US 20150337275A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0337275 A1 Pearlman et al. (43) Pub. Date: Nov. 26, 2015 (54) BOCONVERSION PROCESS FOR Publication Classification PRODUCING NYLON-7, NYLON-7.7 AND POLYESTERS (51) Int. C. CI2N 9/10 (2006.01) (71) Applicant: INVISTATECHNOLOGIES S.a.r.l., CI2P 7/40 (2006.01) St. Gallen (CH) CI2PI3/00 (2006.01) CI2PI3/04 (2006.01) (72) Inventors: Paul S. Pearlman, Thornton, PA (US); CI2P 13/02 (2006.01) Changlin Chen, Cleveland (GB); CI2N 9/16 (2006.01) Adriana L. Botes, Cleveland (GB); Alex CI2N 9/02 (2006.01) Van Eck Conradie, Cleveland (GB); CI2N 9/00 (2006.01) Benjamin D. Herzog, Wichita, KS (US) CI2P 7/44 (2006.01) CI2P I 7/10 (2006.01) (73) Assignee: INVISTATECHNOLOGIES S.a.r.l., (52) U.S. C. St. Gallen (CH) CPC. CI2N 9/13 (2013.01); C12P 7/44 (2013.01); CI2P 7/40 (2013.01); CI2P 13/005 (2013.01); (21) Appl. No.: 14/367,484 CI2P 17/10 (2013.01); CI2P 13/02 (2013.01); CI2N 9/16 (2013.01); CI2N 9/0008 (2013.01); (22) PCT Fled: Dec. 21, 2012 CI2N 9/93 (2013.01); CI2P I3/04 (2013.01); PCT NO.: PCT/US2012/071.472 CI2P 13/001 (2013.01); C12Y 102/0105 (86) (2013.01) S371 (c)(1), (2) Date: Jun. 20, 2014 (57) ABSTRACT Embodiments of the present invention relate to methods for Related U.S. Application Data the biosynthesis of di- or trifunctional C7 alkanes in the (60) Provisional application No. 61/578.265, filed on Dec. presence of isolated enzymes or in the presence of a recom 21, 2011, provisional application No. 61/578,272, binant host cell expressing those enzymes. The di- or trifunc filed on Dec. 21, 2011, provisional application No. tional C7 alkanes are useful as intermediates in the produc 61/578,289, filed on Dec. 21, 2011. tion of nylon-7, nylon-7.X. nylon-X.7, and polyesters. Patent Application Publication Nov. 26, 2015 Sheet 1 of 42 US 2015/0337275 A1 F.G. Ho-oh HON-N-N-N-OH Pinelic acid G 1,7-heptanedio carboxylic acid reductase Sphingomonas 4. aidehyde DH falcohol DH (CAR) EC 1.2.99.- macrogofitabida EC ...- alcoho H EC 1.1.1 - --- Ho-ohO Pimelic acid semialdehyde 7-hydroxyheptanoate (7-oxoheptanoate) co-transaminase EC 2.6.1- Amidohydrolase EC 3.52 Hos--~~ -e O C. 7-aminoheptanoic acid eantholactam Aldehyde DH EC 111 acchio H ~-NH. O 7-aminoheptanal 7-aminoheptanol ()-transaminase EC 261 heptamethylenediamine (1,7-diaminoheptane) Patent Application Publication Nov. 26, 2015 Sheet 2 of 42 US 2015/0337275 A1 FG, 2. Biotin biosynthesis pathway Benzoate degradation pathway Cyclohexane carboxylate pathway Biotin biosynthesis pathway Condensation of 3 X malonyl-CoA - (reaction not assigned to pathway) - Hos---sco Hor-lap O O O O fatA fat ThioesterPimeloy-CoA hydrolase tigase (EC(EC 3.1.2.-)6.2.1.-) %t. CoA transferase (ECW2.83.-) H or-no O O Acyl-(acyl carrier protein) Acyl-CoA reductase Pima lic acid reduct eductase EC 2.50 afooxylic acid reductase VE: 1299.- EC 2.18O G Horn-inO O Pimelic acid semiaidehyde Tetratin degradation pathway CO2 Ec: 4.1.1 - o-keto acid decarboxylase O H* O OH HO (.-Ketosuberate4. O Coenzyme B pathway (Methanogenic archae) Patent Application Publication Nov. 26, 2015 Sheet 3 of 42 US 2015/0337275 A1 FG, 3 Ho-roO O O-ketoguterateO Acetyl-CoA, H2O EC 2.3.3.14 (akSA) EC 4.2.1.114 (aksDiaksE) or EC 4.2.1.36 CoA EC 4.2.1.114 (aksDiaksE) CO2 EC 1.1.1.87 (aksF) O O to-oh -X // > Lysine O-ketoadipate O Acetyl-CoA, H2O EC 2.3.3.14 (akSA) EC 4.2.1.114 (aksDfaksE) or EC 4.2.1.36 CoA EC 4.2.1.114 (aks faksE) CO2 EC 1.1.1.87 (aksF) Ho-n-rotO O O O-ketopimelate Acetyl-CoA, H2O EC 2.3.3.14 (akSA) Coenzyme B EC 4.2.1.114 (aksDfaksE) or EC 4.2.1.36 COA EC 4.2.1.114 (aksOfaksE) CO2 EC 1.1.1.87 (aksF) O O OH O -- CO HO O O-ketoSuberate O H 2 pimetic acid semialdehyde (7-oxoheptanoate) Change: 4.1.3- to 2.3.3.14 (homocitrate synthase) AksD/AksE can be 4.2.1.114 (R)-homocitrate yase only or 4.2.1.114 + 4.2.1.36 (homoisocitrate hydro-iyase AksF hono-isocitrate dehydrogenase 1.1.1.87 Patent Application Publication Nov. 26, 2015 Sheet 4 of 42 US 2015/0337275 A1 FG. 4 O O condensation O O O o'-'s-coa os---is-ca Malonyl-CoA O O 3-oxoglutaryl-CoA 1sul CO2 oxo group elimination O S-CoA CoA Malonyl-CoA O O Condensation & ----- OXO group elimination O O HO S-CoA o'-'s-coa Pimeloyl-CoA Glutaryl-CoA CO2 CoA Malonyl-CoA Patent Application Publication Nov. 26, 2015 Sheet 5 of 42 US 2015/0337275 A1 F.G. S PA5OBO HO J--- S-acp (CYP107H1)s R S-acp Pimeloy-acp (CH2)CH3 long-chain faty acid-acp O y O Ho-oh Pineic acid ATP, CoA BioW EC 62.4 AMP, PP, L-Aia CO2 O O H CoA HO --~ul S-COA “HN O O Pimeoyl-CoA BioF OH EC 2.34f BioC 7-keto-8-aminopelagonate BioH BioGiEOKFBOY 2 Biotin Patent Application Publication Nov. 26, 2015 Sheet 6 of 42 US 2015/0337275 A1 F.G. 6 EC 2.1.19 EC 2.3.18O O O BioC O O FabH O. O. O. -e -> HO S-COA so-sco O acp Madon yW-COA Maory-CoAy newestery 3-OXO-Gutaw-acol9 y- plgety methwester is, EC 1.3.1.10 EC 4.2.1.59 Fag Fabi FaZ Yo is acp Yo usicS. acp - Yo if acp glutary-acp methyl ester enoylglutary-acp methyl ester 3-hydroxy-glutaryl-acp methyl ester EC 2.3141 FabB EC 1.1.1.100 EC 4.289 O O O FacG O CH C. Faby O O YO acp) YO ---sus aCp Yo ---s-s acp 3-oxopimelyl-acp methyester 3-hydroxypimely-acp) methyl ester enoylpinely-acp methyl ester EC 3.110 EC 2.3.47 EC 3.185 Fabi O O BigF O O BioH O O a "HN OH XHO 1s-s-s-s acp Sas-s-s-sO acp 7-keto-8-aminopelargonate pinely-acp pimelyl-acp methyl ester EC 3.12.4 aaaS Fata, FatB Lipase or Biotin O O esterase O O HO ----- OH N O --~us OH pimelic acid pimelate monomethyl ester EC 2.180 O Acyl-acp reductase -----> HO SO Pimelic acid semialdehyde Patent Application Publication Nov. 26, 2015 Sheet 7 of 42 US 2015/0337275 A1 F.G. 7 coO Benzoate EC 6.2.1 2. C’soO O OH Benzoyl-CoA COA-S Cyclohexane carboxylate O O 2-ketocyclohexane-1-carboxyl-CoA H D. 2-ketocyclohexanecarboxyCoA hydrolase (Rp-bad) O O g O Ho-r-r-sca -X-> Ho-sca Pimeloyl-CoA B-Oxidation Glutary-CoA Patent Application Publication Nov. 26, 2015 Sheet 8 of 42 US 2015/0337275 A1 FG, 8 -/- CO -t, - O OH OH OH Naphtalene Tetrain O (benzocyclohexane) 2,4-dihydrodec-2-ene-1,10-dioic acid O OH h X-K O HC3 O HO --~sSO Puruvate Pimelic acid semialdehyde ThG | Ho-n-n-oh Pinetic acid Patent Application Publication Nov. 26, 2015 Sheet 9 of 42 US 2015/0337275 A1 FEG, 9 O O O O O sy- os---sca O SCoA O OH O O O n’ 2H) O ---sul SCOA Crotorate -s-so O ls-s-sS-1 scoA N-H2O APCOA r -CO2 O O O O crotonyl-CoA -> "sca -s-s SCCA 2H ho Enoyl-CoA O r O 3-hydroxy- OH O hydrataseEC 4.2.1.17 to- O ----- SCOA butyryl-CoA -lul OH. O. Pimeoyl-CoA SCOA --scoA. /y 2H 3-hydroxybutyryl- 2H EC 1157 t aceto- O O O O acetyl-CoA Y CoA--sca O --sca O OO O - sco O-. acetyl-CoAy 1 sco 1sca EC 239COA 'year w- carboxylate N- COASH ATP O Patent Application Publication Nov. 26, 2015 Sheet 10 of 42 US 2015/0337275 A1 F.G. 10A Diaminopimelate NH2 NH2 decarboxylase Ho-oh 6 Lysine O O meso-2,6-diaminopimelate Ammonia iyase NH Ho-s-s-to O O - - - - - - - - - - - - - - - - 6-amino-2-heptenedioic acid Enoate Reductase y - - - - - - - - - - - - - - - NH , Annonia Ho-oh - - - - - - - - A ------- lyase O O O K s t O 3 di p 3.t e 2-amino-heptanedioic acid - - - ---------------- Amino goes O as O HO OH ~~~ HO OH AkSA HO -oh O O -o- HO s 2-heptenedioic acid Oo-ketopimel ate? O CoA transferase - - - Hydro-lyase Carbonyl reductase HO YO acid-thiol ligase OH trihomocitrate Hos---is-CoAs, Ho-oh O t AKSD/AKSE O O O d O 2-heptenedioic acid-CoA c-hydroxypimelate HO 2 Enoate Enoate , OH Reductase Reductase, HO. O. Enoyl-CoA , Cis-(homo)3-aconitate dehydrogenas w t Aks), AkSE HO S-CoA HO OH -e- O O O O O Pimeloyl-CoA hioesterase Pineic acid HO OH HO YO Fatty-acyl-CoA Carboxylic acid (-)-threo-iso(homo)scitrate reductase reductase AkSF Decarboxylase O HO 2O -- HO O O O Pimelic acid semialdehyde O-ketosuberate Amino transferase Anino tances O Decarboxylase HO Hos----NH. se- r-r-no O O NH2 7-aminoheptanoic acid C-aminOSuberate Patent Application Publication Nov. 26, 2015 Sheet 11 of 42 US 2015/0337275 A1 FG, 10B CoA transferase OH acid-thiol gase CH Ho-oh - - Ho----> S-CoA O O O O CoA-SH O-hydroxypimelate C O-hydroxypimeoyl-CoA Hydro-lyase Hydro-lyase CoA transferase Ho-oh acid-thioi Eigase Hos---sca O O 7 O O 2-heptenedioic acid COA-SH 2-heptenedioic acid-CoA Enoate Enoate Reductase Reductase COA transferase Enoyl-CoA dehydrogenase acid-thiol ligase or Ho-oh hioesterase Hos---is-CoA O O O O Pinneic acid CoA-SH Pimeoyl-CoA Fig.