesterase is classified under EC The method of embodiment 1 1 , wherein said alcohol dehydrogenase is classified under EC 1.1.1.-, such as EC 1.1.1 .61 or EC 1.1.1.258.

The method of embodiment 11 , wherein said aldehyde dehydrogenase is classified under EC 1.2.1.3, EC 1 .2.1.16, EC 1.2.1.20, EC 1.2.1.24, EC 1.2.1.63, or EC 1.2.1 ,79, wherein said aldehyde dehydrogenase classified under EC 1.2. 1 , 16, EC 1 .2.1.24, or EC 1.2.1.79 is a succinate-semialdehyde dehydrogenase, wherein said aldehyde dehydrogenase classified under EC 1.2.1.20 is a 5-oxopentanoate dehydrogenase, wherein said aldehyde dehydrogenase classified under EC 1.2, 1 .63 is a 6-oxohexanoate dehydrogenase, and wherein said aldehyde dehydrogenase classified under EC 1.2.1 .- is a 7-oxoheptanoate dehydrogenase. The method of embodiment 9, wherein said non-3 -enal is converted to azelaic acid using one or more polypeptides having the enzymatic activities of an enoate reductase, an aldehyde dehydrogenase, a monooxygenase, and/or an alcohol dehydrogenase,

The method of embodiment 18, wherein said aldehyde dehydrogenase is classified under EC 1.2.1 ,3, EC 1.2.1.4, EC 1.2.1.5, or EC 1.2.1.48.

The method of embodiment 18, wherein said alcohol dehydrogenase is classified under EC 1.1.1.-, such as EC 1.1.1.61 or EC 1.1.1.258.

The method of embodiment 18, wherein said aldehyde dehydrogenase is classified under EC 1.2.1 .3, EC 1 .2.1.16, EC 1.2.1 .20, EC 1.2.1 .24, EC 1 .2.1.63, or EC 1.2.1.79, wherein said aldehyde dehydrogenase classified under EC 1.2.1. 16, EC 1.2, 2.24, or EC 1.2, 1 .79 is a succinate-semialdehyde dehydrogenase, wherein said aldehyde dehydrogenase classified under EC 1.2.1.20 is a 5-oxopentanoate dehydrogenase, wherein said aldehyde dehydrogenase classified under EC 1.2.1.63 is a 6-oxohexanoate dehydrogenase and wherein said aldehyde dehydrogenase classified under EC 1.2.1.- is a 7-oxoheptanoate dehydrogenase.

The method of embodiment 9, wherein said 9-oxononanoate is converted to azelaic acid using a polypeptide having the enzymatic activity of an aldehyde dehydrogenase.

The method of embodiment 22, wherein said aldehyde dehydrogenase is classified under EC 1.2.1.3, EC 1.2.1.16, EC 1.2.1.20, EC 1.2.1.24, EC 1 .2.1.63, or EC 1.2.1 ,79, wherein said aldehyde dehydrogenase classified under EC 1.2.1.16, EC 1.2.1.24, or EC 1.2.1.79 is a succinate-semialdehyde dehydrogenase, wherein said aldehyde dehydrogenase classified under EC 1.2.1.20 is a 5-oxopentanoate dehydrogenase, wherein said aldehyde dehydrogenase classified under EC 1.2.1 ,63 is a 6-oxohexanoate dehydrogenase, and wherein said aldehyde dehydrogenase classified under EC 1.2.1.- is a 7-oxoheptanoate dehydrogenase.

The method of any of embodiments 11 , 18, or 22, wherein said azelaic acid is converted to pimeloyl-CoA using one or more polypeptides having the enzymatic activities of a CoA lig se, an acyl-CoA dehydrogenase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, and/or a β-ketothiolase.

The method of embodiment 24, wherein said Ca.4 / ^' gase is classified under EC 6,2.1.-,

The method of embodiment 24, wherein said acyl-CoA dehydrogenase is classified under EC 1.3.8.·-.

The method of embodiment 26, wherein said acyl-CoA dehydrogenase is classified under EC 1.3.8.6, EC 1 .3.8.7, or EC 1 .3.8.8.

The method of embodiment 24, wherein said enoyl-CoA hydratase is classified under EC 4.2.3.17 or EC 4.2.1.139,

The method of embodiment 24, wherein said 3-hydroxyacyl-CoA dehydrogenase is classified under EC 1 , 3 .3.35, EC 1 , 3.1.36, or EC 3.1.1 .357.

The method of embodiment 24, wherein said β-ketothioiase is classified under EC 2.3, 3 .16 or EC 2.3.1.174.

A method for biosynthesizmg azelaic acid, said method comprising enzymatically synthesizing non-3 -enal and 9-oxononanoate from 9-hydroxyperoxy octadec- 10, 12 dienoate using a polypeptide having the activity of a hydroperoxide lyase classified under EC 4.2,99.- and enzymatically converting non-3 -enal and/or 9-oxononanoate to azelaic acid.

The method of embodiment 31, wherein said non-3-enal is converted to non-3 - enoate using a polypeptide having the activity of an aldehyde dehydrogenase; non- 3-enoate is converted to non-3-enoyl-CoA using a polypeptide having the activity of a CoA ligase; non-3-enoyl-CoA is converted to non-2-enoyi-CoA using a polypeptide having the activity of a dodecenoyl-CoA isomer ase; non-2-enoyl-CoA is converted to nonanoyl-CoA using a polypeptide having the activity of a trans-2- enoyl-CoA reductase; nonanoyl-CoA is converted to nonanoic acid using a polypeptide having the activity of a thioesterase; nonanoic acid is converted to 9- hydroxynonanoic using a polypeptide having the activity of a nwnooxygenase; 9- hydroxynonanoic is converted to 9-oxononanoate using a polypeptide having the activity of an alcohol dehydrogenase; and 9-oxononanoaie is converted to azelaic acid using a polypeptide having the activity of an aldehyde dehydrogenase.

The method of embodiment 31 , wherein non-3 -enal is converted to nonanai using a polypeptide having the activity of an enoate reductase; nonanai is converted to nonanoic acid using a polypeptide having the activity of an aldehyde dehydrogenase; nonanoic acid is converted to 9-hydroxynonanoic acid using a polypeptide having the activity of a monooxygenase; 9-hydroxynonanoic acid is converted to 9-oxononanoic acid using a polypeptide having the activity of an alcohol dehydrogenase; and 9-oxononanoic acid is converted to azelaic acid using a polypeptide having the activity of an aldehyde dehydrogenase .

The method of embodiment 31, wherein said 9-oxononanoate is converted to azelaic acid using a polypeptide having the activity of an aldehyde dehydrogenase.

The method of any one of embodiments 31 to 34, further comprising the following steps to convert azelaic acid to pimeloyl-CoA: azelaic acid is converted to azelaoyl- CoA using a polypeptide having the activity of a Co A ligase; azelaoyl-CoA is converted to 2,3-dehydro-azelaoyl-CoA using a polypeptide having the activity of an acy!-CoA dehydrogenase; 2,3-dehydro-azelaoyi-CoA is converted to 3-hydroxy- aze!aoyl-CoA using a polypeptide having the activity of an enoyl-CoA hydra!ase; 3- hydroxy-azelaoyi-CoA is converted to 3 -oxo-azelaoyl-Co A using a polypeptide having the activity of a 3-hydroxyacyl-CoA dehydrogenase; and 3 -oxo-azelaoyl-Co A is converted to pimeloyl-CoA using a polypeptide having the activity of a β- keiothiolase .

A method of producing one or more of pirnelic acid, 7-aminoheptanoate, 7- hydroxyheptanoate, heptamethyienediamine, or 1 ,7-heptanediol, or corresponding salts thereof, said method comprising the step of converting 9- hydroxyperoxyoctadec- 10,12-dienoate to non-3 -enal and 9-oxononanoate using a polypeptide having the enzymatic activity of a hydroperoxide lyase and subsequently converting non-3 -enal or 9-oxononanoate to pimeloyl-CoA.

The method of embodiment 36, wherein said non-3 -enal is converted to pimeloyl - CoA using an enzymatic pathway comprising a polypeptide having the activity of a dodecenoyl-CoA isomerase classified under EC 5.3.3,8 and a polypeptide having the activity of a monooxygenase classified under EC 1.14.14.- or EC 1 .14.15.-.

The method of embodiment 36. wherein said non-3 -enal is converted to pime!oyl- CoA using an enzymatic pathway comprising a polypeptide having the activity of an enoale reductase classified under EC 1.3.1.31 and a polypeptide having the activity of a monooxygenase classified under EC 1.14.14.- or EC 1.14.15.-.

The method of embodiment 36, wherein said pimeloyl-CoA is converted to pimelate semialdehyde using one or more polypeptides having the activity of an acetylating aldehyde dehydrogenase.

The method of embodiment 39, wherein said pimeloyl-CoA is converted to pimelate semialdehyde using one or more polypeptides having at least 70%, at least 80%, or at least 85% sequence identity to an acetylating aldehyde dehydrogenase encoded by pduB from Salmonella (yphimurium or encoded by pduP from Klebsiella pneumoniae.

The method of embodiment 36, wherein said pimelate is converted to pimelate semialdehyde using one or more polypeptides having the activity of a carboxylase reductase classified under EC 1.2.99,6,

The method of embodiment 41, wherein said one or more polypeptides have at least 70%, at least 80%, or at least 85% sequence identity to a polypeptide having the activity of a carboxylate reductase classified under EC 1.2.99,6.

The method of embodiment 36, wherein said pimeloyl-CoA is converted to one or more of pimelic acid, 7-amiiioheptanoate, 7~hydroxyheptanoate, hepiamethylenediamine, or 1,7-heptanediol, or corresponding salts thereof, in one or more steps,

The method of embodiment 43, wherein said pimeloyl-CoA is converted to pimelic acid using at least one polypeptide having the enzymatic activity of a ihioesterase encoded by any one of yciA from Escherichia coh, acotlS from Mus musculus, or lesB from Escherichia coli.

The method of embodiment 44, wherein said pimeloyl-CoA is converted to pimelic acid using a polypeptide having at least 70%, at least 80%, or at least 85% sequence identity to a polypeptide encoded by any one of yciA from Escherichia colt, acoU ^' 3 from Mus miisculus or tesB from Escherichia coll

. The method of embodiment 43, wherein said pime!oyl-CoA is converted to pimelie acid using a polypeptide having the activity of a CoA ligase classified under EC 6.2.1.- or a polypeptide having the activity of a CoA transferase classified under EC 2.8.3.-,

, The method of embodiment 46, wherein said pimeloyl-CoA is converted to pimelie acid using a polypeptide having at least 70%, at least 80%, or at least 85% sequence identity to a CoA ligase classified under EC 6.2.1 .- or at least 70%, at least 80%, or at least 85%) sequence identity to a CoA transferase classified under EC 2.8.3.-.. The method of embodiment 39 or 40, wherein said pimelate semialdehyde is converted to pimelie acid using one or more polypeptides having the activity of an aldehyde dehydrogenase classified under EC 1 ,2.1.3, EC 1 ,2.1.16, EC 1.2.1.20, EC 1.2.1.24, EC 1 ,2, 1.63, or EC 1 ,2.1 .79, wherein said aldehyde dehydrogenase classified under EC 1.2.1 , 16, EC 1.2.1.24, or EC 1.2.1.79 is a succinate- semialdehyde dehydrogenase, wherein said aldehyde dehydrogenase classified under EC 1.2.1.20 is a 5-oxopentanoate dehydrogenase, wherein said aldehyde dehydrogenase classified under EC 1 ,2.1.63 is a 6-oxohexanoate dehydrogenase and wherein said aldehyde dehydrogenase classified under EC 1.2.1.- is a 7- oxohepianoate dehydrogenase .

, The method of embodiment 48, wherein said pimelate semialdehyde is converted to pimelie acid using one or more polypeptides having at least 70%, at least 80%, or at least 85% sequence identity to an aldehyde dehydrogenase classified under EC 1.2.1.3, EC 1.2.1.16, EC 1.2.1.20, EC 1 .2.1.24, EC 1.2.1.63, or EC 1.2.1.79.

. The method of any one of embodiments 39 to 41 , wherein said pimelate semialdehyde is converted to 7-aminoheptanoate using one or more polypeptides having the activity of a ω -transaminase classified under EC 2.6.1.-.

, The method of embodiment 50, wherein said pimelate semialdehyde is converted to 7-aminoheptanoate using one or more polypeptides having a!, least 70%, at least 80%, or at least 85% sequence identity to a polypeptide having the activity of a co- iransaminase classified under EC 2,6.1.-.

The method of any one of embodiments 39 to 41, wherein said pimelate se niaidehyde is converted to 7-hydroxyheptanoate using one or more polypeptides having the enzymatic activity of an alcohol dehydrogenase, wherein said alcohol dehydrogenase is a 4-hydroxybulanoate dehydrogenase, a 5~hydroxypentanoate dehydrogenase, or a 6-hydroxyhexanoate dehydrogenase.

The method of embodiment 52, wherein said alcohol dehydrogenase is encoded by any one of chnD from Acinetobacter sp. NCIMB9871, cpnD from Comamonas sp., or gabD from Escherichia coll,

The method of embodiment 53, wherein said alcohol dehydrogenase is encoded by a polypeptide having at least 70%, at least 80%, or at least 85% sequence identity to a polypeptide encoded by any one chnD from Acinetobacter sp. NCIMB9871, cpnD from Comamonas sp., or gabD from Escherichia coli.

The method of embodiment 50 or 51 , wherein said 7-aminoheptanoate is converted to 7-aminoheptanal using one or more polypeptides having the activity of a carboxylase reductase classified under EC 1 .2.99.6 ,and wherein said 7- aminoheptanai is converted to heptamethylenediamine using one or more polypeptides having the activity of a ω --transaminase classified under EC 2.6.1.-. The method of embodiment 55, wherein said carboxylase reductase classified under EC 1.2.99.6 is encoded by griC or griD from Streptomyces griseus.

The method of embodiment 56, wherein said carboxylase reductase classified under EC 1.2.99.6 is a polypeptide having at least 70%, at least 80%>, or at least 85% sequence identity to a polypeptide encoded by griC or griD from Streptomyces griseus,

The method of embodiment 55, wherein said 7-aminoheptanal is converted to heptamethylenediamine using one or more polypeptides having at least 70%, at least 80%), or at least 85% sequence identity to a polypeptide having the activity of a co- transaminase classified under EC 2.6.1.-. The method of embodiment 50 or 51 , wherein said 7-aminoheptanoate is converted to N7-acetyl-7-aminoheptanoate using one or more polypeptides having the activity of an N-acetyltransferase classified under EC 2.3.1 .32; wherein N7-acetyI-7- arninobeptanoate is converted to N7-acetyl - 7- aminoheptanal using one or more polypeptides having the activity of a carboxylase reductase classified under EC 1 .2.99.6; wherein N7-acetyl-7-aminoheptanal is converted to N7-acetyl-l ,7- diaminoheptane using one or more polypeptides having the activity of a co- transaminase classified under EC 2.6.1 .-; and wherein N7-acetyi- i ,7- diaminoheptane is converted to heptamethylenediamine using one or more polypeptides having the activity of a deacylase classified under EC 3.5.1.-.

The method of any one of embodiments 52 to 54. wherein said 7-hydroxyheptanoate is converted to 7-hydroxyheptanai using one or more polypeptides having the activity of a carboxylase reductase classified under EC 1.2.99.6; wherein 7- hydroxyheptanal is converted to 7-aminoheptanol using one or more polypeptides having the activity of a ω-transaminase classified under EC 2.6.1.18, EC 2.6.1.19, or EC 2.6.1.48; wherein 7-aminoheptanol is converted to 7-aminoheptanal using one or more polypeptides having the activity of an alcohol dehydrogenase classified under EC 1.1.1.-; and wherein said 7-aminoheptanal is converted to heptamethylenediamine using one or more polypeptides having the activity of a co- transaminase classified under EC 2.6.1.··.

The method of embodiment 60, wherein said alcohol dehydrogenase has at -least 70%, at least 80%, or at least 85% sequence identity to a polypeptide encoded by yqhD from Escherichia coll .

The method of embodiment 61, wherein said alcohol dehydrogenase has at least 70% sequence identity to a polypeptide encoded by yqhD from Escherichia coli . The method of any one of embodiments 39 to 41 , wherein said pimelate semialdehyde is converted to heptanedial using one or more polypeptides having the activity of a carboxylase reductase classified under EC 1.2.99.6; wherein heptanedial is converted to 7-aminoheptanal using one or more polypeptides having the activity of a ^-transaminase classified under EC 2.6.1.18, EC 2.6.1.19, EC 2,6.1 ,48, EC 2.6.1 ,29, or EC 2.6.1.82; and wherein 7-aminoheptanal is converted to heptamethylenediamine using one or more polypeptides having the activity of a co- transaminase classified under EC 2.6.1.-.

64. The method of any of embodiments 52 to 54, wherein said 7-hydroxyheptanoate is converted to 7-hydroxyheptana) using a carboxylase reductase classified under EC 1.2,99.6; and wherein 7-hydroxyheptana! is converted to 1,7 he tanedioi using one more polypeptides having the activity of an alcohol dehydrogenase classified under EC i .1 , 1. - .

65. The method of embodiment 1 , wherein said 9-hydroxyperoxyoctadec- 10, 12- dienoate is enzymaticaily produced from octadecanoyl-CoA,

66. The method of embodiment 65, wherein said 9-hydroxyperoxyoctadec- 10,12- dienoate is enzymaticail produced from octadecanoyl-CoA using one or more polypeptides having the activity of a delta9-desaturase, a dehal 2-desalurase, a thioesterase, and/or a 9-lipoxygenase.

67. The method of embodiment 66, wherein said polypeptide having the activity of a stearoyl-CoA delta9-desaturase is classified under EC 1.14.19.1.

68. The method of embodiment 66, wherein said polypeptide having the activity of a delt l 2-desaturase is classified under EC 1 , 14, 19.6.

69. The method of embodiment 66, wherein said polypeptide having the activity of a thioesterase is classified under EC 3.1.2.-.

70. The method of embodiment 66, wherein said polypeptide having the activity of a 9- lipoxygenase is classified under EC 1.13.11.58, EC 1.13.11.60, EC 3.13.11 .61 , or EC 1.13.11.62.

71 . The method of any of the preceding embodiments, wherein said method is performed in a recombinant microorganism.

72. The method of embodiment 71 , wherein said microorganism is subjected to a cultivation strategy under aerobic, anaerobic or micro-aerobic cultivation conditions.

73. The method of embodiment 71 or 72, wherein said microorganism is cultured under conditions of nutrient limitation. 74. The method according to any one of embodiments 71 to 73, wherein said microorganism is retained using a ceramic membrane to maintain a high cell density during fermentation.

75. The method of any one of embodiments 71 to 74, wherein the principal carbon source fed to the fermentation derives from a biological feedstock.

76. The method of embodiment 75, wherein the biological feedstock is, or derives from, monosaccharides, disaccharides, lignoceHulose, hemiceliulose, cellulose, lignin, levulinic acid, formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, plant oils, or municipal waste.

77. The method of any one of embodiments 71 to 74, wherein the principal carbon source fed to the fermentation derives from a non-biological feedstock.

78. The method of embodiment 77, wherein the non-biological feedstock is, or derives from, natural gas, syngas, Cl ^' VtT, methanol, ethanol, benzoate, non-volatile residue (NVR) caustic wash waste stream from cycloheptane oxidation processes, or terephthalic acid / isophthalic acid mixture waste streams.

79. The method of embodiment 71 , wherein the microorganism is a prokaryote.

80. The method of embodiment 79, wherein said prokaryote is from a genus selected from Escherichia, Clostridia,Corynebacteria, Cupriavidus, Pseudomonas, Delftia, Bacillus, Lactobacillus, Lactococcus, and Rhodococcus.

81. The method of embodiment 80. wherein said prokaryote is selected from Escherichia coli, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium kluyveri, Corynebacterium glutamicuin, Cupriavidus necator, Cupriavidus metallidurans. Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas oleavorans, Delftia acidovorans, Bacillus subtillis, Lactobacillus delbrueckii, Lactococcus lactis, and Rhodococcus equi.

82. The method of embodiment 71 , wherein the microorganism is a eukaryole.

83. The method of embodiment 82, wherein said eukaryote is from a germs selected from Aspergillus, Saccharomyces, Pichta, Yarrowia, Issatchenkia, Debaryomyces, Arxula, and Kluyveromyces. 84. The method of embodiment 83, wherein said eukaryote is selected from Aspergillus niger, Saccharomyces cerevisiae, Pichia pastoris, Yarrow ia lipolytica, Issalhenkia orientalis, Debaryomyces hansenii, Arxula adenoinivorans, and Kluyveromyces lac/is.

85. The method of embodiment 71 , wherein the microorganism's tolerance to high concentrations of a C7 building block is improved relative to a wild type organism.

86. The method of embodiment 71, wherein the microorganism's tolerance to high concentrations of a C7 building block is improved relative to a wild type organism through continuous cultivation in a selective environment.

87. The method of embodiment 71 , wherein said microorganism comprises an attenuation to one or more of the following enzymes: a polyhydroxyalkanoate synthase, an aceiyl-CoA ihioestera.se, a phosphotransacetylase forming acetate, an acetate kinase, a lactate dehydrogenase, a menaquinol-fiimarate oxidoreductase, an alcohol dehydrogenase forming ethanol, a triose phosphate isomer ase, a pyruvate decarboxylase, a glucose-6-phosphate isomerase, an NADH-consuming transhydrogenase, an NADH-specific glutarnate dehydrogenase, an NADH/NADPH-utilizing glutamate dehydrogenase, a pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenase accepting C7 building blocks and central precursors as substrates: a hutaryl-CoA dehydrogenase; or an adipyl-CoA synthetase.

88. The method of any one of embodiments 71 to 87, wherein said microorganism overexpresses one or more genes encoding: an acetyl-CoA synthetase, a 6- phospho gluconate dehydrogenase; a transketolase; a puridine nucleotide transhydrogenase; a glyceraldehyde-3 F '-dehydrogenase; a malic enzyme; a glucose- 6-phosphate dehydrogenase; a glucose dehydrogenase; a fructose 1.6 diphosphatase; a L-alanine dehydrogenase; a L-glutamate dehydrogenase; a formate dehydrogenase; a L-glutamine synthetase; a diamine transporter; a dicarboxylate transporter; and/or a multidrug transporter.

89. A recombinant microorganism comprising at least one exogenous nucleic acid encoding a polypeptide having the enzymatic activity of (i) a hydroperoxide lyase, (ii) an aldehyde dehydrogenase, (iii) a CoA ligase, (iv) a dodecenoyl-CoA isomerase, (v) a lrans-2-enoyl-CoA reductase, (vi) a thioesterase, (vii) a rnonooxygenase, and/or (viii) an alcohol dehydrogenase, said microorganism producing azelaic acid.

90. A recombinant microorganism comprising at least one exogenous nucleic acid encoding a polypeptide having the enzymatic activity of (i) a hydroperoxide lyase, (ii) an enoate reductase, (iii) an aldehyde dehydrogenase, (iv) a rnonooxygenase, and/or (v) an alcohol dehydrogenase, said microorganism producing azelaic acid.

91. A recombinant microorganism comprising at least one exogenous nucleic acid encoding a polypeptide having the enzymatic activity of (i) a hydroperoxide lyase, and or (ii) an aldehyde dehydrogenase, said microorganism producing azelaic acid.

92. The recombinant microorganism of any of embodiments 89 to 91, said microorganism further comprising one or more exogenous polypeptides having the enzymatic activity of: (i) a CoA ligase, (ii) an acyl-CoA dehydrogenase, (iii) an enoyl-CoA hydratase, (iv) a 3-hydroxyacyl-CoA dehydrogenase or a 3-oxoacyl ACP reductase, and/or (v) a β-ketothiolase, said microorganism further producing pimeloyl-CoA.

93. The recombinant microorganism of embodiment 92, said microorganism further comprising one or more exogenous polypeptides having the enzymatic activity of a thioesterase, a CoA ligase, a CoA transferase, an acetylating aldehyde dehydrogenase, and/or an aldehyde dehydrogenase, said microorganism further producing pimelic acid.

94. The recombinant microorganism of embodiment 92 or 93, said microorganism further comprising one or more polypeptides having the activity of an aldehyde dehydrogenase and/or a ω-transaminase, said microorganism further producing 7- am inoheptano ate .

95. The recombinant microorganism of embodiment 92 or 93, said microorganism further comprising one or more exogenous polypeptides having the activity of a carboxylate reductase and/or a ω-transaminase, said microorganism further producing 7-aminoheptanoate.

r

J O 96. The recombinant microorganism of embodiment 92 or 93, said microorganism further comprising one or more of the following exogenous enzymes: a carboxylate reductase, an alcohol dehydrogenase, or an acetylating aldehyde dehydrogenase, said microorganism further producing 7-h droxyheptanoate.

97. The recombinant microorganism of any one of embodiments 92 to 96. said microorganism comprising one or more of the following exogenous enzymes: a carboxylate reductase, a ω-transaminase, an alcohol dehydrogenase, an N- acetyltransferase, or a deacylase, said microorganism further producing heptamethyienediamine.

98. The recombinant microorganism of embodiment 92 or 93, wherein said pimeloyl-

CoA or said pimelic acid is converted to pimelate semialdehyde using an acetylating aldehyde dehydrogenase encoded by pduB from Salmonella typhimurivm or pduP from Klebsiella pneumoniae or one or more polypeptides having the activity of a carboxylate reductase classified under EC 1.2.99.6.

99, The recombinant microorganism of embodiment 98, said microorganism comprising polypeptides having the activity of a carboxylate reductase and one or more ω- transarninases, said microorganism producing heptamethyienediamine.

100. The recombinant microorganism of embodiment 96, said microorganism further comprising polypeptides having the activity of a carboxylate reductase and an alcohol dehydrogenase, said microorganism further producing 1 ,7 heptanediol.

101 . The recombinant microorganism of any one of embodiments 89 to 100, said microorganism further comprising one or more exogenous enzymes: a delta.9- desaturase, a delta 12-desaturase, a thioestera.se, or a 9-Upoxygenase.

102. A non-naturaily occurring microorganism comprising at least one exogenous nucleic acid encoding at least one polypeptide having the activity of at least one enzyme, at least one substrate, and at least one product, depicted in any one of FIG. 1 to 7.

1 03. A nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having carboxylate reductase activity, wherein the polynucleotide is operabiy linked to one or more heterologous control sequences thai direct production of the polypeptide and wherein the polypeptide having carboxylate reductase activity is selected from: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 1 : (b) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 2: (c) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 3; (d) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ) ID NO: 4, (e) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 5 and (f) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 6.

104. A nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having co- transaminase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having ω -transaminase activity is selected from: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ) ID NO: 7; (b) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 8; (c) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 9; (d) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 10; (e) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 1 1 or SEQ ID NO: 48; and (f) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 12.

105. A composition comprising the nucleic acid construct or expression vector of embodiment 103 or 104.

106. A culture medium comprising the nucleic acid construct or expression vector of embodiment 103 or 104.

107. A non-naturally occurring biochemical network comprising a 9- hydroxyperoxyoctadec- 10, 2-dienoate, an exogenous nucleic acid encoding a polypeptide having the activity of a hydroperoxide lyase classified under EC 4.2.99.-, and non-3-enal and 9-oxononanoate. 108. A non-naturally occurring biochemical network comprising non- 3 -enoyl-Co A, an exogenous nucleic acid encoding a polypeptide having the activity of a dodecenoyl- CoA isomerase classified under EC 5.3.3.8 and non-2-enoyl-CoA.

109. A non-naturally occurring biochemical network comprising non-3-enal, an exogenous nucleic acid encoding a polypeptide having the activity of an eno te reductase classified under EC 1.3.1.31 and nonanal.

1 10. A non-naturally occurring biochemical network comprising nonanoic acid, an exogenous nucleic acid encoding a polypeptide having the activity of a monooxygenase classified under EC 1.14.14.- or EC 1.14.15.- and 9- hydroxynonanoic acid.

1 1 1. Means for producing pimeloyl-CoA, comprising culturing a non-natural !y occurring microorganism comprising at least one exogenous nucleic acid encoding a polypeptide having the enzymatic activity of (i) a hydroperoxide lyase, (ii) an aldehyde dehydrogenase, (iii) a CoA ligase, (iv) a dodecenoyh CoA isomerase, (iv) a trans-2-enoyl-CoA reductase, (v) a thioesterase, (vi) an enoate reductase, (vii) a monooxygenase, (viii) an alcohol dehydrogenase, (ix) an acyl-CoA dehydrogenase, (x) an enoyl-Co A hydratase, (xi) a 3 -hydroxyacyl-CoA dehydrogenase and (xii) a β- ketothiolase, expressed in a sufficient amount in said microorganism to produce pimeloyl-CoA.

1 12. A bio-derived, bio-based, or fermentation-derived product, wherein said product comprises:

(i) a composition comprising at least one bio-derived, bio-based, or fermentation-derived compound according to any one of embodiments 6-8, 36, or 39 or any combination thereof,

(ii) a bio-derived, bio-based, or fermentation-derived polymer comprising the bio-derived, bio-based or fermentation-derived composition or compound of (i), or any combination thereof,

(iii) a bio-derived, bio-based, or fermentation-derived resin comprising the bio-derived, bio-based, or fermentation-derived compound or bio-derived, bio-based, or fermentation-derived composition of (i) or any combination thereof or the bio-derived, bio-based, or fermentation-derived polymer of (ii) or any combination thereof,

(iv) a molded substance obtained by molding the bio-derived, bio-based, or fermentation-derived polymer of (ii) or the bio-derived, bio-based, or fermentation- derived resin of (iii), or any combination thereof,

(v) a bio-derived, bio-based, or fermentation-derived formulation comprising the bio-derived, bio-based, or fermentation-derived composition of (i), bio- derived, bio-based, or fermentation-derived compound of (i), bio-derived, bio-based, or fermentation-derived polymer of (ii), bio-derived, bio-based, or fermentation-derived resin of (iii), or bio-derived, bio-based, or fermeniation-derived molded substance of (v), or any combination thereof, or

(vi) a bio-derived, bio-based, or fermentation-derived semi-solid or a non-semi-solid stream, comprising the bio-derived, bio-based, or fermentation- derived composition of (i), bio-derived, bio-based, or fermentation-derived compound of (i), bio- derived, bio-based, or fermentation-derived polymer of (ii), bio-derived, bio-based, or fermentation-derived resin of (iii ), bio-derived, bio-based, or fermentatio -derived formulation of (v), or bio-derived, bio-based, or fermentation-derived molded substance of (iv), or any combination thereof,

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein ca be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control, in addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the application, including the written description and drawings, and the claims. The word "comprising" in the claims may be replaced by "consisting essentially of or with "consisting of," according to standard practice in patent law.

DESCRIPTION OF DRAWINGS

FIG, 1 is a schematic of exemplary biochemical pathways leading to pimeloyl- CoA via isomerase activity using octadecanoyl-CoA as a central metabolite.

FIG. 2 is a schematic of further exemplary biochemical pathways leading to pimeloyl-CoA via enoaie reductase activity using octadecanoyl-CoA as a central metabolite.

FIG. 3 is a schematic of exemplary biochemical pathways leading to pimelic acid using pimeloyl-CoA as a central precursor.

FIG. 4 is a schematic of exemplary biochemical pathways leading to 7- aminoheptanoate using pimeloyl-CoA and pimelate as central precursors.

FIG. 5 is a schematic of exemplary biochemical pathways leading to heptamethylenediamine using 7-aminoheptanoate, 7-hydroxyheptanoate, and pimelate semialdehyde as central precursors.

FIG. 6 is a schematic of exemplary biochemical pathways leading to 7- hydroxyheptanoate using pimeloyl-CoA and pimelate as central precursors.

FIG. 7 is a schematic of an exemplary biochemical pathway leading to 1,7- hepianediol using 7-hydroxyheptanoate as a central precursor.

FIG. 8 contains the amino acid sequences of a Mycobacterium marinum carboxylate reductase (see GenBank Accession No. ACC40567.1 , SEQ ID NO: 1), a Mycobacterium smegmatis carboxylate reductase (see GenBank Accession No. ABK71854.1, SEQ ID NO: 2), a Segniliparus rugosus carboxylate reductase (see GenBank Accession No. EFV1 1917.1, SEQ ID NO: 3), a Mycobacterium smegmatis carboxylate reductase (see GenBank Accession No, ABK75684.1, SEQ ID NO: 4), a Mycobacterium massiliense carboxylate reductase (see GenBank Accession No. EIV1 1 143.1 , SEQ ID NO: 5), a Segniliparus rotundus carboxylate reductase (see GenBank Accession No. ADG98140.1, SEQ ID NO: 6), a Chromobacteriurn violaceum ω-transaminase (see GenBank Accession No. AAQ59697.1, SEQ ID NO: 7), a Pseudomonas aeruginosa co-transaminase (see GenBank Accession No. AAG08191.1, SEQ ID NO: 8), a Pseudomonas syringae co-transaminase (see GenBank Accession No. AAY39893.1 , SEQ ID NO: 9), a Rhodobacter sphaeroides co-transaminase (see GenBank Accession No. ABA81 135.1 , SEQ ID NO: 10), an Escherichia coli o> transaminase (see GenBank Accession No. AAA57874.1 , SEQ ID NO: 1 1 , SEQ ID NO: 48), a Vibrio fluvialis co-transaminase (see GenBank Accession No. AEA39183.1 , SEQ ID NO: 1.2), a Cuc mis sativus hydroperoxide lyase (see GenBank Accession No. AAF6404L 1 , SEQ ID NO: 13), a Oryza sativa hydroperoxide lyase (see GenBank Accession No. BAG97978.1, SEQ ID NO: 14), a Lactobacillus casei enoate reductase (see GenBank Accession No. AGP69310.1 , SEQ ID NO: 15), a Pseudomonas putida enoate reductase (see GenBank Accession No. AAN66878.1 , SEQ ID NO: 16), a Saccharomyces cerevisiae isomerase (see GenBank Accession No. AAC83700. L SEQ ID NO: 17), a Clostridium perfringens thioesterase (see GenBank Accession ABG82470.1 , SEQ ID NO: 1 8), a Saccharomyces cerevisiae isomeras (see GenBank Accession No. AAC83700.1, SEQ ID NO: 19), a Bacter -aides thetaiotaomicron thioesterase (see GenBank Accession No. AA077182.1, SEQ ID NO: 20), a Geobacillus stearothermophilus alcohol dehydrogenase (see GenBank Accession No. CAA83612.1 , SEQ ID NO: 21), a Lactobacillus planiar m WCFS1 thioesterase (see GenBank Accession No. CCC78182.1 , SEQ ID NO: 22), and an Anaerococcus tetradius ATCC 35098 thioesterase (see GenBank Accession No. EEI82564.1, SEQ ID NO: 23). In addition, gene ID and gene sequences (SEQ ID NOs: 24 - 46) corresponding to the amino acid sequences are provided in FIG. 8,

FIG. 9 is a bar graph summarizing the change in absorbance at 340 nm after 20 minutes, which is a measure of the consumption of NADPH and activity of six carboxylase reductase preparations in enzyme only controls (no substrate).

FIG. 10 is a bar graph of the change in absorbance at 340 nm after 20 minutes, which is a measure of the consumption of NADPH and the activity of two carboxylaie reductase preparations for converting pime!ate to pimelate semialdehyde relative to the empty vector control. FIG. 1 1 is a bar graph of the change in absorbance at 340 nm after 20 minutes, which is a measure of the consumption of NADPH and the activity of six carboxylate reductase preparations for converting 7-hydroxyheptanoate to 7-hydroxyheptanal relative to the empty vector control.

FIG. 12 is a bar graph of the change in absorbance at 340 nm after 20 minutes, which is a measure of the consumption of NADPH and the activity of three carboxylate reductase preparations for converting N7-acety[-7-aminoheptanoate to N7-acetyl-7- aminoheptanal relative to the empty vector control.

FIG. 13 is a bar graph of the change in absorbance at 340 nm after 20 minutes, which is a measure of the consumption of NADPH and activity of a carboxylate reductase preparation for converting pimelate semia!dehyde to heptanedial relative to the empty vector control.

FIG. 14 is a bar graph summarizing the percent conversion of pyruvate to L- alanine (rnol/mol) as a measure of the ay-transaminase activity of the enzyme only controls (no substrate).

FIG. 15 is a bar graph of the percent conversion after 4 hours of pyruvate to L- alanine (rnol/mol) as a measure of the a>transaminase activity of four ay-transaminase preparations for converting 7-aminoheptanoate to pimelate semialdehyde relative to the empty vector control.

FIG. 16 is a bar graph of the percent conversion after 4 hours of L-alanine to pyruvate (mol/mol) as a measure of the ay-transaminase activity of three oy-transaminase preparations for converting pimelate semialdehyde to 7-aminoheptanoate relative to the empty vector control.

FIG. 1 7 is a bar graph of the percent conversion after 4 hours of pyruvate to L- alanine (mol/mol) as a measure of the ay-transaminase activity of six ay-transaminase preparations for converting heptamethylenedi amine to 7-aminoheptanal relative to the empty vector control.

FIG. 1 8 is a bar graph of the percent conversion after 4 hours of pyruvate to L- alanine (mol/mol) as a measure of the ^transaminase activity of six ay-transaminase preparations for converting N7-acelyl-l ,7-diaminoheptane to N7-acetyf-7-aminoheptana{ relative to the empty vector control.

FIG. 19 is a bar graph of the percent conversion after 4 hours of pyruvate to L- aianine (raol/mol) as a measure of the ω-trcmsaminase activity of three <o-transaminase preparations for converting 7-aminoheptano! to 7-oxoheptanol relative to the empty vector control.

DETAILED DESCRIPTION

In general, this document provides enzymes, non-natural pathways, cultivation strategies, feedstocks, microorganisms, and attenuations to the microorganism's biochemical network, for producing pimeloyl-CoA or one or more of pimelic acid, 7- aminoheptanoate, 7-hydroxyheptanoic acid, heptamethylenediamine, or 1 ,7-heptanediol, or corresponding salts thereof, all of which are referred to as C7 building blocks herein.

As used herein, a "bio-based product" is a product in which both the feedstock (e.g. , sugars from sugar cane, corn, wood; biomass; waste streams from agricultural processes) and the conversion process to the product are biologically based (e.g., fermentation/enzymatic transformation involving a biological host/organism/enzyme). As used herein, a "bio-derived product" is a product in which one of the feedstocks (e.g., sugars from sugar cane, corn, wood; biomass; waste streams from agricultural processes) or the conversion process to the product is biologically based (e.g., fermentation/enzymati c transformation involving a biological host/organism/enzyme).

As used herein, a "fermentation-derived product" is a product produced by fermentation involving a biological host or organism.

The term "C7 building block" is used to denote a seven (7) carbon chain aliphatic backbone. As used herein, the term "central precursor" is used to denote any metabolite in any metabolic pathway shown herein leading to the synthesis of a C7 building block. The term "central metabolite" is used herein to denote a metabolite that is produced in all microorganisms to support growth.

Microorganisms described herein can include endogenous pathways that can be manipulated such that pimeloyl-CoA or one or more other C7 building blocks can be produced. In an endogenous pathway, the microorganism naturally expresses all of the enzymes catalyzing the reactions within the pathway. A microorganism containing an engineered pathway does not naturally express all of the enzymes catalyzing the reactions within the pathway but has been engineered such that all of the enzymes within the pathway are expressed in the microorganism.

The term "exogenous" as used herein with reference to a nucleic acid (or a protem) and a microorganism refers to a nucleic acid that does not occur in (and cannot be obtained from) a cell of that particular type as it is found in nature or a protein encoded by such a nucleic acid. Thus, a non-naturally-occurring nucleic acid is considered to be exogenous to a microorganism once in the microorganism. It is important to note that non-natural ly-oeeurrmg nucleic acids can contain nucleic acid subsequences or fragments of nucleic acid sequences that are found in nature provided the nucleic acid as a whole does not exist in nature. For example, a nucleic acid molecule containing a genomic DNA sequence within an expression vector is a non-naturally- occurring nucleic acid, and thus is exogenous to a microorganism once introduced into the microorganism, since that nucleic acid molecule as a whole (genomic DNA plus vector DNA) does not exist in nature. Thus, any vector, autonomously replicating plasmid, or virus (e.g. , retrovirus, adenovirus, or herpes virus) that as a whole does not exist in nature is considered to be non-naturally-occurring nucleic acid. It follows that genomic DNA fragments produced by PGR or restriction endonuciease 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 a non-natural ly-occurring nucleic acid. A nucleic acid that is naturally-occurring can be exogenous to a particular microorganism. For example, an entire chromosome isolated from a cell of yeast x is an exogenous nucleic acid with respect to a ceil of yeast y once that chromosome is introduced into a cell of yeast y.

In contrast, the term "endogenous" as used herein with reference to a nucleic acid (e.g. , a gene) (or a protein) and a microorganism refers to a nucleic acid (or protein) that does occur in (and can be obtained from) that particular microorganism as it is found in nature. Moreover, a ceil "endogenously expressing" a nucleic acid (or protein) expresses that nucleic acid (or protein) as does a microorganism of the same particular type as it: is found in nature. Moreover, a microorganism "endogenously producing" or that "endogenously produces" a nucleic acid, protein, or other compound produces that nucleic acid, protein, or compound as does a microorganism of the same particular type as it is found in nature,

For example, depending on the microorganism and the compounds produced by the microorganism, one or more polypeptides having the following specific enzymatic activities may be expressed in the microorganism in addition to a hydroperoxide lyase: an acetylating aldehyde dehydrogenase, a CoA ligqse, a dodecenoyl-CoA isomerase or an enoate reductase, a. trans-2-enoyl-CoA reductase, a thioesterase, a monooxygenase, an enoyl-CoA hydratase, a deacetylase, an acyl-CoA dehydrogenase, an enoyl-CoA hydratase, an alcohol dehydrogenase, a 4-hydroxybutanoate dehydrogenase, a 6- hydroxyhexanoate dehydrogenase, an aldehyde dehydrogenase, a succinate-semialdehyde dehydrogenase, a 5-oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 7~oxoheptanoate dehydrogenase, an acyl-CoA dehydrogenase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, a 3-oxoacyl ACP reductase, a β-ketothiolase, a CoA transferase, a carhoxylate reductase, a ω-transa inase , an N-acetyltransferase. and/or a deacylase. in recombinant microorganisms expressing a polypeptide having the activity of a carhoxylate reductase, a polypeptide having the activity of a phosphopantetheinyl transferase also can be expressed as it enhances activity of the carhoxylate reductase.

For example, a recombinant microorganism can include a polypeptide having the activity of an exogenous hydroperoxide lyase and produce non-3 -enal and 9- oxononanoate from 9-hydroxyperoxyoctadec- 10, 12-dienoate. The non-3 -enal and 9- oxononanoate can be converted enzymaticaily to pimeloyl-CoA and subsequently to one or more of pirnelic acid, 7-aminoheptanoate, 7-hydroxyheptanoic acid, heptamethylenediamine, or 1 ,7-heptanediol, or corresponding salts thereof.

For example, a recombinant microorganism producing pimeloyl-CoA can include one or more of exogenous polypeptides having the enzymatic activity of: a thioesterase, a CoA ligase, a CoA transferase, an acetylating aldehyde dehydrogenase, a succinate- semialdehyde dehydrogenase, a 5-oxopentiinoate dehydrogenase, a 6-oxohexanoate dehydrogenase, and/or a 7-oxoheptanoate dehydrogenase, and further produce pimeiic acid. See FIG. 3.

For example, a recombinant microorganism producing pimeloyl-CoA can include an exogenous polypeptide having the activity of a thioesterase and produce pimeiic acid. For example, a recombinant microorganism producing pimeloyl-CoA can include an exogenous polypeptide having the activity of a CoA ligase or a CoA transferase, and further produce pimeiic acid. For example, a recombinant microorganism producing pimeloyl-CoA can include an exogenous polypeptide having the activity of an acetylating aldehyde dehydrogenase and one or more polypeptides having the enzymatic activity of: an aldehyde dehydrogenase, a succinate-semialdehyde dehydrogenase, a 5- oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, and/or a 7- oxoheptanoate dehydrogenase, and produce pimeiic acid. See FIG. 3.

For example, a recombinant microorganism can include one or more exogenous polypeptides having the enzymatic activity of an aldehyde dehydrogenase, a ω- transaminase, and/or a carhoxylate reductase, and produce 7-aminoheptanoate. See FIG. 4.

For example, a recombinant microorganism producing pimeloyl-CoA can include an exogenous polypeptide having the activity of an acetylating aldehyde dehydrogenase and an exogenous polypeptide having the activity of a ω-transaminase, and produce 7- aminoheptanoate. For example, a recombinant microorganism producing pimelate (see FIG. 3) can include an exogenous polypeptide having the activity of a carboxylase reductase and an exogenous polypeptide having the activity of a ω-transammase, and produce 7-aminoheptanoate. See FIG. 4.

For example, a recombinant microorganism producing pimeloyl-CoA can include one or more exogenous polypeptides having the enzymatic activity of a carhoxylate reductase, an alcohol dehydrogenase, a 4-hydroxyhntanoate dehydrogenase, a 5- hydroxypentanoate dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, and/or an aldehyde dehydrogenase and further produce 7-hydroxyheptanoate. See FIG. 6. For example, a recombinant microorganism producing pimeloyl-CoA can include an exogenous polypeptide having the activity of a carhoxylate reductase and an exogenous polypeptide having the activity of a 4-hydroxybutanoate dehydrogenase, and produce 7-hydroxyheptanoate. For example, a recombinant microorganism producing pimeloyl-CoA can include an exogenous polypeptide having the activity of a carhoxylate reductase and an exogenous polypeptide having the activity of a 5-hydroxypentanoate dehydrogenase, and produce 7-hydroxyheptanoate. For example, a recombinant microorganism producing pimeloyl-CoA can include an exogenous polypeptide having the activity of a carhoxylate reductase and an exogenous polypeptide having the activity of a. 6-hydroxyhexanoate dehydrogenase, and produce 7-hydroxyheptanoate. For example, a recombinant microorganism producing pimelate (see FIG. 3) can include an exogenous polypeptide having the activity of a carhoxylate reductase and an exogenous polypeptide having the activity of either a 4-hydroxybutanoate dehydrogenase, a 5- hydroxypenianoate dehydrogenase, or a 6-hydroxyhexanoate dehydrogenase, and produce 7-hydroxyheptanoate. See FIG. 6.

For example, a recombinant microorganism producing pimeloyi-CoA can include one or more exogenous polypeptides to produce 7-aminoheptanoate or 7- bydroxyheptanoate. See FIG. 4 and FIG. 6. A recombinant microorganism producing 7- aminoheptanoate or 7-hydroxyheptanoate can include one or more exogenous polypeptides having the activity of: a carhoxylate reductase, a ω-transaminase, an alcohol dehydrogenase, an N-acetyllransferase, and/or a deacylase, and produce heptamethylenediamine. See FIG. 5.

For example, a recombinant microorganism producing pimeloyl-CoA can include the polypeptides necessary to convert pimeioyl-CoA to 7-aminoheptanoate and can include an exogenous polypeptide having the acti vity of a carhoxylate reductase and one or more exogenous polypeptides having the activity of ω -transaminases {e.g. , one transaminase or two different transaminases) and produce heptamethylenediamine. For example, a recombinant microorganism producing pimeloyl-CoA can include the polypeptides necessary to convert pimeloyl-CoA to 7-aminoheptanoate and can include one or more exogenous polypeptides having the activity of an N-acetyltransferase, a carboxylate reductase, a ω -transaminase, and/or a deacylase, and produce heptamethylenediamine. For example, a recombinant microorganism producing phneloyi-CoA can include the polypeptides necessary to convert pimeloyl-CoA to 7- hydroxyheptanoate and can include one or more exogenous polypeptides having the activity of a carboxylate reductase, a ω-transaminase {e.g. , one transaminase or two different transaminases), and/or an alcohol dehydrogenase, and produce heptamethylenediamine. See FIG. 5.

For example, a recombinant microorganism producing pimeloyl-CoA can include the polypeptides having the necessary enzymatic activity for conversion of pimeloyl-CoA to 7-hydroxyheptanoate (see FIG. 6) as described above and can also include one or more exogenous polypeptides having the enzymatic activity of a carboxylate reductase and/or an alcohol dehydrogenase, and further produce 1 ,7-heptanediol from 7- hydroxyheptanoate. See FIG. 7.

In any of the recombinant microorganisms, the recombinant microorganism also can include one or more (e.g., one, two, or three) of the following exogenous enzymes used to convert either octadecanoyl-CoA to 9-hydroxyperoxyoctadec- 10,12-dienoate: a delta9-desaturase, a delta! 2-desat rase, a thioesterase, or a 9-lipoxygenase. For example, a recombinant microorganism can include a delta9~desaturase, a deltaH- desaturase, a thioesterase, and a 9-lipoxygenase.

Within an engineered pathway, the enzymes can be from a single source, i.e., from one species or genera, or can be from multiple sources, i.e. , different species or genera. Nucleic acids encoding the enzymes described herein have been identified from various organisms and are readily available in publicly available databases such as GenBank or EMBL. Enzyme Commission (EC) numbers for many enzymes are also provided. EC numbers are well known in the art and provide a numerical classification scheme for enzymes based on the chemical reactions they catalyze. An enzyme classified with an EC number to the fourth level is discretely and specifically classified on the basis of the reactions that its members are able to perform. Well known nomenclature databases such as ENZYME, maintained by the Swiss Institute of Bioinformatics, provide examples of specific enzymes corresponding to specific EC numbers. Any of the enzymes described herein thai can be used for production of one or more C7 building blocks can have at least 70% sequence identity (homology) (e.g. , at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of the corresponding wild-type enzyme. It will be appreciated that the sequence identity can be determined on the basis of the mature enzyme (e.g., with any signal sequence removed) or on the basis of the immature enzyme (e.g. , with any signal sequence included). It also will be appreciated that the initial methionine residue may or may not be present on any of the enzyme sequences described herein.

For example, a polypeptide having the activity of a carboxylate reductase described herein can have at least 70% sequence identity (homology) (e.g. , at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Mycobacterium marin m (see GenBank Accession No. ACC40567.1 , SEQ ID NO: 1), a Mycobacterium smegmatis (see GenBank Accession No. ABK71854. I, SEQ ID NO: 2), a Segniliparus rugosus (see GenBank Accession No. EFVI 1917.1 , SEQ ID NO: 3), a Mycobacterium smegmatis (see GenBank Accession No. ABK75684.1 , SEQ ID NO: 4). a Mycobacterium massiliense (see GenBank Accession No. EIV 1 1 143.1 , SEQ ID NO: 5), or a Segniliparus roiundus (see GenBank Accession No. ADG98140.1 , SEQ ID NO: 6) carboxylate reductase. See FIG. 8.

For example, a polypeptide having the activity of a ω-iransaminase described herein can have at least 70%;· sequence identity (homology) (e.g. , at least 75%), 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Chromobacterium violaceum (see GenBank Accession No. AAQ59697.1, SEQ ID NO: 7), a Pseudomonas aeruginosa (see GenBank Accession No. AAG08191.1, SEQ ID NO: 8), a Pseudomonas syringae (see GenBank Accession No. AAY39893.1, SEQ ID NO: 9), a Rhodobacter sphaeroides (see GenBank Accession No. ABA81 135.1 , SEQ ID NO: 10), an Escherichia coli (see GenBank Accession No. AAA57874.1, SEQ ID NO: 1 1 , SEQ ID NO: 48), or a Vibrio fluvialis (see GenBank Accession No. AEA391 83.1 , SEQ ID NO: 12) ω-transaminase. Some of these (^-transaminases are diamine o> transaminases . See FIG. 8. For example, a polypeptide having the activity of a hydroperoxide lyase described herein can' have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Cucumis sativus (see GenBank Accession No. AAF64041.1 , SEQ ID NO: 13) or a Oryza sativa hydroperoxide lyase (see GenBank Accession No. BAG97978.1, SEQ ID NO: 14), See FIG. 8.

For example, a polypeptide having the activity of an enoate reductase described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 300%) to the amino acid sequence of a Lactobacillus casei (see GenBank Accession No. AGP69310.1 , SEQ ID NO: 15) or a Pseudomonas pulida enoate reductase (see GenBank Accession No. AAN66878.1 , SEQ ID NO: 16). See FIG. 8.

For example, a polypeptide having the activity of an isomerase described herein can have at least 70% sequence identity (homology) (e.g. , at least 75%, 80%, 85%, 90%, 93%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Saccharomyces cerevisiae isomerase (see GenBank Accession No. AAC83700.1, SEQ ID NO: 17 and SEQ ID NO: 19). See FIG. 8.

For example, a polypeptide having the activity of a ihioesterase described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Clostridium perfringens (see GenBank Accession No. ABG82470.1 , SEQ ID NO: 18), a Bacteroides thetaiotaomicron VPI-5482 (see GenBank Accession No. AA077182.1, SEQ ID NO: 20), a Lactobacillus plantarum WCFS1 (see GenBank Accession No. CCC78182. I , SEQ ID NO: 22), or a Anaerococcus tetradius ATCC 35098 (see GenBank Accession No. EEI82564.1 , SEQ ID NO: 23). See FIG. 8.

For example, a polypeptide having the activity of an alcohol dehydrogenase described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Geobacillus stearothermophUus (see GenBank Accession No. CAA81612.1 , SEQ ID NO: 21 ). See FIG. 8. The percent identity (homology) between two amino acid sequences can be determined as follows. First, the amino acid sequences are aligned using the BLAST 2 Sequences (B12seq) program from the stand-alone version of BLASTZ containing BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained from Fish & Richardson's web site (e.g. , www.fr.com/biast/) or the U.S. government's National Center for Biotechnology Information web site (www.ncbi.nlm.nih.gov). Instructions explaining how to use the B12seq program can be found in the readme file accompanying BLASTZ. B12seq performs a comparison between two amino acid sequences using the BLASTP algorithm. To compare two amino acid sequences, the options of B12seq are set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g. , C:\seq i .txt); -j is set to a file containing the second amino acid sequence to be compared (e.g. , C:\seq2.txt); -p is set to blastp; -o is set. to any desired file name (e.g. , C:\output.txt); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\B12seq -i c:\seql .txt -j c:\seq2.txt -p blastp -o c:\output.txt. If the two compared sequences share homology (identity), then the designated output file will present those regions of homology as aligned sequences, if the two compared sequences do not share homology (identity), then the designated output file will not present aligned sequences. Similar procedures can be following for nucleic acid sequences except that blastn is used.

Once aligned, the number of matches is determined by counting the number of positions where an identical amino acid residue is presented in both sequences. The percent identity (homology) is determined by dividing the number of matches by the length of the full-length polypeptide amino acid sequence followed by multiplying the resulting value by 100. It is noted that the percent identity (homology) value is rounded to the nearest tenth. For example, 78.1 1 , 78.12, 78.13, and 78.14 is rounded down to 78.1 , while 78.15, 78.16, 78, 17, 78.18, and 78.19 is rounded up to 78.2. It also is noted that the length value will always be an integer.

It will be appreciated that a number of nucleic acids can encode a polypeptide having a particular amino acid sequence. The degeneracy of the genetic code is well known to the art; i.e.. for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid. For example, codons in the coding sequence for a given enzyme can be modified such that optimal expression in a particular species (e.g. , bacteria or fungus) is obtained, using appropriate codon bias tables for that species.

Functional fragments of any of the enzymes described herein can also be used in the methods of the document. The term "functional fragment" as used herein refers to a peptide fragment of a protein that has at least 25% (e.g. , at least: 30%; 40%; 50%; 60%; 70%; 75%; 80%; 85%; 90%; 95%; 98%; 99%; 100%; or even greater than 100%») of the activity of the corresponding mature, full-length, wild-type protein. The functional fragment can generally, but not always, be comprised of a continuous region of the protein, wherein the region has functional activity.

This document also provides (i) functional variants of the enzymes used in the methods of the document and (ii) functional variants of the functional fragments described above. Functional variants of the enzymes and functional fragments can contain additions, deletions, or substitutions relative to the corresponding wild-type sequences. Enzymes with substitutions wi ll generally have not more than 100 (e.g., not more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 100) amino acid substitutions (e.g. , conservative substitutions). This applies to any of the enzymes described herein and functional fragments. A conservative substitution is a substitution of one amino acid for another with similar characteristics. Conservative substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoieucine: aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The nonpolar hydrophobic amino acids include alanine, leucine, isoieucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic or acidic groups by another member of the same group can be deemed a conservative substitution. By contrast, a nonconservative substitution is a substitution of one amino acid for another with dissimi lar characteristics.

Deletion variants can lack one, two, three, four, five, six, seven, eight, nine, ten, I I , 12, 13, 1 4, 15, 16, 17, 1 8, 19, 20, 30, 40, or 50 amino acid segments (of two or more amino acids) or non-contiguous single amino acids. Additions (addition variants) include fusion proteins containing: (a) any of the enzymes described herein or a fragment thereof; and (b) internal or terminal (C or N) irrelevant or heterologous amino acid sequences, in the context of such fusion proteins, the term 'lieterologous amino acid sequences" refers to an amino acid sequence other than (a). A heterologous sequence can be, for example a sequence used for purification of the recombinant protein (e.g. , FLAG, po!yhistidine (e.g. , hepta istidine (SEQ ID NO: 47)), hemagglutinin (HA), giutathione-S-transferase (GST), or maltosebinding protein (M BP)). Heterologous sequences also can be proteins useful as detectable markers, for example, luciferase, green fluorescent protein (GFP), or chloramphenicol acetyl transferase (CAT). In some embodiments, the fusion protein contains a signal sequence from another protein, in certain microorganisms (e.g. , yeast cells), expression and/or secretion of the target protein can be increased through use of a heterologous signal sequence. In some embodiments, the fusion protein can contain a carrier (e.g. , KLH) useful, e.g. , in. eliciting an immune response for antibody generation) or ER or Golgi apparatus retention signals. Heterologous sequences can be of varying length and in some cases can be a longer sequences than the full-length target proteins to which the heterologous sequences are attached.

Engineered microorganisms can naturally express none or some (e.g. , one or more, two or more, three or more, four or more, five or more, or six or more) of the enzymes of the pathways described herein. Thus, a pathway within an engineered microorganism can include ail exogenous enzymes, or can include both endogenous and exogenous enzymes. Endogenous genes of the engineered microorganisms also can be disrupted to prevent the formation of undesirable metabolites or prevent the loss of intermediates in the pathway through other enzymes acting on such intermediates. Engineered microorganisms can be referred to as recombinant, microorganisms or recombinant cells. As described herein recombinant microorganisms can include nucleic acids encoding one or more of a hydroperoxide lyase, an aldehyde dehydrogenase, a CoA ligase, a dodecenoyl-CoA isomerase or an enoate reductase, a trans-2-enoyl-CoA reductase, a thioesierase, a monooxygenase, an enoyl-CoA hydratase, a deacetylase, an acyl-CoA dehydrogenase, an enoyl-CoA hydratase, an alcohol dehydrogenase, a 4'- hydroxybutanoaie dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, an aldehyde dehydrogenase, a succinate-semialdehyde dehydrogenase a 5-oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, an acyl-CoA dehydrogenase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, a 3-oxoacyl ACP reductase, a β-ketothiolase, a delta9-desaturase, a delta! 2-desaiurase, a thioesierase, or a 9-Upoxygenase, as described herein.

in addition, the production of C7 building blocks can be performed in y/iro using the isolated enzymes described herein, using a lysate (e.g. , a ceil lysate) from a microorganism as a source of the enzymes, or using one or more lysates irorn different microorganisms as the source of the enzymes.

The reactions of the pathways described herein can be performed in one or more microorganisms (a) naturally expressing one or more relevant enzymes, (b) genetically engineered to express one or more relevant enzymes, or (c) naturally expressing one or more relevant enzymes and genetically engineered to express one or more relevant enzymes. Alternatively, relevant enzymes can be isolated, purified or extracted from of the above types of microorganism cells and used in a purified or semi-purified form, Moreover, such extracts include lysates (e.g. , cell lysates) that can be used as sources of relevant enzymes, in the methods provided by the document, all the steps can be performed in microorganism cells, all the steps can be performed using extracted enzymes, or some of the steps can be performed in ceils and others can be performed using extracted enzymes,

Esizymes

Esizym s generating pimeloyl-CoA

As depicted in FIG. 1 and FIG. 2, 9-hydroxyperoxyoctadec- 10,12-dienoate can be enzymaticaily synthesized from the central metabolite octadecanoyl-CoA using one or more exogenous polypeptides having the enzymatic activity of: a delta9-desaturase, a deltal 2-desaturase, a thioester se, and/or a 9-lipoxygenase.

In some embodiments, a polypeptide having the activity of a delta9-desalurase may be classified under EC 1.14.19.3 , such as, for example, the gene product of Le-FADl from Lentinula edodes (UniProtKB Accession No. Q76C19), the gene product of SCD1 from Mesocricelus auratus (UniProtKB Accession No. A7LCI9), an acyl-CoA-delta9-3a- desaturase from Dendrolim s punctatus (UniProtKB Accession No. B7SB75), the gene product of scdl from Rattus norvegicus (UniProtKB Accession No, P07308), the gene product of PF3D7 0511200 from Plasmodium falciparum (UniProtKB Accession No. Q8I0W9), or the gene product of desBl from Bombus lucoriim (UniProtKB Accession No. A5CK.E1).

A polypeptide having the activity of a deltal 2-desaturase may be classified under EC 1 .14.19.6, such as, for example, the gene product of D12Des from Acheta domesticus (UniProtKB Accession No. B7SB91), the gene product of FAD2 from Gossypium hirsutum (UniProtKB Accession No. Q8W2B9), the gene product of CFad6 from Chlorella vulgaris (UniProtKB Accession No, D3U658), a deltal 2 fatty acid desaturase from Triadica sebifera (UniProtKB Accession No. A5J295), the gene product of Pc-fad2 from Phanerochaete chrysosporium (UniProtKB Accession No. D4Q8H2), the gene product of Cs~fad2 from Ceriporiopsis subvermispora (UniProtKB Accession No. D4Q8S6), or the gene product of AN1037.2 from Emericella nidulans (UniProtKB Accession No. Q5BEJ3).

A polypeptide having the activity of a thioesterase may be classified under EC 3.1.2.-, such as, for example, the gene product of BT_2075 from Bacteroides thetaioiaomicron (strain ATCC 29148 / DSM 2079 / NCTC 10582 / E50 / VPj-5482) (GenBank Accession No. AA077182.1 , SEQ ID NO: 20), the gene product of lp 0708 from Lactobacillus plantarum (strain ATCC BAA-793 / NCIMB 8826 / WCFS1) (GenBank Accession No. CCC78182.1 , SEQ ID NO: 22), the gene product of HMPREF0077J 17 from Anaerococcus tetradius ATCC 35098 (GenBank Accession No. EEI82564.1, SEQ ID NO: 23), or the gene product of CPF 954 from Clostridium perfringens (strain ATCC 13124 / DSM 756 / JCM 1290 / NCIMB 6125 / NCTC 8237 / Type A) (GenBank Accession No. ABG82470J , SEQ ID NO: 18).

A polypeptide having the activity of a 9-lipoxygenase may be classified, for example, under EC 1.13.1 1.58, EC 1.1 3.1 1 .60, EC 1 .13.1 1.61 , or EC 1.13.1 1 .62, such as, for example, an allene oxide synthase-lipoxygenase protein from Plexaura homomalla (UniProtKB Accession No, 016025), a Psi-producing oxygenase A from Emericella nid lans (UniProtKB Accession No. Q6RET3), a 5,8-linoleate diol synthase from Aspergillus fumigatus (UniProtKB Accession No. C1KH66), or a linoleate diol synthase from Gaeumannomyces graminis (UniProtKB Accession No. Q9UUS2).

As further depicted in FIG. I and FIG. 2, 9-hydroxyperoxyoctadec- 10, 12-dienoate may be enzymatically cleaved into non-3-enal and 9-oxononanoate using a polypeptide having the activity of a hydroperoxide lyase. In some embodiments, a polypeptide having the activity of a hydroperoxide lyase may be classified under EC 4.2.99.-, such as, for example, the gene product of Cucumis sativus (GenBank Accession No. AAF64041.1, SEQ ID NO: 13) or the gene product of Oryza sativa (GenBank Accession No. BAG97978.1 , SEQ ID NO: 14).

As shown in FIG. 1 and FIG. 2, non-3 -enal and 9-oxononanoate may be converted by separate enzymatic pathways to azelaic acid. Azelaic acid, regardless of its origination from non-3-enal or 9-oxononanoate, may then be converted to pimeloyl-CoA. The enzymes involved in the conversion of non-3 -enal to azelaic acid, 9-oxononanoate to azelaic acid and azelaic acid to pimeloyl-CoA are as described in the following paragraphs.

As shown in FIG. 1 , non-3 -enal may be converted to azelaic acid by one or more exogenous polypeptides having the enzymatic activities of: (i) an aldehyde dehydrogenase, (ii) a Co A ligase, (iii) a dodecenoyl-CoA isomerase, (iv) a trans-2-enoyl- CoA reductase, (v) a thioeslerase, (vi) a monooxygenase, (vii) an alcohol dehydrogenase, and/or (viii) a succinate-semialdehyde dehydrogenase, a 5-oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, and/or a 7-oxoheptanoate dehydrogenase. As shown in FIG. 2, non-3-enal may be converted to azelaic acid by one or more exogenous polypeptides having the enzymatic activities of: (i) an enal isomer ase, (ii) an enoate reductase, (iii) an aldehyde dehydrogenase, (iv) a monooxygenase , (v) an alcohol dehydrogenase, and/or (vi) a succinate-semialdehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, and/or a 5-oxopentanoate dehydrogenase.

In some embodiments, a polypeptide having the activity of an aldehyde dehydrogenase may be classified under EC 1.2.1.-, such as EC 1.2.1.3, EC 1.2.1.4, EC 1.2.1 .5, or EC 1.2.1.48, such as, for example, the gene product of Bt~aldh from Geobacillus thermoleovorans B23 (UniProtKB Accession No. Q9FAB1), the gene product of dhaS from Bacillus subtilis (UniProtKB Accession No. 034660), the gene product of ALD5 from Saccharomyces cerevisiae (UniProtKB Accession No. A6ZR27), the gene product of ALDH2C4 from Arabidopsis thaliana (UniProtKB Accession No. Q56YU0), the gene product of aldhl from Rhodococcus ruber (UniProtKB Accession No. Q840S9), the gene product of alkH from Pseudomonas oleovorans (UniProtKB Accession No. PI 2693), the gene product of aldl from Acinetobacter sp. M-l (UniProtKB Accession No. Q9FDS1), or the gene product of acoD from Ralstonia eutropha (UniProtKB Accession No. P46368).

In some embodiments, a polypeptide having the activity of an enoate reductase may be classified, for example, under EC 1.3.1 .31 , such as, for example, the gene product of xenA from Pseudomonas putida (GenBank Accession No. AAN66878.1, SEQ ID NO: 16) or the gene product of LOCK919 2632 from Lactobacillus cased (GenBank Accession No. AGP69310.1 , SEQ ID NO: 1 5).

In some embodiments, a polypeptide having the activity of a Co A ligase may be classified under EC 6.2.1.-, such as, for example, the gene product of acs6 from Brassica nanus (UniProtKB Accession No. Q9FNT6), the gene product of PCS60 from Saccharomyces cerevisiae (UniProtKB Accession No. P38137), the gene product oialkK from Pseudomonas oleovorans (UniProtKB Accession No. Q00594), the gene product of ACSM5 from Homo sapiens (UniProtKB Accession No. Q6NUN0), or the gene product of alkK ixom Aeropyrum perriix (UniProtKB Accession No. Q9YF45). In some embodiments, a polypeptide having the activity of a dodecenoyl-CoA isomerase may be classified under EC 5.3.3.8, such as, for example, the gene product of ECU from Saccharomyces cerevisiae (GenBank Accession No. AAC83700.1, SEQ ID NO: 17 and SEQ ID NO: 19, Geisbrecht ··· al J. Biol. ( ^" hem. 1 998 273 (50) 331 84- 33191 ).

In some embodiments, a polypeptide having the activity of a trans-2-enoyl-CoA reductase may be classified under EC 1.3.1 .38 or EC 1.3.1.44, such as, for example, the gene product of ter from Escherichia coli, Fibrobacter succinogenes, or Treponema denticola (Nishimaki ei al, J. Biochem., 1984, 95: 1315 - 1321 ; Shen et ai, 201 1 , supra) or idler from Treponema denticola (Bond-Watts et al, Biochemistry, 2012, 51 :6827 - 6837) or EC 1.3.1.8 (Inui et al , Eur. J. Biochem, , 1984, 142, 121 - 126).

In some embodiments, a polypeptide having the activity of a thioesterase may be classified under EC 3.1.2.-, such as, for example, the gene product of BT 2075 from Bacteroides thetaiotaomicron (strain ATCC 29148 / DSM 2079 / NCTC 10582 / E50 / VPI-5482) (GenBank Accession No. AA077182.1 , SEQ ID NO: 20), the gene product of Ip 0708 from Lactobacillus plantarum (strain ATCC BAA-793 / NCIMB 8826 / WCFS1) (GenBank Accession No. CCC78182.1, SEQ ID NO: 22), the gene product of HMPREFOO 77 J 317 from Anaerococcus tetradvus ATCC 35098 (GenBank Accession No..EEI82564.1, SEQ ID NO: 23), or the gene product of CPFJ954 from Clostridium perfringens (strain ATCC 13124 / DSM 756 / JCM 1290 / NCIMB 6125 / NCTC 8237 / Type A) (GenBank Accession No. ABG82470.1, SEQ ID NO: 18).

In some embodiments, a polypeptide having the activity of a monooxygenase may be classified in the cytochrome P450 family under EC 1.14.14.- or EC 1.14.15.-, such as EC 1.14.14.1, EC 1.14.14.3, EC 1 .14.15. i , or EC 1.14.15.3 or as the gene products of alkBGT from Pseudomonas putida, CYP153A from Polaromorias sp., or CVP52A3 from Saccharomyces cerevisiae.

In some embodiments, a polypeptide having the activity of an alcohol dehydrogenase may be classified under EC 1.1.1.-, such as a 6-hydroxyhexanoate dehydrogenase classified, for example, under EC 1.1.1.258, such as, for example, the gene product of chnD from Acineiobacter sp. NCIMB9871 (Donoghue ei al, Eur. J. Biochern, 1975, 60: 1 -7); or a 4-hydroxybutano ie dehydrogenase classified, for example, under EC 1.1.1.61 such as, for example, the gene product of gbd (e.g. , from Sorangium cellulosum) or gabD from, for example, Escherichia coli (Bartsch el al , J. Bacterial , 1990, 172(12), 7035). In some embodiments, a polypeptide having the activity of an aldehyde dehydrogenase may be classified under, for example, EC 1.2.1.-, such as a 7-oxoheptanoate dehydrogenase (e.g. , the gene product of thnG from Sphingomorias tnacrogolitabida), a 6-oxohexanoate dehydrogenase (e.g., the gene product of chnE from Acinetobacter sp.) classified, for example, under EC 1.2.1.63, a 5-oxopentanoate dehydrogenase classified, for example, under EC 1.2.1.20 (e.g. , the gene product of cpnE Comamonas sp.), a succinate-sernialdehyde dehydrogenase classified, for example, under EC 1.2.1.16, EC 1.2.1.24, or EC 1.2.1.79 (e.g. , the gene product of ALDH5F1 from Arabidopsis thaliana (UniProtKB Accession No. Q9SAK4), the gene product of araE from AzospirUlum hrasHense (UniProtKB Accession No. Q1JUP4), the gene product of Ssadh from Drosophila melanogaster (UniProtKB Accession No. Q9VBP6), the gene product of ALDH5A1 from. Gorilla gorilla (UniProtKB Accession No. Q6A2H1), the gene product of ALDH5A1 from Hylohates tar (UniProtKB Accession No. Q3MSM3), the gene product of ssadh from Lucilia cuprina (UniProtKB Accession No. B0JFD4), the gene product of ALDH5A 1 from Pan paniscus (UniProtKB Accession No. Q3MSM4), the gene product of ALDH5A 1 from Pan iroglodyies (UniProtKB Accession No. Q6A2H0), the gene product of ALDH5 A 1 from Pongo abelii (UniProtKB Accession No. Q6A2H2), the gene product of ALDH5A1 from Pongo pygmaeus (UniProtKB Accession No. Q6A2H2), or the gene product of gapN-1 from Sulfolohus solfataricus (UniProtKB Accession No. Q97XS9)), or an aldehyde dehydrogenase classified under EC 1.2.1.3.

As shown in FIG. 1 and FIG. 2, 9-oxononanoate may be converted to azelaic acid by one or more exogenous polypeptides having the following enzymatic activities; for example, a 7-oxoheptanoate dehydrogenase (e.g. , the gene product of thnG from Sphingornonas tnacrogolitabida) classified under EC 1.2.1.-, a 6-oxohextanoate dehydrogenase (e.g. , the gene product of chnE from Acinetobacter sp.) classified, for example, under EC 1.2.1.63, a 5-oxopentanoate dehydrogenase classified, for example, under EC 1.2.1.20 (the gene product of cpnE from Comamonas sp.), a succinate- semialdehyde dehydrogenase classified, for example, under EC 1.2.1 .16, EC 1.2.1.24, or EC 1.2.1.79, and/or an aldehyde dehydrogenase classified under EC 1.2.1.3.

As shown in FIG. 1 and FIG. 2, azelaic acid may be converted to pimeloyl-CoA by one or more exogenous polypeptides having the enzymatic activities of: a CoA iigase, an acyl-CoA dehydrogenase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, a 3-oxoacyl-ACP reductase, and/or a β-ketothiolase. in some embodiments, a polypeptide having the activity of a CoA iigase may be classified under, for example, EC 6.2.1.-.

In some embodiments, a polypeptide having the activity of an acyl-CoA dehydrogenase may be classified under, for example, EC 1.3.8,-, such as EC 1.3.8.6, EC 1.3.8.7, or EC 1.3.8.8.

in some embodiments, a polypeptide having the activity of an enoyl-CoA hydratase may be classified under, for example, EC 4.2.1.17, such as. for example, the gene product of crt from Clostridium acetobutylicum, or classified under EC 4.2, 1 .1 19, such as, for example, the gene product of phaJ from Pseudomonas aeruginosa. In some embodiments, a polypeptide having the activity of a 3-hydroxyacyl-CoA dehydrogenase may be classified for example, under EC 1 , 1 .1.- , such as EC 1.1.3.35 (e.g. , the gene product oifadB from Escherichia coli), EC 1.1.1.36 (e.g. , the gene product of phaB from Cupriavidus necator), or EC 1.1.1.157 (e.g. , the gene product of hbd from Clostridium acetobutylicum), and a polypeptide having the activity of a 3-oxoacyl-ACP reductase may be classified, for example, under EC 1.1 .1.100, such as. for example, the gene product offabG from Escherichia coli.

In some embodiments, a polypeptide having the activity of a β-ketothiolase may ^¬ be classified, for example, under EC 2.3.1.16 or EC 2.3, 1.174 such as, for example, the gene product of bktB from Cupriavidus necator or paaJ kom Escherichia coli.

Enzymes generating the terminal carboxyl groups in the biosynthesis of pimelk acid As depicted in FIG. 3, pimcioyi-CoA can be enzymatically converted to pimelie acid. The terminal carboxyl group leading to the production of pimeiic acid can be enzymatically formed using polypeptides having the activity of a thioesierase, a CoA ligase, a CoA transferase, an aldehyde dehydrogenase, a succinate-semialdehyde dehydrogenase, a 5-oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, and/or a 7-oxoheptanoate dehydrogenase.

In some embodiments, the second terminal carboxyl group leading to the synthesis of pimeiic acid can be enzymaticaliy formed in pimeloyl-CoA by a polypeptide having the activity of a thioesterase classified under EC 3, 1 ,2.-. The polypeptide having the activity of a thioesterase can be, for example, the gene product of yciA from Escherichia coli or acotlS from Mus musculus (Cantu et a!.. Protein Science. 2010, 19, 1281 - 1295; Zhuang el a!. , Biochemistry, 2008, 47(9):2789 - 2796; Naggert et al , J. Biol Chem. , 1991, 266(17): 1 1044 - 1 1050), or tesB from Escherichia coli, or the gene product of BT 2075 from Bacleroides thetaiotaomicron (strain ATCC 29148 / DSM 2079 / NCTC 10582 / E50 / VPI-5482) (GenBank Accession No. AA077182.1 , SEQ ID NO: 20), the gene product of Ip 0708 from Lactobacillus plantar um (strain ATCC BAA- 793 / NCIMB 8826 / WCFS l) (GenBank Accession No. CCC78182.1 , SEQ ID NO: 22), the gene product of HMPREF0077J317 from Anaerococcus tetradius ATCC 35098 (GenBank Accession No. EEI82564.1, SEQ ID NO: 23), or the gene product of CPFJ954 from Clostridium perfringens (strain ATCC 13124 / DSM 756 / JCM 1290 / NCIMB 6125 / NCTC 8237 / Type A) (GenBank Accession No. ABG82470. I , SEQ ID NO: 18)).

In some embodiments, the second terminal carboxyl group leading to the synthesis of pimeiic acid can be enzymaticaliy formed in pimeloyi-CoA by a polypeptide having the activity of a CoA ligase classified under EC 6.2.1 .-, such as EC 6.2.1.5 or EC 6.2.1.15. or a polypeptide having the activity of a CoA transferase classified under EC 2.8.3.-, such as EC 2,8.3.8 or EC 2.8.3.12 {e.g. , a succinyl-CoA: acetate CoA-transferase from Acetobacter aceti (UniProtKB Accession No. B3 EY95), the gene product of ANACAC_ 01149 from Anaerostipes caccae (UniProtKB Accession No. B0MC58), a hutyryl-CoA: acetate CoA-transferase from Butyrivibrio fibrisolvens (UniProtKB Accession No. D2WEY7), a butyryl-CoA : acetate CoA-transferase from Eubacterium hallii (UniProtKB Accession No. D2WEY8), the gene product of FAEPRAA2165 01575 from Faecalibacterium prausnitzii (UniProtKB Accession No. C7H5K4), a hutyryl- CoA: acetate CoA-transferase from Faecalibacterium prausnitzii (UniProtKB Accession No. D2WEZ2), the gene product of FAEPRAM212 J) 2812 from Faecalibacterium prausnitzii (UniProtKB Accession No. A8SFP6), a butyryl-CoA transferase from Roseburia hominis (Uni ProtKB Accession No. Q2TME9), or a butyryl-CoA : acetate CoA- transferase from Roseburia inulinivorans (UniProtKB Accession No. D2WEY6)).

in some embodiments, pimeloyl-CoA can be enzymatically converted to pimelate semialdehyde by a polypeptide having the activity of an aldehyde dehydrogenase classified under, for example, EC 1.2.1.10, such as an acetaldehyde dehydrogenase encoded by pduB from Salmonella typhimurium. The second terminal carboxyi group leading to the synthesis of pime!ic acid can be enzymatically formed in pimelate semialdehyde by a polypeptide having the activity of an aldehyde dehydrogenase classified under EC 1.2.1.3 (Guerrillot & Vandecasteele, Eur. J. Biochem. , 1977, 81 , 185 - 192); a 7-oxoheptanoate dehydrogenase (e.g. , the gene product of thnG from Sphingomonas macrogolitabida; Lopez-Sanchez et ai , Appl. Environ. Microbiol , 2010, 76( 1 ), 1 10 - 1 18) classified under EC 1 ,2.1.-; a 6-oxohextanoate dehydrogenase (e.g. , the gene product of chnE from Acinetobacter sp. ) classified, for example, under EC 1.2.1.63; a 5-oxopentanoate dehydrogenase classified, for example, under EC 1.2.1.20 (e.g. , the gene product of cpnE from Comamonas sp.) or a succinate-semi ^' aldehyde dehydrogenase classified, for example, under EC 1.2.1 .16, EC 1.2.1.24, or EC 1.2.1.79 (e.g. , the gene product of ALDH5F1 from Arabidopsis thaiiana (UniProtKB Accession No. Q9SAK4), the gene product of araE from AzospiriUum brasUense (UniProtKB Accession No. Q1JUP4), t e gene product of Ssadh from Drosophila melanogaster (UniProtKB Accession No. Q9VBP6), the gene product of ALDH5A I from Gorilla gorilla (UniProtKB Accession No. Q6A2H1), the gene product of ALDH5A1 from Hylobates lar (UniProtKB Accession No. Q3MSM3), the gene product of ssadh from Lucilia cuprina (UniProtKB Accession No. B0JFD4), the gene product of ALDH5A1 from Pan paniscus (UniProtKB Accession No. Q3MSM4), the gene product of ALDH5A1 from Pan troglodytes (UniProtKB Accession No. Q6A2H0), the gene product of ALDH5A I from Pongo abelii (UniProtKB Accession No. Q6A2H2), the gene product of ALDH5A1 from Pongo pygmaeus (UniProt JB Accession No. Q6A2H2), or the gene product of gapN-1 from Sulfolobus solfataricus (UniProtKB Accession No. Q97XS9)).

Enzymes generating 7-aminoheptanoate

As depicted in FIG. 4, pimeloyi-CoA is converted to pimelate semiaidehyde by a polypeptide having the enzymatic activity of an aldehyde dehydrogenase classified under, for example, EC 1.2.1 .10, such as an acetaldehyde dehydrogenase encoded by pdiiB from Salmonella typhimurium or pduP from Klebsiella pneumoniae. A terminal amine group may then be enzymaticaily formed or removed using one or more polypeptides having the activity of a ω-transaminase classified under, for example, EC 2.6.1.-, e.g., EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82, such as, for example, that obtained from Chromobacterium violaceum (GenBank Accession No, AAQ59697.1, SEQ ID NO: 7), Pseudomonas aeruginosa (GenBank Accession No. AAG08191.1 , SEQ ID NO: 8), Pseudomonas syringae (GenBank Accession No. AAY39893.1 , SEQ ID NO: 9), Rhodobacter sphaeroides (GenBank Accession No. ABA81 135.1 , SEQ ID NO: 10), Escherichia coli (GenBank Accession No. AAA57874. L SEQ ID NO: 1 1, SEQ ID NO: 48), Vibrio fluvialis (GenBank Accession No. AEA39183.1, SEQ ID NO: 12), Streptomyces griseus, or Clostridium viride. See FIG. 8. The reversible o)-transaminase from Chromobacterium violaceum (GenBank Accession No. AAQ59697.1 , SEQ ID NO: 7) has demonstrated analogous activity accepting 7- aminoheptanoic acid as amino donor, thus forming the first terminal amine group in pimelate semiaidehyde (Kaulniann el al . Enzyme and Microbial Technology, 2007, 41 , 628 - 637).

Alternatively, pimelate (pimelic acid) as shown in FIG. 3, may be enzymaticaily converted to pimelate semiaidehyde by a polypeptide having the activity of a carboxylase reductase classified, for example, under EC 1.2.99.6 such as polypeptides represented by the following GenBank Accession Nos: EFVl 1917.1 (SEQ ID NO: 3) and ADG98140.1 (SEQ ID NO: 6). A terminal amine group may then be enzymaticaily formed or removed from pimelate semiaidehyde using one or more polypeptides having the activity of a co- transaminase classified under, for example, EC 2.6.1.-, e.g. , EC 2.6.1.18, EC 2.6.1 .19, EC 2.6, 1.29, EC 2.6.1.48, or EC 2.6.1.82, such as, for example, thai obtained from Chromobacterium violaceum (GenBank Accession No. AAQ59697.1, SEQ ID NO: 7), Pseudomonas aeruginosa (GenBank Accession No. AAG08191.1 , SEQ ID NO: 8), Pseudomonas syringae (GenBank Accession No. AAY39893.1 , SEQ ID NO: 9), Rhodobacter sphaeroides (GenBank Accession No. ABA81 135.1, SEQ ID NO: 10), Vibrio fluvialis (GenBank Accession No. AEA39183.1 , SEQ ID NO: 12), Streptomyces griseus, or Clostridium viride. See FIG. 8. The reversible ω-transaminase from Chromobacterium violaceum (GenBank Accession No. AAQ59697.1, SEQ ID NO: 7) has demonstrated analogous activity accepting 7-aminoheptanoic acid as amino donor, thus forming the first terminal amine group in pimelate semialdehyde (Kaulmann et al.. Enzyme and Microbial Technology, 2007, 41 , 628 - 637).

An additional ω-transaminase that can be used in the methods and microorganisms described herein is from Escherichia coli (GenBank Accession No. AAA57874.1, SEQ ID NO: 1 1 , SEQ ID NO: 48). Some of the ω-tramaminases classified, for example, under EC 2.6.1.29 or EC 2.6.1.82 are diamine ω-transaminases (e.g. , SEQ ID NO: 1 1 , SEQ ID NO: 48).

The reversible co-iransaminase from Chromobacterium violaceum (GenBank Accession No. AAQ59697.1, SEQ ID NO: 7) has demonstrated analogous activity accepting 7-aminoheptanoic acid as amino donor, thus forming the first terminal amine group in pimelate semialdehyde (Kaulmann et al . Enzyme and. Microbial Technology, 2007, 41 , 628 - 637).

Enzymes generating the terminal amine groups in the biosynthesis of Heptamethylenediamine

As depicted in FIG. 5, terminal amine groups can be enzymatically formed or removed using polypeptides having the activity of a ω-transaminase or a deacylase.

In some embodiments, a terminal amine group leading to the synthesis of 7- aminoheptanoic acid is enzymatically formed in 7-aminoheptanal by a polypeptide having the activity of a co-iransaminase classified, for example, under EC 2.6.1.-, e.g. , EC 2.6.1.1.8, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82, such as that obtained, for example, from Chromobacterium violace m (GenBank Accession No. AAQ59697.1 , SEQ ID NO: 7), Pseudomonas aeruginosa (GenBank Accession No. AAG08191.1, SEQ ID NO: 8), Pseudomonas syringae (GenBank Accession No. AAY39893.1, SEQ ID NO: 9), Rhodobacter sphaeroides (GenBank Accession No. ABA81135.1, SEQ ID NO: 10), Vibrio fluvialis (GenBank Accession No. AEA39183.1, SEQ ID NO: 12), Streptomyces griseus, or Clostridium viride. See FIG. 8.

An additional polypeptide having the activity of a ω-tramaminase that can be used in the methods and microorganisms described herein is from Escherichia coli (GenBank Accession No. AAA57874.1 , SEQ ID NO: 1 1 , SEQ ID NO: 48). Some of the polypeptides having the activity of ω-transaminases classified, for example, under EC 2.6.1.29 or EC 2.6.1.82 are diamine co-transaminases (e.g. , SEQ ID NO: 1 1 , SEQ ID NO: 48).

The reversible ω-transaminase from Chromobacterium violaceum (GenBank Accession No. AAQ59697.1, SEQ ID NO: 7) has demonstrated analogous activity accepting 7-aminoheptanoic acid as amino donor, thus forming the first terminal amine group in pimelate semialdehyde (Kaulmann et αί , Enzyme and Microbial ^' Technology, 2007, 41 , 628 - 637).

The reversible 4 ^' -amino buby ate : 2-oxoadipate transaminase from Streptomyces griseiis has demonstrated activity for the conversion of 7-aminoheptanoate to pimelate semialdehyde (Yonaha et a!. , Eur. J. Biochem. , 1985, 146, 101 - 106).

The reversible 5-aminovalerate transaminase from Clostridium viride has demonstrated activity for the conversion of 7-aminoheptanoate to pimelate semialdehyde (Barker et al , J. Biol. Chem. , 1987, 262(19), 8994 - 9003).

In some embodiments, the second terminal amine group leading to the synthesis of heptamethylenediamine is enzymatically formed in 7-aminoheptanal by a polypeptide having the activity of a diamine transaminase classified, for example, under EC 2.6, 1.29 or classified, for example, under EC 2.6.1.82, such as, for example, the gene product of yg G from E. coli (GenBank Accession No. AAA57874.1 , SEQ ID NO: 1 1 , SEQ ID NO: 48). The polypeptides having the activity of a transaminase set forth in SEQ ID NOs: 7 - 10 and 12 also can be used to produce heptamethylenediamine. See FIG. 8. The gene product oiygjG from Escherichia coli accepts a broad range of diamine carbon chain length substrates, such as putrescine, cadaverine and spermidine (Samsonova et al. , BMC Microbiology, 2003, 3:2),

The diamine transaminase from E.coli strain B has demonstrated activity for 1 ,7 diamine heptane (Kim, The Journal of Chemistry, 1964, 239(3), 783 - 786).

In some embodiments, the second terminal amine group leading to the synthesis of heptamethylenediamine is enzymatically formed in N7-acetyI-l ,7-diaminoheptane by a polypeptide having the activity of a deacylase classified, for example, under EC 3.5.1.-, such as, for example, EC 3.5.1.62 or EC 3.5.1.82.

Enzymes generating the terminal hydroxy! groups in the biosynthesis of 7- hydroxyheptanoate

As depicted in FIG. 6, pimeiic acid, as shown in FIG. 3, may be converted to pimelate semialdehyde by a polypeptide having the activity of a carboxylate reductase classified, for example, under EC 1 ,2.99.6, such as, for example, the polypeptides represented by the following GenBank Accession Numbers; EFV1 1917.1 (SEQ ID NO: 3) or ADG98140.1 (SEQ ID NO: 6). Having formed pimelate semialdehyde, a terminal hydroxy! group can then be enzymatically formed (or removed) using one or more polypeptides having the activity of an alcohol dehydrogenase classified, for example, under EC 1.1.1 ,-, such as, for example, a 5-hydroxypentanoate dehydrogenase, such as, for example, the gene product of cpnD from Comamonas sp (Iwaki et al. , Appl, Environ. Microbiol. , 1999, 65(1 1):5158 - 5162), or a 4-hydroxyhutanoate dehydrogenase, such as, for example, the gene product of gabD from Escherichia coli (Bartsch et al , J, Bacteriol., 1990, 172(12), 7035). The polypeptide having the activity of an alcohol dehydrogenase may also be classified under EC 1.1.1.258, such as a 6-hydroxyhexanoate dehydrogenase , such as, for example, the gene product of chnD from Acineiobacter sp. NC1MB9871 (Donoghue et al, Eur, J. Biochem, 1975, 60: 1-7).

Alternatively, as shown in FIG. 6, pimeloyl-CoA may be converted to pimelate semialdehyde by an polypeptide having the activity of an alcohol dehydrogenase classified, for example, under EC 1.2.1 .10, such as a polypeptide having the activity of an acetaldehyde dehydrogenase encoded by pduB from Salmonella iyphimurium or pduP from Klebsiella pneumoniae. Having formed pimelate semialdehyde, a terminal hydroxyl group can then be enzymatically formed (or removed) using one or more polypeptides having the activity of an alcohol dehydrogenase classified, for example, under EC 1.1.1.- such as a 5-hydroxypenlanoate dehydrogenase, such as, for example, the gene product of cpnD from Comamonas sp. (Iwaki et al , Appl. Environ. Microbiol , 1999, 65(1 1 ) : 5158 - 5162), or a 4-hydroxybutanoate dehydrogenase, such as, for example, the gene product of gabD from Escherichia coll (Bartsch et al , J. Bacteriol., 1990, 172(12), 7035). The polypeptide having the activity of an alcohol dehydrogenase may also be classified under EC 1 .1.1.258, such as a 6-hydroxyhexanoale dehydrogenase, such as, for example, the gene product of chnD from Acineiobacter sp. NCIMB9871 (Donoghue et al, Eur. J. Biochem, 1975, 60: 1 -7).

Enzymes generating the terminal hydroxy! groups in the biosynthesis of 1,7 heptanediol

As depicted in FIG. 7, the terminal hydroxy! group can be enzymatically formed using a polypeptide having the activity of an alcohol, dehydrogenase. For example, the second terminal hydroxyl group leading to the synthesis of 1 ,7 heptanediol can be enzymatically formed in 7-hydroxyheptana! by a polypeptide having the activity of an alcohol dehydrogenase classified under EC 1.1.1.- {e.g., EC 1.1.1.1, EC 1 .1.1.2, EC 1.1.1.21 , or EC 1.1.1 .184), such as, for example, the gene product of YMR318C from Saccharomyces cerevisiae oxyqhD from Escherichia coli (Liu et al , Microbiology, 2009, 155, 2078 - 2085; Larroy et al , 2002, Biochem I , 361(Pt 1 ), 163 - 172; Jarboe, 201 1 , Appl. Microbiol. Biotechnol , 89(2), 249 - 257) or the polypeptide represented by GenBank Accession No. CAA81612.1 (SEQ ID NO; 21). The alcohol dehydrogenase encoded by YMR318C has broad substrate specificity, including the oxidation of C7 alcohols. The polypeptide having the activity of an alcohol dehydrogenase classified under EC 1.1 .1.- may also be a. polypeptide represented by GenBank Accession CAA81612.1 (SEQ ID NO: 21). Biochemical pathways

Pathways to pimeloyl-CoA

In some embodiments, and as shown in FIG. 1 and FIG. 2, the central metabolite octadecanoyl-CoA is converted to octadec-9-enoyl-CoA using a poiypeptide having the enzymatic activity of a delta9-desaturase classified under, for example, EC 1.14.19.1 ; followed by conversion of octadec-9-enoyi-CoA to octadec-9,12-dienoyl-CoA using a polypeptide having the enzymatic activity of a deltal 2-desaturase classified under, for example, EC 1.14.19.6: followed by conversion of octadec-9,12-dienoyl-CoA to linoleic acid using a polypeptide having the enzymatic activity of a thioesterase classified under, for example, EC 3.1.2-; followed by conversion of linoleic acid using a polypeptide having the enzymatic activity of a 9-lipoxygenase classified under, for example, EC 1.13.1 1 .58, EC 1.13.1 1.60, EC 1.13.1 1.61 , or EC 1.13.1 1 .62.

In some embodiments, and as shown in FIG. 1 and FIG. 2, 9~ hydroxyperoxyoctadec- 10,12-dienoate is cleaved by a polypeptide having the enzymatic activity of a hydroperoxide lyase classified, for example, under EC 4.2.99.-, such as, for example, a polypeptide represented by GenBank Accession No. AAF64041.1 (SEQ ID NO: 13) or a polypeptide represented by GenBank Accession No. BAG97978.1 (SEQ ID NO: 14), to produce non-3-enal and 9-oxononanoate.

In some embodiments, and as shown in FIG. 1 , non-3 -enal is converted to non-3 - enoate by one or more polypeptides having the enzymatic activity of an aldehyde dehydrogenase classified, for example, under EC 1.2.1 .-. such as EC 1.2.1 .3, EC 1.2.1 .4, EC 1 .2.1.5, or EC 1.2.1.48, such as, for example, the gene product of Bt-aldh from Geobacillus ther mole ov or arts B23 (UniProtKB Accession No. Q9FAB1 ), the gene product of dhaS from Bacillus suhtilis (UniProtKB Accession No. 034660), the gene product of ALD5 from Saccharomyces cerevisiae (Uni ProtK B Accession No, A6ZR27), the gene product of ALDH2C4 from Arabidopsis thaliana (UniProtKB Accession No. Q56YU0), the gene product of aldhl from Rhodococcus ruber (UniProtKB Accession No. Q840S9), the gene product of alkH from Pseudomonas oleovorans (UniProtKB Accession No. P I 2693), the gene product of aldl from Acinetobacter sp. M- l (UniProtKB Accession No. Q9FDS 1), or the gene product of acoD from Ralstonia euiropha (UniProtKB Accession No. P46368); followed by conversion of non-3-enoate to non-3 -enoyl-Co A by a polypeptide having the enzymatic activity of a Co A ligase classified, for example, under EC 6.2, 1.-, such as, for example, the gene product of acs6 from Brassica napus (Uni ProtKB Accession No. Q9FNT6), the gene product of PCS60 from Saccharomyces cerevisiae (UniProtKB Accession No. P38137), the gene product of alkK from Pseudomonas okovorans (UniProtKB Accession No. Q00594), the gene product of ACSM5 from Homo sapiens (UniProtKB Accession No. Q6NUN0), or the gene product of alkK from Aeropyrum pernix (UniProtKB Accession No. Q9YF45); followed by conversion of non-3-enoyi-CoA to non-2-enoyl-CoA by a polypeptide having the enzymatic activity of a dodecenoyl-CoA isornerase classified, for example, under EC 5.3.3.8; followed by conversion of non-2-enoyi-CoA to nonanoyi-CoA by a irarts-2-enoyl-CoA reductase classified, for example, under EC 1.3.1.38, EC 1.3, 1 .44 such as, for example, the gene product of ler from Escherichia colt, Fibrobacter succinogenes, or Treponema denticola (Nishimaki el al., J. Blochem. , 1984, 95: 13 15 - 1 321 ; Shen et al , 201 1 , supra) or tdter from Treponema denticola (Bond- Waits el al , Biochemistry, 2012, 51 :6827 - 6837) or EC 1.3.1.8 (Imii et al , Eur. J. Biochem., 1984, 142, 121 - 126); followed by conversion of nonanoyi-CoA to nonanoic acid using a polypeptide having the enzymatic activity of a thioesterase classified, for example, under EC 3.1.2.-, such as, for example, the gene product of BT 2075 from Bacteroides thetaiotaomicron (strain ATCC 29148 / DSM 2079 / NCTC 10582 / E50 / VPI-5482) (GenBank Accession No. AA077182.1 , SEQ ID NO: 20), the gene product of lp 07 8 from Lactobacillus plantarum (strain ATCC BAA-793 / NCIMB 8826 / WCFS 1) (GenBank Accession No. CCC78182.1, SEQ ID NO: 22), the gene product of HMPREF0077 1317 from Anaerococcus telradius ATCC 35098 (GenBank Accession No. EE182564.1 , SEQ ID NO: 23), or the gene product of CPF_2954 from Clostridium perfringens (strain ATCC 13 124 / DSM 756 / JCM 1290 / NCIMB 6125 / NCTC 8237 / Type A) (GenBank Accession No. ABG82470.1), SEQ ID NO: 18); followed by conversion of nonanoic acid to 9-hydroxynonanoic acid using a polypeptide having the enzymatic activity of a monooxygenase classified, for example, under EC 1.14, 14- or EC 1.14.15.-, such as EC 1 .14.14.1 , EC 1 .14.14.3, EC 1.14.1 5.1 or EC 1.14.15.3 and as encoded by alkBGT from Pseudomonas putida, CYP153A from Polaromonas sp., or CYP52A3 from Saccharomyces cerevisiae; followed by conversion of 9- hydroxynonanoic acid to 9-oxononanoate using a polypeptide having the enzymatic activity of an alcohol dehydrogenase classified under EC 1.1.1 .-, a 6-hydroxyhexanoaie dehydrogenase classified under EC 1.1.1.258, such as, for example, the gene product of chnD from Acinetohacter sp. NC1MB9871 (Donoghue et al, Eur. J. Biochem, 1975, 60: 1 -7); or a 4-hydroxybiiianoate dehydrogenase classified under EC 1.1.1.61, such as, for example, the gene product of gabD from Escherichia coli (Bartsch et al. , J. Bacterial. , 1990, 172(12), 7035); followed by conversion of 9-oxononanoate to azeiaic acid using a polypeptide having the enzymatic activity of an aldehyde dehydrogenase classified under, for example, EC 1.2.1.- such as a 7-oxoheptanoate dehydrogenase (e.g. , the gene product of IhnG from Sphingomonas macrogol itabida), a 6-oxohextanoate dehydrogenase {e.g. , the gene product of chnE from Acinetohacter sp.) classified, for example, under EC 1 .2.1.63, a 5-oxopentanoate dehydrogenase classified, for example, under EC 1.2.1.20 (e.g. , the gene product of cpnE from Comamonas sp.), a succinate- semialdehyde dehydrogenase classified, for example, under EC 1.2.1.16, EC 1.2.1 ,24, or EC 1.2.1.79, or an aldehyde dehydrogenase classified under EC 1.2.1.3.

in some embodiments, and as shown in FIG. 2, non-3-enal is converted to nonrenal by a polypeptide having the enzymatic activity of an enal isomerase classified, for example, under EC 5.3.3.-; followed by conversion of non-2-al to nonanal by a polypeptide having the enzymatic activity of an enoate reductase classified, for example, under EC 1.3.1.31; followed by conversion of nonanal to nonanoic acid by a polypeptide having the enzymatic activity of an aldehyde dehydrogenase classified, for example, under EC 1.2.1.-. such as EC 1.2.1.3, EC 1.2.1.4, EC 1.2.1.5, or EC 1.2.1.48, such as, for example, the gene product of Bt-aldh from GeobaciUus thermoleovorans B23 (UniProtKB Accession No. Q9FAB1), the gene product of dhaS from Bacillus subtilis (UniProtKB Accession No. 034660), the gene product of ALD5 from Saccharomyces cerevisiae (UniProtKB Accession No. A.6ZR27), the gene product of ALDH2C4 from Arabidopsis thaliana (UniProtKB Accession No. Q56YU0), the gene product of aldh? from Rhodococcus ruber (UniProtKB Accession No. Q840S9), the gene product of alkH from Pseudomonas oleovorans (UniProtKB Accession No. P12693), the gene product of aldl from Acinetobacter sp. M-l (UniProtKB Accession No. Q9FDS1 ), or the gene product of acoD from Ralsionia euiropha (UniProtKB Accession No. P46368); followed by conversion of nonanoic acid to 9-hydroxynonanoic acid by a monooxygenase classified, for example, under EC 1.14.14- or EC 1.14.15.-, such as EC 1.14.14.1, EC 1.14, 14.3, EC 1.14.15.1 , or EC i .14.15.3 or encoded by alkBGT from Pseudomonas putida, CYP153A from Polaromonas sp., or CYP52A3 from Saccharomyces cerevisiae; followed by conversion of 9-hydroxynonanoic acid to 9-oxotionanoic acid by a polypeptide having the enzymatic activity of an alcohol dehydrogenase classified under EC 1 , 1.1.-, a 6 -hydroxy hexanoate dehydrogenase classified under EC 1.1.1.258, such as, for example, the gene product of chnl) from Acinetobacter sp. NC1MB9871 (Donoghue et al., Eur. J, Biochem, 1975, 60: 1-7); or a 4-hydroxybutanoate dehydrogenase classified under EC 1.1.1.61 , such as, for example, the gene product of gab D from Escherichia coli (Bartsch et al , J. Bacteriol., 1990, 172( 32), 7035); followed by conversion of 9- oxononanoate to azelaic acid using a polypeptide having the enzymatic activity of an aldehyde dehydrogenase classified under, for example, EC 1 .2.1.-, such as a 7- oxohepianoate dehydrogenase (e.g. , the gene product of thriG from. Sphingomonas macrogolitabida), a 6-oxohextanoate dehydrogenase (e.g. , the gene product oi ^' chnE from Acinetobacter sp.) classified, for example, under EC 1.2.1 .63, a 5-oxopentanoate dehydrogenase classified, for example, under EC 1.2.1 .20 (e.g. , the gene product of cpnE from Comamonas sp.), a succinate-semialdehyde dehydrogenase classified, for example, under EC 1.2.1 .16, EC 1 .2.1.24, or EC 1.2.1.79 (e.g. , the gene product of ALDH5F1 from Arabidopsis thaliana (UniProtKB Accession No, Q9SAK4), the gene product of araE from Azospir ilium brasiiense (UniProtKB Accession No. Q1 JUP4), the gene product of Ssadh from Drosophila melanogaster (UniProtKB Accession No. Q9VBP6), the gene product of ALDH5A1 from Gorilla gorilla (UniProtKB Accession No. Q6A2H1), the gene product of ALDUS A 1 from Hylobates lar (UniProtKB Accession No. Q3MSM3), the gene product of ssadh from Lucilia cuprina (UniProtKB Accession No. BQJFD4), the gene product of A LDH5 A 1 from Pan paniscus (UniProtKB Accession No. Q3MSM4), the gene product of ALDUS Al from Pan troglodytes (UniProtKB Accession No. Q6A2H0), the gene product of ALDH5A1 from Pongo abelii (UniProtKB Accession No, Q6A2H2), the gene product of ALDHSAI from Pongo pygmaeus (Uni ProtKB Accession No. Q6A2H2), or the gene product of gapN-1 from Su!jbiobus solfataricus (UniProtKB Accession No. Q97XS9)), or an aldehyde dehydrogenase classified under EC 1 ,2.1.3.

In some embodiments, and as shown in FIG, 1 and 2, 9-oxononanoate is converted to azelaic acid using a polypeptide having the activity of an aldehyde dehydrogenase classified under, for example, EC 1.2.1 .-, such as a 7-oxoheptanoate dehydrogenas {e.g. , the gene product of thnG from Sphingomonas macrogolitabida ), a 6-oxohextanoate dehydrogenase {e.g., the gene product of chnE from Acinetobacter sp.) classified, for example, under EC 1 .2.1.63, a 5-oxopentanoate dehydrogenase classified, for example, under EC 1.2, 1 ,20 {e.g., the gene product of cpnE from Comamonas sp.), a succinate-semialdekyde dehydrogenase classified, for example, under EC 1.2, 1 .16, EC 1.2.1.24, or EC 1.2.1 .79 {e.g. , the gene product of ALDH5F1 from Arahidopsis thaliana (UniProtKB Accession No. Q9SAK4), the gene product of araE from Azospirilh n bra ilense (UniProtKB Accession No, Q1JUP4), the gene product of Ssadh from Drosophila melanogaster (UniProtKB Accession No, Q9VBP6), the gene product of ALDHSA I from Gorilla gorilla (UniProtKB Accession No. Q6A2H1), the gene product of ALDHSAI from Hylobates lar (UniProtKB Accession No. Q3MSM3), the gene product of ssadh from Lucilia c prina (UniProtKB Accession No. B0JFD4), the gene product of ALDHSAI from Pan paniscus (UniProtKB Accession No. Q3MSM4), the gene product of ALDHSAI from Pan troglodytes (UniProtKB Accession No. Q6A2H0), the gene product of ALDHSAI from Pongo abelii (UniProtKB Accession No. Q6A2H2), the gene product of ALDHSAI from Pongo pygmaeus (UniProtKB Accession No, Q6A2H2), or the gene product of gapN-1 from Sulfolobus solfataricus (UniProtKB Accession No. Q97X89)), or an aldehyde dehydrogenase classified under EC 1.2.1.3.

In some embodiments, and as shown in FIG. 3 and FIG. 2, azelaic acid is converted to azelaoyl-CoA by a polypeptide having the enzymatic activity of a CoA ligase classified, for example, under EC 6.2.1.-, such as, for example, the gene product of acs6 from Brassica napus (UniProtKB Accession No. Q9FNT6), the gene product of PCS6Q from Saccharomyces cerevisiae (UniProtKB Accession No. P38137), the gene product of alkK from Pseudomonas oleovorans (UniProtKB Accession No. Q00594), the gene product of ACSM5 from Homo sapiens (UniProtKB Accession No. Q6NUN0). or the gene product of alkK from Aeropyrum pernix (UniProtKB Accession No. Q9YF45); followed by conversion of azelaoyl-CoA to 2,3-dehydro-azelaoyl-CoA using a polypeptide having the enzymatic activity of an acyl-CoA dehydrogenase classified, for example, under EC 13.8.-, such as EC 1.3.8.6, EC 1 .3.8.7 or EC 1.3.8.8; followed by conversion of 2,3-dehydro-azelaoyl-CoA to 3-hydroxy-azelaoyl-CoA by a polypeptide having the enzymatic activity of an enoyl-CoA hydrata.se classified, for example, under EC 4.2.1.17, such as, for example, the gene product of crt from Clostridium acelobutylicum, or classified under EC 4.2.1.1 19, such as, for example, the gene product of phaJ from Pseudomonas aeruginosa; followed by conversion of 3-hydroxy-azelaoyl- CoA to 3-oxo-azelaoyl-CoA by a polypeptide having the enzymatic activity of a 3- hydroxyacyl-CoA dehydrogenase classified for example, under EC 1.1.1.-, such as EC 1.1.1.35 (e.g. , the gene product of fadB from Escherichia coli), EC 1.1.1.36 (e.g. , the gene product of phaB from Cupriavidus necator), or EC 1 , 1 .1.157 (e.g. , the gene product of hbd from Clostridium acetobutylicum) or a 3-oxoacyl-ACP reductase classified, for example, under EC 1.1.1.100, such as, for example, the gene product of fabG from Escherichia coli; followed by conversion of 3-oxo-azelaoyl-CoA to pimeloyl-CoA by a polypeptide having the enzymatic activity of a β-ketoihiolase classified, for example, under EC 2.3.1.16 or EC 2.3.1.174 such as, for example, the gene product of bktB from Cupriavidus necator or paaJ from Escherichia coli.

Pathways using pimeloyl-CoA as central precursor to pimdic acid

In some embodiments, pimelic acid is synthesized from pimeloyl-CoA by a polypeptide having the activity of a thioesterase classified under, for example, EC 3.1.2.-. The polypeptide having the activity of a thioesterase can be the gene product of yciA from Escherichia coli or acot!3 from Mus mus cuius (Cantu et al , Protein Science, 2010, 19, 1281 - 1295; Zhuang et al, Biochemistry, 2008, 47(9):2789 - 2796; Naggert et al. , J. Biol. Chem., 1991, 266(17):11044 - 11050), or tesB from Escherichia coli or a polypeptide represented by one of the following GenBank accession numbers: AA077182.1 (SEQ ID NO: 20): CCC78182.1 (SEQ ID NO: 22); EEI82564.1 (SEQ ID NO: 23); or ABG82470.1 (SEQ ID NO: 18).

In some embodiments, pimelic acid is synthesized from pimeioyl-CoA by a polypeptide having the activity of a CoA ligase classified under, for example, EC 6,2.1.-, such as EC 6.2.1.5 or EC 6.2.1.15, or a CoA transferase classified under, for example, EC 2.8.3.-, such as EC 2.8.3.8 or EC 2.8.3.12 (e.g. , a succinyl-CoA: acetate CoA-transferase from Acetobacier aceti (UniProtKB Accession No. B3EY95), the gene product of ANACAC )l 149 from Anaerostipes caccae (UniProtKB Accession No. B0MC58), a butyryl-Co A: acetate CoA-transferase from Butyrivibrio fibrisolvens (UniProtKB Accession No. D2WEY7), a butyryl-CoA: acetate CoA-transferase from Eubacterium hallii (UniProtKB Accession No. D2WEY8), the gene product of FAEPRAA2165 1575 from Faecalibacterium prausnUzii (UniProtKB Accession No. C7H5K4), a butyryl- Co A: acetate CoA-transferase from Faecalibacterium prausnUzii (UniProtKB Accession No. D2WEZ2), the gene product of FA EPRA M212JJ2812 from Faecalibacterium prausnUzii (UniProtKB Accession No. A8SFP6), a butyryl-CoA transferase from Roseburia hominis (Uni ProtKB Accession No. Q2TME9), or a butyryl-Co A: acetate CoA- transferase from Roseburia inulinivorans (UniProtKB Accession No. D2WEY6)).

In some embodiments, pimeioyl-CoA is converted to pimelate semiaidehyde by a polypeptide having the activity of an aldehyde dehydrogenase, such as an acetaldehyde dehydrogenase classified under, for example, EC 1.2.1 , 10, such as that encoded by pduB from Salmonella typhimurium.

Pimelate semiaidehyde is then converted to pimelic acid by a polypeptide having the activity of an aldehyde dehydrogenase classified under, for example, EC 1.2.1.-, such as a 7-oxoheptanoate dehydrogenase (e.g. , the gene product of thnG from Sphingomonas macrogolitabida), a 6-oxohextanoate dehydrogenase (e.g. , the gene product of chnE from Acinetobacter sp.) classified, for example, under EC 1 .2.1.63, a 5-oxopentanoate dehydrogenase classified, for example, under EC 1.2.1.20 (e.g. , the gene product of cpnE from Comamonas sp.,. ⁾ , a succinate-semialdehyde dehydrogenase classified, for example, under EC 1.2.1.16, EC 1 .2.1.24, or EC 1.2, 1 .79, or an aldehyde dehydrogenase classified under EC 1.2.1.3. See FIG. 3. Pathways using pinieioyl-CoA as central precursor to 7-aminoheptanoate

In some embodiments, pimeloyl-CoA is converted to pimelate semialdehyde using a polypeptide having the enzymatic activity of an aldehyde dehydrogenase classified under, for example, EC 1.2, 1.10, such as an acetaldehyde dehydrogenase encoded by pduB from Salmonella typhimurium or pduP from Klebsiella pneumoniae, Pimelate semialdehyde is then converted to 7-aminoheptanoate using a polypeptide having the enzymatic activity of a ω-transaminase classified, for example, under EC 2.6.1.-, e.g., EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82, such as, for example, that obtained from Chromobacterium violaceum (GenBank Accession No. AAQ59697.1 , SEQ ID NO: 7), Pseudomonas aeruginosa (GenBank Accession No. AAG08191.1 , SEQ ID NO: 8), Pseudomonas syringae (GenBank Accession No. AAY39893.1 , SEQ ID NO: 9), Rhodobacter sphaeroides (GenBank Accession No. ABA81 135.1 , SEQ ID NO: 10), Vibrio fluvialis (GenBank Accession No. AEA39183.1, SEQ ID NO: 12), Streptomyces griseus, or Clostridium viride.

In some embodiments, pimelate (see FIG. 3) is converted to pimelate semialdehyde using a polypeptide having the enzymatic activity of a carboxylate reductase classified, for exampie, under EC 1.2.99.6 such as, for example, the gene product of car in combination with a phosphopantetheine transferase enhancer (e.g. , encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia) or the gene products of griC and griD from Streptomyces griseus (Suzuki et at , J Antibiot. , 2007, 60(6), 380 - 387). The polypeptide having the activity of a carboxylate reductase can be obtained, for exampie, from Mycobacterium marinum (GenBank Accession No. ACC40567.1 , SEQ ID NO: 1 ), Mycobacterium smegmaiis (GenBank Accession No. ABK71854.1, SEQ ID NO: 2), Segniliparus rugosus (GenBank Accession No, EFV1 1917.1 , SEQ ID NO: 3), Mycobacterium smegmatis (GenBank Accession No. ABK75684.1 , SEQ ID NO: 4), Mycobacterium massiliense (GenBank Accession No. EIV1 1 143.1 , SEQ ID NO: 5), or Segniliparus rotundus (GenBank Accession No. ADG98140.1, SEQ ID NO: 6). Pimelate semialdehyde is then converted to 7- aminoheptanoate using a polypeptide having the enzymatic activity of a ω -transaminase classified, for example, under EC 2.6.1.-, e.g. , EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6, 1.48, or EC 2.6.1.82, such as that obtained from Chromobacterium violaceum (GenBank Accession No. AAQ59697.1 , SEQ ID NO: 7), Pseudomonas aeruginosa (GenBartk Accession No. AAG08191.1 , SEQ ID NO: 8), Pseudomonas syringae (GenBank Accession No. AAY39893.1, SEQ ID NO: 9), Rhodobacter sphaeroides (GenBank Accession No. ABA81 135.1 , SEQ ID NO: 10), Vibrio fiuvialis (GenBank Accession No. AEA39183.1, SEQ ID NO: 12), Streptomyces griseus, or Clostridium viride. See FIG. 4.

Pathways using pimeioyi-CoA as central precursor to 7-hydroxyheptanoate

In some embodiments, 7-hydroxyheptanoate is synthesized from the central precursor, pimeloyl-CoA using a polypeptide having the enzymatic activity of an aldehyde dehydrogenase classified under, for example, EC 1 .2.1.10, such as an acetaldehyde dehydrogenase encoded by pduB from Salmonella typhimurium or pduP from Klebsiella pneumoniae; followed by conversion of pimelate semialdehyde to 7- hydroxyheptanoate by a polypeptide having the activity of an alcohol dehydrogenase classified, for example, under EC 1.1.1.2 such as, for example, the gene product of YMR318C from Saccharomyces cerevisiae, a 6-hydroxyhexanoate dehydrogenase classified, for example, under EC 1.1.1.258, a 5-hydroxypentanoate dehydrogenase classified, for example, under EC 1.1.1.-, such as, for example, the gene product of cpnD from Comamonas sp. (Iwaki et al, , Appl. Environ. Microbiol, 1999, 65(1 1):5158 - 5162), or a 4-hydroxybutanoate dehydrogenase classified, for example, under EC 1.1.1.- such as, for example, the gene product of gabD from Escherichia coli (Bartsch et al., J. Bacteriol, 1990, 172(12), 7035). The alcohol dehydrogenase encoded by YMR318C has broad substrate specificity, including the oxidation of C7 alcohols. See FIG. 6.

Pathway using 7-aminoheptanoate, 7-hydroxyheptanoate, pimelate semialdehyde, or 1,7-heptanedioI as a central precursor to heptamethylenediamine

In some embodiments, heptamethylenediamine is synthesized from the central precursor, 7-aminoheptanoate (which can be produced as described in FIG. 4), by conversion of 7-aminoheptanoate to 7-aminoheptanal by a polypeptide having the activity of a carboxylate reductase classified, for example, under EC 1.2.99.6, such as, for example, the gene product of car in combination with a phosphopantetheine transferase enhancer (e.g. , encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia) or the gene products of griC and griD from Streptomyces griseus (Suzuki et al , J. AntihioL , 2007, 60(6), 380 - 387); followed by conversion of 7-aminoheptanal to heptamethylenediamine by a polypeptide having the activity of a ω-transaminase such as a s-transarninase classified under EiC 2.6, 1 .- (e.g. , EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.48, or EC 2.6.1.82, such as, for example, SEQ ID NOs: 7 - 12). The polypeptide having the activity of a carboxylase reductase can be obtained, for example, from Mycobacterium marinum (GenBank Accession No. ACC40567.1 , SEQ ID NO: 1 ), Mycobacterium smegmalis (GenBank Accession No. ABK71854.1 , SEQ ID NO: 2), Segniliparus rugosus (GenBank Accession No. EFV1 1917.1, SEQ ID NO: 3), Mycobacterium smegmatis (GenBank Accession No. ABK75684.1 , SEQ ID NO: 4), Mycobacterium massiliense (GenBank Accession No. EJVl 1143.1, SEQ ID NO: 5), or Segniliparus rotundas (GenBank Accession No. ADG98140.1, SEQ ID NO: 6).

The carboxylate reductase encoded by the gene product of car and enhancer npt from Nocardia or sfp from Bacillus subtilis has broad substrate specificity, including terminal difunctional C4 and C5 carboxvlic acids (Venkitasubramanian et al , Enzyme and Microbial Technology, 2008, 42, 130 - 137).

In some embodiments, heptamethylenediamine is synthesized from the central precursor, 7-hydroxyheptanoate (which can be produced as described in FIG. 6), by ^¬ conversion of 7-hydroxyheptanoate to 7-hydroxyheptanal by a polypeptide having the activity of a carboxylate reductase classified, for example, under EC 1.2.99.6, such as, for example, the gene product of car in combination with a polypeptide having the activity of a phosphopantetheine transferase enhancer (e.g. , encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia) or the gene product of griC & griD from Streptomyces griseus (Suzuki et al. , J. Antibiot , 2007, 60(6), 380 - 387); followed by conversion of 7-aminoheptanal to 7-aminoheptanol by a polypeptide having the activity of a ω-transaminase classified, for example, under EC 2.6.1.18. EC 2.6.1.19, EC 2.6.1 .29, EC 2.6.1.48, or EC 2.6.1.82 such as, for example, SEQ ID NOs: 7 - 32, see above; followed by conversion to 7-aminoheptanal by a polypeptide having the activity of an alcohol dehydrogenase classified, for example, under EC 1.1.1.- (e.g. , EC 1.1.1 .1 , EC 1.1.1.2, EC 1.1.1.21 , or EC 1.1.1.184) such as, for example, the gene product of YMR318C from S ccharomyces cerevisiae or yqhD from Escherichia coli (Liu et al , Microbiology, 2009, 155, 2078 - 2085; Larroy el al , 2002, Biochem J. , 361 (Ft 1 ), 163 -· 172: Jarboe, 201 1, Appl. Microbiol. Biotechnol , 89(2), 249 - 257) or a polypeptide represented by GenBank Accession No. CAA81612.1 (SEQ D NO: 21 ); followed by conversion to heptamethylenediamine by a polypeptide having the activity of a ω- transaminase classified, for example, under EC 2,6.1.18, EC 2.6, 1 .19, EC 2.6.1.29, EC 2.6.1 .48, or EC 2.6.1.82, such as, for example, SEQ ID NOs: 7 - 12, see above. See FIG. 5.

In some embodiments, heptamethylenediamine is synthesized from the centra! precursor, 7-aminoheptanoate (which can be produced as described in FIG. 4), by conversion of 7-aminoheptanoate to N7-acetyl-7-aminoheptanoate by a polypeptide having the activity of an N-acetyltransferase such as a lysine N-acetyltransferase classified, for example, under EC 2.3.1.32, such as, for example, the gene product of LYC1 from Yarrowia Hpolyiica (UniProtKB Accession No. P41929), the gene product of ablB from Methanococcus maripaludis (UniProtKB Accession No. Q6LYX3), or the gene product of ablB from Methanosarcina mazei (UniProtKB Accession No. Q8PYC8); followed by conversion to N7-acetyl-7-aminoheptanal by a polypeptide having the activity of a carboxylase reductase classified, for example, under EC 1.2.99.6 such as, for example, the gene product of car (see above, e.g. , SEQ ID NOs: 1 - 6) in combination with a polypeptide having the activity of a phosphopantetheine transferase enhancer (e.g. , encoded by a sfp gene from Bacillus subtil is or npt gene from Nocardia) or the gene product of griC & griD from Streptomyces griseus (Suzuki et al , J. Antibiot., 2007, 60(6), 380 - 387; followed by conversion to N7-acetyl-l ,7-diaminoheptane by a polypeptide having the activity of a ω-transaminase classified, for example, under EC 2.6.1 .18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82, such as SEQ ID NOs: 7 - 12, see above: followed by conversion to heptamethylenediamine by a polypeptide having the activity of a deacyiase classified, for example, under EC 3.5.1.-, such as, for example, EC 3.5.1.62 or EC 3.5.1.82. See FIG. 6. In some embodiments, heptamethylenediamine is synthesized from the centra! precursor, pimelate semialdehyde, by conversion of pimelate semialdehyde to heptanedial by a polypeptide having the activity of a carboxyiale reductase classified, for example, under EC 1 .2,99.6 such as, for example, the gene product of car (see above, e.g. , SEQ ID NO: 5) in combination with a phosphopantetheine transferase enhancer {e.g. , encoded by a sfp gene from Bacillus siibtilis or npt gene from Nocardia) or the gene product of griC & griD from Streptomyces griseus (Suzuki et al , J. Antihiot. , 2007, 60(6), 380 - 387) followed by conversion to 7-aminoheptanai by a polypeptide having the activity of a ω-transaminase classified, for example, under EC 2.6.1.1 8, EC 2.6, 1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82; followed by conversion to heptamethylenediamine by a polypeptide having the activity of a to-iransaminase classified, for example, under EC 2,6.1.18, EC 2.6, 1.19, EC 2.6.1.29, EC 2.6, 1.48, or EC 2.6.1.82, such as, for example, SEQ ID NOs: 7 - 12. See FIG. 5.

Pathways using 7-hydroxyheptanoate as centra! precursor to 1,7-heptanediol

In some embodiments, 1 ,7 heptanediol is synthesized from the central precursor,

7-hydroxyheptanoate (which can be produced as described in FIG. 6), by conversion of 7-hydroxyhepta.noate to 7-hydroxyheptanal by a polypeptide having the activity of a carboxylate reductase classified, for example, under EC 1.2.99.6 such as, for example, the gene product of car (see above, e.g. , SEQ ID NOs: 1 - 6) in combination with a polypeptide having the activity of a phosphopantetheine transferase enhancer {e.g. , encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia) or the gene products of griC and griD from Streptomyces griseus (Suzuki et al , J. Antibiot. , 2007, 60(6), 380 - 387); followed by conversion of 7-hydroxyheptanal to 1 ,7 heptanediol by a polypeptide having the activity of an alcohol dehydrogenase (classified, for example, under EC 1.1.1.- such as EC 1.1.1 .1 , EC 1.1.1.2, EC 1 .1.1.21 , or EC 1.1.1.184) such as, for example, the gene product of YMR318C from Saccharomyces cerevisiae or yqhD from Escherichia coli (see, e.g. , Liu et al, Microbiology, 2009, 155, 2078 - 2085; Larroy et al , 2002, Biochem J. , 361 (Pt 1), 163 - 172; or Jarboe, 201 1, Appl Microbiol. BiotechnoL, 89(2), 249 - 257), or a polypeptide represented by GenBank Accession No, CAA81612.1 (SEQ ID NO: 21) (from Geobacillus stear other mophilus). See FIG. 7.

Cultivation strategy

In some embodiments, one or more C7 building blocks are biosynthesized in a recombinant microorganism using anaerobic, aerobic or micro-aerobic cultivation conditions. In some embodiments, the cultivation strategy entails nutrient limitation such as, for example, nitrogen, phosphate, or oxygen limitation.

In some embodiments, a cell retention strategy using, for example, ceramic hollow fiber membranes can be employed to achieve and maintain a high cell density during either fed-batch or continuous fermentation.

in some embodiments, the principal carbon source fed to the fermentation in the synthesis of one or more C7 building blocks can derive from biological or non-biological feedstocks.

In some embodiments, the biological feedstock can be or can derive from, monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levuiinic acid and formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, plant oils, or municipal waste.

The efficient catabolism of crude glycerol stemming from the production of biodiesel has been demonstrated in several microorganisms such as Escherichia col Cupriavidus necaior, Pseudomonas oleavorans, Pse domonas putida, and Yarrowia. lipolytica (Lee et ai , Appl. Biochem. BiotechnoL , 2012, 166: 1801 - 1813; Yang ei ai ,

Biotechnology for Bio fuels, 2012, 5: 13; Meijnen ei ai , Appl, Microbiol BiotechnoL ,

201 1 , 90:885 - 893).

The efficient catabolism of lignocellulosic-derived levuiinic acid has been demonstrated in several organisms such as Cupriavidus necaior and Pseudomonas putida in the synthesis of 3 -hydroxy valerate via the precursor propanoyl-CoA (Jaremko and Yu, 201 1, supra; Martin and Prather, J, BiotechnoL , 2009, 139:61 - 67).

The efficient catabolism of iignin-derived aromatic compounds such as benzoate analogues has been demonstrated in several microorganisms such as Pseudomonas putida, Cupriavidus necator (Bugg et al, Current Opinion in Biotechnology, 2011, 22, 394 - 400; Perez-Pant ja et al. , FEMS Microbiol Rev. , 2008, 32, 736 - 794).

The efficient utilization of agricultural waste, such as olive mil! waste water has been demonstrated in several microorganisms, including Yarrowia lipolytica (Papanikolaou et al. , Bioresour. Techno!, , 2008, 99(7):2419 - 2428).

The efficient utilization of fermentable sugars such as monosaccharides and disaccharides derived from cellulosic, hemicellulosic, cane and beet molasses, cassava, corn, and other agricultural sources has been demonstrated for several microorganism such as Escherichia coli, Corynebacterium gluiamicum, Lactobacillus delbrueckii, and Lactococcus I act is (see, e.g., Hermann et al, J. Bioiechnol. , 2003, 104: 155 - 172; Wee et al.. Food Technol. Bioiechnol. , 2006, 44(2): 163 - 172; Ohashi et al, J. Bioscience and Bioengineering, 1999, 87(5):647 - 654).

The efficient utilization of furfural, derived from a variety of agricultural lignoce!lulosic sources, has been demonstrated for Cupriavidus necator (Li et al, Biodegradation, 201 1 , 22: 1215 - 3225).

In some embodiments, the non-biological feedstock can be or can derive from natural gas, syngas, CO ₂ /H ₂ , methanol, ethanol, benzoate, non-volatile residue (NVR) or a caustic wash waste stream from cycloheptane oxidation processes, or terephthalic acid / isophthalic acid mixture waste streams.

The efficient catabolism of methanol has been demonstrated for the methylotrophic yeast Pichia pastoris.

The efficient catabolism of ethanol has been demonstrated for Clostridium Muyveri (Seedorf et al. , Proc. Natl. Acad. Sci. USA, 2008, 105(6) 2128 - 2133).

The efficient catabolism of C0 ₂ and ¾, which may be derived from natural gas and other chemical and petrochemical sources, has been demonstrated for Cupriavidus necator (Prybylski et al, Energy, Sustainability and Society, 2012, 2: 1 1).

The efficient catabolism of syngas has been demonstrated for numerous microorganisms, such as Clostridium ljungdahlii and Clostridium autoethanogenum (Kopke et al , Applied and Environmental Microbiology, 201 1 , 77(15): 5467 - 5475). The efficient catabolism of the non- volatile residue waste stream from cycloheptane processes has been demonstrated for numerous microorganisms, such as Delfiia acidovorans and Cupriavidus necaior (Ramsay et al. , Applied and Environmental Microbiology, 1 986, 52(1 ): 152 - 1 56).

In some embodiments, the microorganism is a prokaryote. For example, the prokaryote can be a bacterium from the genus Escherichia such as Escherichia coli; from the genus Clostridia such as Clostridium ljungdahlii, Clostridium autoethanogenum, or Clostridium kluyveri; from the genus Corynebacteria such as Corynebacterium glutamicum; from the genus Cupriavidus such as Cupriavidus necator or Cupriavidus metallidurans; from the genus Pseudomonas such as Pseudomonas fluoresceins, Pseudomonas putida, or Pseudomonas oleavorans; from the genus Delfiia such as Delfiia acidovorans; from the genus Bacillus such as Bacillus sub till is; from the genus Lactobacillus such as Lactobacillus delhrueckii or from the genus Lactococcus such as Lactococcus lactis. Such prokaryotes also can be a source of genes to construct recombinant microorganisms described herein that are capable of producing one or more C7 building blocks.

In some embodiments, the microorganism is a eukaryote, For example, the eukaryote can be a filamentous fungus, e.g., one from the genus Aspergillus such as Aspergillus niger. Alternatively, the eukaryote can be a yeast, e.g. , one from the genus Saccharomyces such as Saccharomyces cerevisiae; from the genus Pichia such as Pichia pastoris; or from the genus Yarrow ia such as Yarrowia lipolytica; from the genus Issatchenkia such as Issathenkia orientalis; from the genus Debaryomyces such as Debaryomyces hanseniv, from the genus Arxula such as Arxula adenoinivorans; or from the genus Kluyveromyces such as Kluyveromyces lactis. Such eukaryotes also can be a source of genes to construct recombinant microorganisms described herein that are capable of producing one or more C7 building blocks.

Metabolic engineering

The present document provides methods involving less than all the steps described for all the above pathways. Such methods can involve, for example, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more of such steps. Where less than all the steps are included in such a method, the first, and in some embodiments the only, step can be any one of the steps listed.

Furthermore, recombinant microorganisms described herein can include any combination of the above enzymes such that one or more of the steps, e.g. , one, two, three, four, five, six, seven, eight, nine, ten, or more of such steps, can be performed within a recombinant microorganism. This document provides cells of any of the genera and species listed and genetically engineered to express one or more (e.g. , one, two, three, four, five, six, seven, eight, nine, 10, 1 1 , 12 or more) recombinant forms of any of the enzymes recited in the document. Thus, for example, the cells can contain exogenous nucleic acids encoding enzymes catalyzing one or more of the steps of any of the pathways described herein.

In addition, this document recognizes that where enzymes have been described as accepting CoA-aciivated substrates, analogous enzyme activities associated with [acp]- bound substrates exist that are not necessarily in the same enzyme class.

Also, this document recognizes that where enzymes have been described as accepting (R)-enantiomers of substrate, analogous enzyme activities associated with (S)- enantiomer substrates exist that are not necessarily in the same enzyme class.

This document also recognizes that where an enzyme is shown to accept a particular co-factor, such as NADPH, or co-substrate, such as acetyl-CoA. many enzymes are promiscuous in terms of accepting a number of different co-factors or co-substrates in catalyzing a particular enzyme activity. Also, this document recognizes that where enzymes have high specificity for e.g. , a particular co-factor such as NADH, an enzyme with similar or identical activity that has high specificity for the co-factor NADPH may be in a different enzyme class.

In some embodiments, the enzymes in the pathways outlined herein are the result of enzyme engineering via non-direct or rational enzyme design approaches with aims of improving activity, improving specificity, reducing feedback inhibition, reducing repression, improving enzyme solubility, changing stereo-specificity, or changing co- factor specificity. In some embodiments, the enzymes in the pathways outlined here can be gene dosed, i. e. , overexpressed, into the resulting genetically modified organism via episomal or chromosomal integration approaches.

In some embodiments, genome-scale system biology techniques such as Flux Balance Analysis can be utilized to devise genome scale attenuation or knockout strategies for directing carbon flux to a C7 building block.

Attenuation strategies include, but are not limited to; the use of transposons, homologous recombination (double cross-over approach), mutagenesis, enzyme inhibitors and RNAi interference.

Attenuation strategies have been employed to increase the yield of desired end products of engineered metabolic pathways. For example, genetic manipulations previously studied to make succinate the major fermentation product in E. coli include deletion of the fermentative lactate dehydrogenase (LDH) pathway (Mat-Jan el al , 1989). deietion of both the LDH and pyruvate formate lyase (PFL) pathways (Bunch et al. , 1997), and deletion of multiple pathways including PFL and LDH pathways with an additional ptsG mutation (Donnelly ei al , 1998; Chatterjee et al , 2001). Overexpression of phosphoenolpyruvate carboxylase (PEPC) (Millard et al , 1996), overexpression of the malic enzyme (Stols and Donnelly, 1997; Hong and Lee, 2000), overexpression of pyruvate carboxylase (PYC) (Gokarn et al , 1998; Gokarn et al , 2000; Vemuri et al , 2002a), and overexpression of the heterologous Actinobacill s saccinogenes phosphoenolpyruvate car 'box 'kinase in a PEPC E. coli mutant (Kirn et al , 2004) have also been studied to improve succinate yield from recombinant E. coli.

In some embodiments, fluxomic, metaboloraic and transcriptonial data can be utilized to inform or support genome-scale system biology techniques, thereby devising genome scale attenuation or knockout strategies in directing carbon flux to a C7 building block.

In some embodiments, the microorganism's tolerance to high concentrations of a C7 building block can be improved through continuous cultivation in a selective environment. In some embodiments, the microorganism's endogenous biochemical network can be attenuated or augmented to (1) ensure the intracellular availability of acetyl-CoA or malonyl-CoA, (2) create an NADH or NADPH imbalance that may only be balanced via the formation of one or more C7 building blocks, (3) prevent degradation of central metabolites, central precursors leading to and including one or more C7 building blocks, and/or (4) ensure efficient efflux from the cell.

In some embodiments requiring intracellular availability of acetyl-CoA for C7 building block synthesis, endogenous enzymes catalyzing the hydrolysis of acetyl-CoA such as short-chain length thioesterases can be attenuated in the microorganism.

In some embodiments requiring the intracellular availability of acetyl-CoA for C7 building block synthesis, an endogenous gene encoding a phosphotransacetylase generating acetate such as pta can be attenuated (Shen et al , Appl. Environ, Microbiol , 201 1 , 77(9):2905-2915).

in some embodiments requiring the intracellular availability of acetyl-CoA for C7 building block synthesis, an endogenous gene in an acetate synthesis pathway encoding an acetate kinase, such as ach can be attenuated.

In some embodiments requiring the intracellular availability of acetyl-CoA and NADH for C7 building block synthesis, an endogenous gene encoding an enzyme that catalyzes the degradation of pyruvate to lactate such as lactate dehydrogenase encoded by IdhA can be attenuated (Shen et al. , 201 1, supra).

in some embodiments, enzymes that catalyze anapleurotic reactions such as PEP carboxylase and/or pyruvate carboxylase can be overexpressed in the microorganism.

In some embodiments requiring the intracellular availability of acetyl-CoA and NADH for C7 building block synthesis, endogenous genes encoding enzymes, such as menaquinol-fumarate oxidoreductase, that catalyze the degradation of phophoenolpyruvate to succinate such as frdBC can be attenuated {see, e.g. , Shen et al. , 201 1 , supra).

In some embodiments requiring the intracellular availability of acetyl-CoA and NADH for C7 building block synthesis, an endogenous gene encoding an enzyme that catalyzes the degradation of acetyl-CoA to ethanoi such as, for example, the alcohol dehydrogenase encoded by adhE from Clostridium acetobutylicum can be attenuated (Shen et al, 201 1 , supra). In some embodiments, where pathways require excess NADH co-factor for C7 building block synthesis, a recombinant formate dehydrogenase gene, e.g. ,fdhl from Candida boidinii, can be overexpressed in the microorganism (Shen et al, 201 1 , supra).

In some embodiments, where pathways require excess NADH co-factor for C7 building block synthesis, a recombinant NADH-consuming trans hydro genase can be attenuated.

In some embodiments, an endogenous gene encoding an enzyme that catalyzes the degradation of pyruvate to ethanoi such as pyruvate decarboxylase can be attenuated.

In some embodiments requiring the intracellular availability of acetyl-CoA for C7 building block synthesis, a recombinant acetyl-CoA synthetase such as, for example, the gene product of acsA from Cupriavidus necator can be overexpressed in the microorganism (Satoh et al , J, Bioscience and Bioengineering, 2003, 95(4):335 - 341).

In some embodiments, carbon flux can be directed into the pentose phosphate cycle to increase the supply of NADPH by attenuating an endogenous glucose-6- phosphate isomerase (EC 5.3.1.9).

In some embodiments, carbon flux can be redirected into the pentose phosphate cycle to increase the supply of NADPH by overexpression a 6-phosphogluconate dehydrogenase and/or a transketolase (Lee et l., 2003, Biotechnology Progress, 19(5), 1444 - 1449) from, for example, Escherichia coli.

In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C7 building block, a gene such as udhA from Escherichia coli encoding a puridine nucleotide transhydrogenase can be overexpressed in the microorganisms (Brigham et al, Advanced Biofuels and Bioproducts, 2012, Chapter 39, 1065 - 1090).

In some embodiments, where pathways require excess NA DPH co-factor in the synthesis of a C7 Building Block, a recombinant glyceraldehyde-3-phosphate- dehydrogena.se gene such as gapN from Sulfolobas solfataricus can be overexpressed in the microorganisms (Brigham et al, 2012, supra). in some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C7 building block, a recombinant malic enzyme gene such as mm A or maeB from Cupriavidus necator can be overexpressed in the microorganisms (Brigham et al , 2012, supra).

In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C7 building block, a recombinant glucose-6-phosphate dehydrogenase gene such as zwf from Escherichia coli can be overexpressed in the microorganisms (Lim et al , J. Bioscience and Bioengineering, 2002, 93(6), 543 - 549).

In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C7 building block, a recombinant fructose 1,6 diphosphatase gene such as fbp from Corynebacterium glutamicum can be overexpressed in the microorganisms (Becker et al, , J. Bioiechnol , 2007, 132:99 - 109),

in some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C7 building block, endogenous triose phosphate isomer ase (EC 5.3.1.1) can be attenuated.

in some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C7 building block, a recombinant glucose dehydrogenase such as, for example, the gene product of gdh from Bacillus subtilis can be overexpressed in the microorganism (Satoh et al, J. Bioscience and Bioengineering, 2003, 95(4):335 - 341).

In some embodiments, endogenous enzymes facilitating the conversion of

NADPH to NADH can be attenuated, such as, for example, the NADH generation cycle that may be generated via inter-conversion of glutamate dehydrogenases classified under EC 1.4.1.2 (NADH-specific) and EC 1.4.1.4 (NADPH-specific).

In some embodiments, an endogenous glutamate dehydrogenase (EC 1.4.1.3) that utilizes both NADH and NADPH as co-factors can be attenuated.

In some embodiments, a membrane-bound cytochrome P450 such as CYP4F3B can be solubilized by only expressing the cytosolic domain and not the N-terminal region that anchors the P450 to the endoplasmic reticulum (Sche!ler et al, J, Biol Chem., 1 994, 269(17): 12779-12783). in some embodiments, an enoyl-CoA reductase can be solubilized via expression as a fusion protein with a small soluble protein, for example, the maltose binding protein (Gloerich el al , FEES Letters, 2006, 580, 2092 - 2096).

in some embodiments using microorganisms that naturally accumulate polyhydroxyalkanoates, the endogenous polymer synthase enzymes can be attenuated in the microorganism strain.

In some embodiments, a L-alanine dehydrogenase can be overexpressed in the microorganism to regenerate L-alanine from pyruvate as an amino donor for ay transaminase catalyzed reactions. For example, the L- lanine dehydrogenase may be from Escherichia coli.

In some embodiments, an L-glutamate dehydrogenase, a L-glutamine synthetase, or an alpha-aminotransaminase can be overexpressed in the microorganism to regenerate L-glutamate from 2-oxoglutarate as an amino donor for co-transaminase catalyzed reactions. For example, the L-glutamate dehydrogenase, the L-gl tamine synthetase, or the alpha-aminotransaminase may be from Escherichia coli.

in some embodiments, enzymes such as a pimeloyl-CoA dehydrogenase classified, for example, under EC 1 ,3.1.62; an acyl-CoA dehydrogenase classified, for example, under EC 1.3,8.7, EC 1.3.8.1 , or EC 1.3.99.-; and/or a butyryl-CoA dehydrogenase classified, for example, under EC 1.3.8.6 that degrade central metabolites and central precursors leading to and including C7 building blocks can be attenuated. Examples of polypeptides having the activity of an acyl-CoA dehydrogenase classified under EC 1.3.99.- include, but are not limited to, the gene product of atuD from Pseuciomonas aeruginosa (UniProt B Accession No. Q9HZV8), the gene product of sen from Drosophila melanogaster (UniProtKB Accession No. 018404), the gene product of fadE26 from Mycobacterium tuberculosis (UniProtKB Accession No. I6YCA3), the gene product of aidB from Escherichia coli (UniProtKB Accession No, P33224), the gene product of acdh-11 from Caenorhahd is elegans (UniProtKB Accession No. Q9XWZ2), and the gene product of Acad I I from M s musculus (UniProtKB Accession No. Q8GXL6). In some embodiments, endogenous enzymes activating C7 building blocks via Coenzyme A esterification such as CoA-llgases (e.g., an adipyl-CoA synthetase) classified under, for example, EC 6.2.1.- can be attenuated.

In some embodiments, the efflux of a C7 building block across the cell membrane to the extracellular media can be enhanced or amplified by genetically engineering structural modifications to the cell membrane or increasing any associated transporter activity for a C7 building block.

The efflux of heptamethylenediamine can be enhanced or amplified by overexpressing broad substrate range multidrug transporters such as Bit from Bacillus suhtilis (Wooiridge et al , 1997, J. Biol. Chem. , 272(14):8864 - 8866); AcrB and AcrD from Escherichia coli (Elkins & Nikaido, 2002, J Bacterial,, 184(23), 6490 - 6499), NorA from Staphylococcus aereus (Ng et al , 1994, Aniimicroh Agents Chemother, 38(6), 1345 - 1355), or Bmr from Bacillus suhtilis (Neyfakh, 1992, Antimicrob Agents Chemother, 36(2), 484 - 485).

The efflux of 7-aminoheptanoate and heptamethylenediamine can be enhanced or amplified by overexpressing the solute transporters such as, for example, the lysE transporter from Corynehacterium. giutamiciim (Bellmann et al , 2001 , Microbiology, 147, 1765 - 1774),

The efflux of pimelic acid can be enhanced or amplified by overexpressing a dicarboxylate transporter such as, for example, the SucE transporter from Corynehacterium glutamicum (Huhn et al, Appl. Microbiol. & Biotech , 89(2), 327 - 335).

Metabolically engineering recombinant hosts with various enzymes to produce final products has been successfully demonstrated by several groups, See, e.g., Blombach B et al , Bioeng Bugs., 201 1 , 2(6): 346-50 (teaching successful metabolic engineering of the last two steps of the Ehrlich pathway (by expression of genes encoding a broad range 2-ketoacid decarboxylase and an alcohol dehydrogenase) in recombinant hosts for the production of higher isobutanol); Adkins, J. et al, Front Microbiol., 2012, 3:313 (summarizing numerous biomonomers (such as polvesier building-blocks) that can be produced as a result of metabolic and pathway engineering in various recombinant hosts): Chan, S, el al , Bioresour Techno!., 2012, 1 03( 1 ):329-36 (teaching production of succinic acid from sucrose and sugarcane molasses by metabolically engineering E. coli with sucrose-utilizing genes (cscKB and cscA)) Lee, S. et al , Appl Biochem BiotechnoL, 2012, 167(l):24-38 (teaching successful metabolic engineering of P. aeruginosa and E. coli for improving long-chain fatty acid production by co-expressing essential enzymes that are involved in the fatty acid synthesis metabolic pathway (accA and fabD) as well as fatty acyl-acyl carrier protein thioesterase gene): Rathnasingh, C. et al , Biotechnol Bioe.ng., 2009, 1 04(4):729-39 (teaching successful metabolic engineering of E. coli for producing 3 -hydroxypropionic acid from glycerol by overexpression of glycerol dehydratase (DhaB) and aldehyde dehydrogenase (AldH) along with glycerol dehydratase reaetivase (GDR)).

Producing C7 Building Blocks Using a Recombinant Microorganism

Typically, one or more C7 building blocks can be produced by providing a microorganism and culturing the provided microorganism with a culture medium containing a suitable carbon source as described above. In general, the culture media and/or culture conditions can be such that the microorganisms grow to an adequate density and produce a C7 building block efficiently. For large-scale production processes, any method can be used such as those described elsewhere (Manual of ndustrial Microbiology and Biotechnology, 2" ^d Edition, Editors: A. L. Demain and J. E. Davies, ASM Press: and Principles of Fermentation Technology, P. F. Stanbury and A. Whitaker, Pergamon). Briefly, a large tank (e.g. , a 100 gallon, 200 gallon, 500 gallon, or greater than 500 gallon tank) containing an appropriate culture medium is inoculated with a particular microorganism. After inoculation, the microorganism is incubated to al low biomass to be produced. Once a desired biomass is reached, the broth containing the microorganisms can be transferred to a second tank, This second tank can be any size. For example, the second tank can be larger, smaller, or the same size as the first tank. Typically, the second tank is larger than the first such that additional culture medium can be added to the broth from the first tank, in addition, the culture medium within this second tank can be the same as, or different from, that used in the first tank. Once transferred, the microorganisms can be incubated to allow for the production of a C7 building block. Once produced, any method can be used to isolate C7 building blocks. For example, C7 building blocks can be recovered selectively from the fermentation broth via adsorption processes, in the case of pimelic acid and 7- aminoheptanoic acid, the resulting eluate can be further concentrated via evaporation, crystallized via evaporative and/or cooling crystallization, and the crystals recovered via centrifugation. In the case of beptamethylenediarnine and 1 ,7-heptanedioh distillation may be employed to achieve the desired product purity.

The invention will be further described in the fo Slowing examples, which do not limit the scope of the invention described in the claims.

Enzyme activity of ω-transamhwse using pime!ate seeiialdeliyde as substrate mid forming 7-aminoheptanoate

A nucleotide sequence encoding an N-terminal His-tag was added to the nucleic acid sequences from Chromobacteriwn violaceum, Pse domonas syringae, Rhodobacter sphaeroides, and Vibrio fluvialis encoding the ω-trans minases of SEQ ID NOs: 7, 9, 10, and 12, respectively (see FIG. 8) such that N-terminal HIS tagged (^transaminases could be produced. Each of the resulting modified genes was cloned into a pET21 a expression vector under control of the T7 promoter and each expression vector was transformed into a BL21 [DE3] E. coli strain. The resulting recombinant E, coli strains were cultivated at 37°C in a 250 n L shake flask culture containing 50 mL LB media and antibiotic selection pressure, with shaking at 230 rpm. Each culture was induced overnight a 16 °C using 1 mM IPTG,

The pellet from each induced shake flask culture was harvested via centrifugation. Each pellet was resuspended and iysed via sonication. The cell debris was separated from the supernatant via centrifugation and the cell free extract was used immediately in enzyme activity assays. Enzyme activity assays in the reverse direction (i.e. , 7-aminohepiaiioate to pirnelate semialdehyde) were performed in a buffer composed of a final concentration of 50 niM HEPES buffer (pH = 7,5), 10 rnM 7-arninoheptanoate, 10 mM pyruvate, and 100 μΜ pyridoxyl 5' phosphate. Each enzyme activity assay reaction was initiated by adding cell free extract of the o transaminase gene product or the empty vector control to the assay buffer containing the 7-aminoheptanoate and incubated at 25°C for 4 h, with shaking at 250 rpra. The formation of L-alanine from pyruvate was quantified via RP- HPLC.

Each enzyme only control without 7-aminoheptanoate demonstrated low base line conversion of pyruvate to [.-alanine. See FIG. 14. The gene product of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 12 accepted 7-aminoheptanoate as a substrate as confirmed against the empty vector control. See FIG. 15.

Enzyme activity in the forward direction (i.e. , pirnelate semialdehyde to 7- aminoheptanoate) was confirmed for the transaminases of SEQ ID NO: 9. SEQ ID NO: 10, and SEQ ID NO: 12. See FIG. 16. Enzyme activity assays were performed in a buffer composed of a final concentration of 50 mM HEPES buffer (pH = 7.5), 10 mM pirnelate semialdehyde, 10 rn L-alanine and 100 μΜ pyridoxyl 5' phosphate. Each enzyme activity assay reaction was initiated by adding a cell free extract of the <¾>- transaminase gene product or the empty vector control to the assay buffer containing the pirnelate semialdehyde and incubated at 25 °C for 4 h, with shaking at 250 rpm. The formation of pyruvate was quantified via RP-HPLC.

The gene products represented by SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 12 accepted pirnelate semialdehyde as substrate as confirmed against the empty vector control. See FIG. 16. The reversibility of the c -transaminase activity was confirmed, demonstrating that the co-transaminases represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ I D NO: 10, and SEQ ID NO: 12 accepted 7-aminoheptanoate as substrate and synthesized pirnelate semialdehyde as a reaction product. See FIG. 15. EXAMPLE 2

Enzyme activity of catboxylate reductase using pimelate as substrate and forming pimelate semia!dehyde

A nucleotide sequence encoding a HIS-tag was added to the nucieic acid sequences from Segniliparus rugosus and Segniliparus rotundus that encode the carboxylate reductases of SEQ ID NOs: 3 (EFV1 1917.1) and 6 (ADG98140.1), respectively (see FIG. 8), such that N-ierminal HIS tagged carboxylate reductases could be produced. Each of the modified genes was cloned into a pET Duet expression vector along with a sfp gene encoding a HIS-tagged phosphopantetheine transferase from Bacillus subtilis, both under the T7 promoter. Each expression vector was transformed into a BL21 [DE3] E. coli strain, and the resulting recombinant E. coli strains were cultivated at 37°C in a 250 mL shake flask culture containing 50 niL LB media and antibiotic selection pressure, with shaking at 230 rpm. Each culture was induced overnight at 37 °C using an auto-induction media,

The pellet from each induced shake flask culture was harvested via centrifugation. Each pellet was re-suspended and lysed via sonication, and the ceil debris was separated from the supernatant via centrifugation. The carboxylate reductases and phosphopantetheine transferases were purified from the supernatant using Ni-affinity chromatography, diluted 10-fold into 50 mM HEPES buffer (pH ^::: 7.5), and concentrated via ultrafiltration.

Enzyme activity assays (i.e. , from pimelate to pimelate semiaidehyde) were performed in triplicate in a buffer composed of a final concentration of 50 mM HEPES buffer (pH = 7.5), 2 mM pimelate, 10 mM MgCi ₂ , 1 mM ATP, and 1 mM NADPH. Each enzyme activity assay reaction was initiated by adding purified carboxylate reductase and phosphopantetheine transferase gene products or the empty vector control to the assay buffer containing the pimelate and then incubated at room temperature for 20 min. The consumption of NADPH was monitored by absorbance at 340 ran. Each enzyme only control without pimelate demonstrated low base line consumption of NADPH. See bars for EFV1 1917.1 and ADG98140.1 in FIG. 9. The gene products represented by SEQ ID NO: 3 (EFV1 1 917.1) and SEQ ID NO: 6 (ADG98140.1), enhanced by the gene product of sfp from Bacillus subli!is, accepted pimelate as a substrate, as confirmed against the empty vector control (see FIG. 10), and synthesized pimelate semialdehyde.

EXAMPLE 3

Enzyme activity of carboxylate reductase using 7-hydroxyheptanoate as substrate and forming 7-hydroxyhepianal

A nucleotide sequence encoding a His-tag was added to the nucleic acids from Mycobacterium marinum, Mycobacterium smegmatis, Segniliparus rugosus, Mycobacterium massiliense, and Segniliparus rotundus that encode the carboxylate reductases of SEQ ID NOs: 1 - 6 respectively (see FIG. 8) such that N~ terminal HIS tagged carboxylate reductases could be produced. Each of the modified genes was cloned into a pET Duet expression vector alongside a sfp gene encoding a His-tagged phosphopantetheine transferase from Bacillus subtilis, both under control of the T7 promoter. Each expression vector was transformed into a BL21 [DE3] E. coli strain along with the expression vectors from Example 2. Each resulting recombinant E. coli strain was cultivated at 37°C in a 250 ml, shake flask culture containing 50 mL LB media and antibiotic selection pressure, with shaking at 230 rpm. Each culture was induced overnight at 37°C using an auto-induction media.

The pellet from each induced shake flask culture, was harvested via centrifugation. Each pellet was resuspended and lysed via sonication. The cell debris was separated from the supernatant via centrifugation. The carboxylate reductases and phosphopantetheine transferase were purified from the supernatant using Ni-affmity chromatography, diluted 10-fold into 50 mM HEPES buffer (pH ^::: 7.5) and concentrated via ultrafiltration.

Enzyme activity (i.e. , 7-hydroxyheptanoate to 7-hydroxyheptana) ) assays were performed in triplicate in a buffer composed of a final concentration of 50 mM HEPES buffer (pH = 7.5), 2 mM 7-hydroxyheptana!, 10 mM gCl ₂ , i mM ATP, and 1 mM NADPH. Each enzyme activity assay reaction was initiated by adding purified carboxylate reductase and phosphopantetheine transferase or the empty vector control to the assay buffer containing the 7-hydrox.yheptanoate and then incubated at room temperature for 20 min. The consumption of NADPH was monitored by absorbance at 340 nrn. Each enzyme only control without 7-hydroxyheptanoate demonstrated low base line consumption of NADPH. See FIG. 9.

The gene products represented by SEQ ID NO 1 - 6 enhanced by the gene product of sfp, accepted 7-hydroxyheptanoate as substrate as confirmed against the empty vector control (see FIG. 1 1), and synthesized 7-hydroxyheptanal .

EXAMPLE 4

Enzyme activity of m-transaminase for 7-aminoheptanol, forming 7~oxoheptanol

A nucleotide sequence encoding an N-terminal His-tag was added to the Chromobacterium violaceum, Pseudomonas syringae, and Rhodobacter sphaeroides nucleic acids encoding the ahtransaminases of SEQ ID NOs: 7. 9, and 10, respectively (see FIG. 8) such that N-terminal HIS tagged co-lransaminases could be produced. The modified genes were cloned into a pET21a expression vector under the T7 promoter. Each expression vector was transformed into a BL21 [DE3] E. coli strain. Each resulting recombinant E. coli strain were cultivated at 37°C in a 250 mL shake flask culture containing 50 mL LB media and antibiotic selection pressure, with shaking at 230 rpm. Each culture was induced overnight at 16°C using 1 niM IPTG.

The pellet from each induced shake flask culture was harvested via centriiiigation. Each pellet was resuspended and lysed via sonication. The cell debris was separated from the supernatant via centrifugation and the cell free extract was used immediately in enzyme activity assays.

Enzyme activity assays in the reverse direction (i.e. , 7-aminoheptanol to 7- oxoheptanol) were performed in a buffer composed of a final concentration of 50 mM HEPES buffer (pH = 7.5), 10 mM 7-aminoheptanol, 10 mM pyruvate, and 100 μΜ pyridoxyl 5' phosphate. Each enzyme activity assay reaction was initiated by adding ceil free extract of the ω-transaminase gene product or the empty vector control to the assay buffer containing the 7-aminoheptanol and then incubated at 25 °C for 4 h, with shaking at 250 rpm. The formation of L-alanine was quantified via RP-HPLC. Each enzyme only control without 7-aminoheptanol had low base line conversion of pyruvate to L-alanine. See FIG. 14.

The gene products represented by SEQ ID NOs: 7, 9, and 10 accepted 7- aminoheptanol as substrate as confirmed against the empty vector control (see FIG, 19) and synthesized 7-oxoheptanol as reaction product. Given the reversibility of the ω- transaminase activity (see Example 1 ), it can be concluded that the gene products of SEQ ID NOs: 7, 9, and 10 accept 7-oxoheptanol as substrate and form 7-aminoheptanol.

EXAMPLE 5

Enzyme activity of eo-transaminase using heptamethylenediamine as substrate and forming 7-aminoheptanal

A nucleotide sequence encoding an N-terminal His-tag was added to the Chromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae, Rhodobacler sphaeroides, Escherichia coli, and Vibrio fiuvialis nucleic acids encoding the (^transaminases of SEQ ID NOs: 7 - 12, respectively (see FIG. 8) such that N- terminai HIS tagged ω-transaminases could be produced. The modified genes were cloned into a pET21a expression vector under the T7 promoter. Each expression vector was transformed into a BL21 [DE3] E. coli strain. Each resulting recombinant E. coli strain were cultivated at 37°C in a 250 mL shake flask culture containing 50 rnL LB media and antibiotic selection pressure, with shaking at 230 rpm. Each culture was induced overnight at 16°C using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested via centrifugation. Each pellet was resuspended and lysecl via sonication. The cell debris was separated from the supernatant via centrifugation, and the cell free extract was used immediately in enzyme activity assays.

Enzyme activity assays in the reverse direction (i.e., heptamethylenediamine to 7- aminoheptanal) were performed in a buffer composed of a final concentration of 50 mM HEPES buffer (pH = 7.5), 10 mM heptamethylenediamine, 10 mM pyruvate, and 100 μΜ pyridoxyl 5' phosphate. Each enzyme activity assay reaction was initiated by adding cell free extract of the ω-transaminase gene product or the empty vector control to the assay buffer containing the heptamethylenediamine and then incubated at 25 °C for 4 h, with shaking at 250 rpm. The formation of L-aiaiiine was quantified via RP-HPLC.

Each enzyme only control without hepiamethyletiediamine had low base line conversion of pyruvate to L-aianine. See FIG. 14.

The gene products of SEQ ID NOs: 7 - 12 accepted heptamethylenediamine as substrate as confirmed against the empty vector control (see FIG. 17) and synthesized 7- aminoheptanal as reaction product. Given the reversibility of the o>- ransaminase activity (see Example 1), it can be concluded that the gene products of SEQ ID NOs: 7 - 12 accept 7-aminoheptanaI as substrate and form heptamethylenediamine. EXAMPLE 6

Enzyme activity of carboxyiate reductase for N7-aeetyl-7-amhioheptanoaie, forming N7-acetyl~7-aminoheptanal

The activity of each of the N-terminal His-tagged carboxyiate reductases of SEQ ID NOs; 2, 5, and 6 (see Examples 2 and 3, and FIG. 8) for converting N7-acetyl-7- aminoheptanoate to N7-acetyl-7-aminoheptanal was assayed in triplicate in a buffer composed of a final concentration of 50 mM HEPES buffer (pH ^:::: 7.5), 2 mM N7-acetyl- 7-aminoheptanoate, 10 mM MgC! ₂ , 1 mM ATP, and 1 mM NADPH. The assays were initiated by adding purified carboxyiate reductase and phosphopanletheine transferase or the empty vector control to the assay buffer containing the N7-acetyl-7-aminoheptanoate then incubated at room temperature for 20 min. The consumption of NADPH was monitored by absorbance at 340 nm. Each enzyme only control without N7-acetyl-7- aminoheptanoate demonstrated low base line consumption of NADPH. See FIG. 9.

The gene products of SEQ ID NOs: 2, 5, and 6, enhanced by the gene product of sfp, accepted N7-acetyl-7-amitioheptatioate as substrate as confirmed against the empty vector control (see FIG. 12), and synthesized N7-acetyi-7-aminoheptanal.

EXAMPLE 7

Enzyme activity of ω-transammase using N7-acetyI-l,7-diammoheptane, and forming N7-acetyl-7-aminoheptanaI The activity of the N-terminal His-tagged (^transaminases of SEQ ID NOs: 7 - 12 (see Example 5, and FIG. 8) for converting N7-acetyl-l ,7-diarninoheptane to N7- acetyl-7-aminoheptanal was assayed using a buffer composed of a final concentration of 50 mM HEPES buffer (pH = 7.5), 10 mM N7-acetyl-l ,7-diaminoheptane, 10 mM pyruvate and 100 μΜ pyridoxyl 5' phosphate. Each enzyme activity assay reaction was initiated by adding a cell free extract of the (^transaminase or the empty vector control to the assay buffer containing the N7-acetyl- 1 ,7-diaminoheptane then incubated at 25 °C for 4 h, with shaking at 250 rpm. The formation of L-alanine was quantified via RP- HPLC.

Each enzyme only control without N7-acety [- 1 ,7-diaminoheptane demonstrated low base line conversion of pyruvate to L-alanine. See FIG. 14.

The gene product of SEQ ID NOs: 7 - 12 accepted N7 -acetyl- 1 ,7-diaminoheptane as substrate as confirmed against the empty vector control (see FIG. 18) and synthesized N7-acetyi-7-aminoheptanai as reaction product.

Given the reversibility of the ω-transaminase activity (see Example I), the gene products represented by SEQ ID NOs: 7 - 12 accept N7-acetyl-7-aminoheptanal as substrate forming N7~acetyl- 1 ,7-diaminoheptane.

EXAMPLE 8

Enzyme activity of airboxylate reductase using pimelate semialdehyde as substrate and forming hepiancdial

The N-terminal His-tagged carboxylate reductase of SEQ ID NO: 6 (see Example 3 and FIG. 8) was assayed using pimelate semialdehyde as substrate. The enzyme activity assay was performed in triplicate in a buffer composed of a final concentration of 50 mM HEPES buffer (pH = 7.5), 2 rnM pimelate semialdehyde, 10 mM Mgi¾, 1 mM ATP and 1 mM NADPH. The enzyme activity assay reaction was initiated by adding purified carboxylate reductase and phospho pantetheine transferase or the empty vector control to the assay buffer containing the pimelate semialdehyde and then incubated at room temperature for 20 min. The consumption of NADPH was monitored by absorbanee at 340 nm. The enzyme only control without pimelate semialdehyde demonstrated low base line consumption of NADPH. See FIG. 9.

The gene product of SEQ ID NO: 6, enhanced by the gene product of sfp from Bacillus subtilis, accepted pimelate semialdehyde as substrate as confirmed against the empty vector control (see FIG. 13) and synthesized heptanedial.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.