# US 2010/0168481 A1 · Green process and compositions for producing poly(5hv) and 5 carbon chemicals > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2010/0168481 A1 - Document ID: US20100168481A1 - Status: Published application - Kind: A1 - Application number: 12/637,706 - Filed: 2009-12-14 - Published: 2010-07-01 - Earliest claimed: 2008-12-12 - Assignees: Metabolix, Inc. - Inventors: William R. Farmer; Jeff Bickmeier; Chenfeng Lu; Dong-Eun Chang; Frank Skraly; Thomas Martin Ramseier - Web page: https://patentgrep.com/patent/US20100168481A1 ## CPC - C12N 15/52: Genes encoding for enzymes or proenzymes - C12N 9/0006: {acting on CH-OH groups as donors (1.1)} - C12N 9/0008: {acting on the aldehyde or oxo group of donors (1.2)} - C12N 9/0069: {acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)} - C12N 9/10: Transferases (2.) (ribonucleases C12N9/22) - C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)} - C12N 9/1096: {transferring nitrogenous groups (2.6)} - C12N 9/80: acting on amide bonds in linear amides {(3.5.1)} - C12N 9/88: Lyases (4.) - C12N 9/93: {Ligases (6)} - C12P 7/04: acyclic - C12P 7/18: polyhydric - C12P 7/42: Hydroxy-carboxylic acids - C12P 7/44: Polycarboxylic acids - C12P 7/625: Polyesters of hydroxy carboxylic acids - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} ## Abstract Recombinant hosts for producing polyhydroxyalkanoates and methods of producing polyhydroxyalkanoates from renewable carbon substrates are provided. Certain recombinant hosts that produce 5 carbon chemicals such as 5-aminopentanoate (5AP), 5-hydroxyvalerate (5HV), glutarate, and 1,5 pentanediol (PDO) are also provided. One embodiment provides a recombinant host expressing a gene encoding a heterologous enzyme selected from the group consisting of a polyhydroxyalkanoate synthase and a 5-hydroxyvalerate-CoA (5HV-CoA) transferase, wherein the host produces a polymer containing 5-hydroxyvalerate. Preferably, the host expresses both a polyhydroxyalkanoate synthase and a 5HV-CoA transferase. The host can be prokaryotic or eukaryotic. A preferred prokaryotic host is E. coli. The polymers produced by the recombinant hosts can be homopolymers or copolymers of 5-hydroxyvalerate. A preferred copolymer is poly(3-hydroxybutyrate-co-5-hydroxyvalerate). ## Claims ### Claim 1 A recombinant organism genetically engineered to convert glutarate semialdehyde or 5-aminopentanoate into a 5 carbon monomer, polymer or copolymer thereof, wherein the recombinant organism expresses at least one or more gene(s) encoding one or more enzymes selected from the group consisting of: lysine 2-monooxygenase, EC 1.13.12.2; 5-aminopentanamidase (δ-aminovaleramidase), EC 3.5.1.30; 5-aminopentanoate transaminase (δ-aminovalerate transaminase), EC 2.6.1.48; lysine decarboxylase, EC 4.1.1.18; succinate semialdehyde reductase (a.k.a. glutarate semialdehyde reductase), EC 1.1.1.61; CoA-transferase, EC 2.8.3.14 and EC 2.8.3.n; Acyl-CoA synthetase, EC 6.2.1.3; PHA synthase, EC 2.3.1.n; β-ketoacyl-CoA thiolase, EC 2.3.1.9; acetoacetyl-CoA reductase, EC 1.1.1.36; glutarate-semialdehyde dehydrogenase, EC 1.2.1.20; propionaldehyde dehydrogenase, EC 1.2.1.3; alcohol dehydrogenase, EC 1.1.1.1; 1,3-propanediol dehydrogenase EC 1.1.1.202 and wherein the recombinant organism produces more glutarate semialdehyde or 5-aminopentanoate than an unmodified organism and wherein the glutarate semialdehyde or 5-aminopentanoate is converted into the 5 carbon monomer, polymer or copolymer thereof by the recombinant organism. ### Claim 2 (depends on 1) The recombinant organism of claim 1 wherein the 5 carbon monomer is selected from the group consisting of glutarate, 1,5 pentanediol, 5-aminovalerate and 5-hydroxyvalerate. ### Claim 3 (depends on 1) The recombinant organism of claim 1 wherein the polymer or copolymer comprises polyhydroxyalkanoate. ### Claim 4 (depends on 1) The recombinant organism of claim 1 wherein the monomer comprises 5-hydroxyvalerate. ### Claim 5 (depends on 1) The recombinant organism of claim 1 wherein the recombinant organism converts 5-aminopentanoate into glutarate semialdehyde. ### Claim 6 (depends on 1) The recombinant organism of claim 1 wherein the recombinant organism converts lysine into 5-aminopentanoate. ### Claim 7 (depends on 6) The recombinant organism of claim 6 wherein the lysine is fed to the organism. ### Claim 8 (depends on 1) The recombinant organism of claim 1 wherein the organism used to construct the recombinant organism has been modified to overproduce lysine relative to an unmodified organism. ### Claim 9 (depends on 1) The recombinant organism of claim 1 wherein the recombinant organism is resistant to the toxic lysine analog S-(2-aminoethyl) cysteine. ### Claim 10 (depends on 1) The recombinant organism of claim 1 wherein the recombinant organism expresses a lysine feedback-resistant dihydrodipicolinate synthase. ### Claim 11 (depends on 1) The recombinant organism of claim 1 wherein the recombinant organism expresses a lysine feedback-resistant aspartate kinase III. ### Claim 12 (depends on 1) The recombinant organism o of claim 1 wherein the organism is fed a renewable carbon substrate. ### Claim 13 (depends on 1) The recombinant organism of claim 1 wherein the recombinant organism is further engineered to inhibit or block lysine export. ### Claim 14 (depends on 1) The recombinant organism of claim 1 wherein the recombinant organism expresses or overexpresses glutarate semialdehyde dehydrogenase and the 5 carbon monomer is glutarate. ### Claim 15 (depends on 1) The recombinant organism of claim 1 wherein the glutarate semialdehyde dehydrogenase is encoded by a gene selected from the group consisting of davD, yneI, and gabD. ### Claim 16 (depends on 1) The recombinant organism of claim 1 wherein the recombinant organism releases glutarate into the extracellular environment. ### Claim 17 (depends on 1) The recombinant organism of claim 1 wherein the organism has been modified to reduce or eliminate glutarate semialdehyde dehydrogenase activity and the 5 carbon monomer is not glutarate. ### Claim 18 (depends on 17) The recombinant organism of claim 17 wherein the glutarate semialdehyde dehydrogenase is reduced or eliminated by deleting or disrupting one or more genes selected from the group consisting of davD, yneI, and gabD or their homologs. ### Claim 19 (depends on 1) The recombinant organism o of claim 1 wherein the recombinant organism releases 5-hydroxyvalerate into the extracellular environment. ### Claim 20 (depends on 17) The recombinant organism of claim 17 wherein the recombinant organism releases 5-hydroxyvalerate in the extracellular environment and the 5-hydroxyvalerate is in equilibrium with delta-valerolactone. ### Claim 21 (depends on 1) The recombinant organism of any of claim 1 wherein the recombinant organism converts 5-hydroxyvalerate into 5-hydroxyvalerate CoA. ### Claim 22 (depends on 1) The recombinant organism of claim 1 wherein the recombinant organism converts 5-hydroxyvalerate-CoA into a polyhydroxyalkanoate. ### Claim 23 (depends on 1) The recombinant organism of claim 1 wherein the recombinant organism converts 5-hydroxyalerate into 1,5 pentanedial. ### Claim 24 (depends on 1) The recombinant organism of claim 1 wherein the recombinant organism converts 5-hydroxyvalerate into poly(5-hydroxyvalerate) or a copolymer thereof. ### Claim 25 (depends on 24) The recombinant organism of claim 24 wherein the copolymer is selected from the group consisting of poly(3-hydroxypropionate-co-5HV), poly(3-hydroxybutyrate-co-5HV) and poly(4-hydroxybutyrate-co-5HV) ### Claim 26 (depends on 1) The recombinant organism of claim 1 wherein the recombinant organism is prokaryotic. ### Claim 27 (depends on 1) The recombinant organism of claim 1 wherein the recombinant organism is E. coli. ### Claim 28 (depends on 1) The recombinant organism of claim 1 wherein the recombinant organism is a eukaryotic microorganism. ### Claim 29 A recombinant organism for producing polymers from lysine wherein the recombinant organism is genetically engineered to express at least one enzyme selected from the group consisting of lysine 2-monooxygenase, EC 1.13.12.2; 5-aminopentanamidase (δ-aminovaleramidase), EC 3.5.1.30; 5-aminopentanoate transaminase (δ-aminovalerate transaminase), EC 2.6.1.48; lysine decarboxylase, EC 4.1.1.18; succinate semialdehyde reductase (a.k.a. glutarate semialdehyde reductase), EC 1.1.1.61; CoA-transferase, EC 2.8.3.14 and EC 2.8.3.n; Acyl-CoA synthetase, EC 6.2.1.3; PHA synthase, EC 2.3.1.n to produce a polyhydroxyalkanoate comprising 5-hydroxyvalerate monomers. ### Claim 30 (depends on 29) The recombinant organism of claim 29 wherein the recombinant organism does not produce lysine. ### Claim 31 (depends on 29) The recombinant organism of claim 29 wherein the recombinant organism does not express a functional glutarate semialdehyde dehydrogenase enzyme activity. ### Claim 32 A recombinant organism for producing 1,5-pentanediol from lysine wherein the recombinant organism is genetically engineered to express at least one enzyme selected from the group consisting of lysine 2-monooxygenase, EC 1.13.12.2; 5-aminopentanamidase (δ-aminovaleramidase), EC 3.5.1.30; 5-aminopentanoate transaminase (δ-aminovalerate transaminase), EC 2.6.1.48; lysine decarboxylase, EC 4.1.1.18; succinate semialdehyde reductase (a.k.a. glutarate semialdehyde reductase), EC 1.1.1.61; CoA-transferase, EC 2.8.3.14 and EC 2.8.3.n; Acyl-CoA synthetase, EC 6.2.1.3, and propionaldehyde dehydrogenase, EC 1.2.1.3; alcohol dehydrogenase, EC 1.1.1.1; 1,3-propanediol dehydrogenase EC 1.1.1.202 ### Claim 33 (depends on 32) The recombinant organism of claim 32 wherein the recombinant organism does not produce lysine. ### Claim 34 (depends on 32) The recombinant organism of claim 32 wherein the recombinant organism does not express a functional glutarate semialdehyde dehydrogenase enzyme activity. ### Claim 35 (depends on 29) A method for producing polymers from lysine comprising feeding the recombinant organism of claim 29 with lysine and other renewable carbon feedstock such that the polymer is produced. ### Claim 36 (depends on 1) A method for producing 5-carbon based monomers, polymers or co-polymers thereof comprising providing lysine or other renewable carbon feedstock to genetically engineered cells according to claim 1, wherein the genetically engineered cells are engineered to produce more glutarate semialdehyde or 5-aminopentanoate than unmodified cells, and wherein the glutarate semialdehyde or 5-aminopentanoate is converted by the genetically engineered cells into a 5 carbon monomer, polymer or copolymer thereof. ### Claim 37 (depends on 36) The method of claim 36 wherein the renewable carbon feedstock is selected from starch, sucrose, glucose, lactose, fructose, xylose, maltose and arabinose, or combinations thereof. ### Claim 38 (depends on 36) The method of claim 36 wherein the monomer is selected from the group consisting of glutarate, 1,5-pentanediol, and 5-hydroxyvalerate. ### Claim 39 (depends on 36) The method of claim 36 wherein the polymer comprises a polyhydroxyalkanoate. ### Claim 40 (depends on 39) The method of claim 39 wherein the polyhydroxyalkanoate comprises 5-hydroxyvalerate. ### Claim 41 (depends on 40) The method of claim 40 wherein the polyhydroxyalkanote is selected from poly(5-hydroxyvalerate), poly(3-hydroxypropionate-co-5HV), poly(3-hydroxybutyrate-co-5HV) and poly(4-hydroxybutyrate-co-5HV), ### Claim 42 (depends on 39) The method of claim 39 further comprising recovering the polyhydroxyalkanote, polymer or copolymer thereof. ### Claim 43 (depends on 42) The method of claim 42 wherein polyhydroxyalkanote polymer or copolymers are recovered by solvent extraction or aqueous processing. ### Claim 44 (depends on 1) A method for overproducing glutarate from renewable carbon substrates is provided where a recombinant organism of claim 1 is fed a renewable carbon substrate selected from lysine, starch, sucrose, glucose, lactose, fructose, xylose, maltose and arabinose, or combinations thereof and glutarate is overproduced, secreted to the medium and recovered therefrom. ### Claim 45 1,5 Pentanediol produced from renewable carbon substrate.