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(12) Patent Application Publication (10) Pub. No.: US 2011/0124911 A1 Burk Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2011/0124911 A1 Burk Et Al US 2011 0124911A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0124911 A1 Burk et al. (43) Pub. Date: May 26, 2011 (54) SEM-SYNTHETICTEREPHTHALIC ACID (57) ABSTRACT VLAMCROORGANISMIS THAT PRODUCE The invention provides a non-naturally occurring microbial MUCONCACID organism having a muconate pathway having at least one exogenous nucleic acid encoding a muconate pathway (76) Inventors: Mark J. Burk, San Diego, CA enzyme expressed in a sufficient amount to produce mucon (US); Robin E. Osterhout, San ate. The muconate pathway including an enzyme selected Diego, CA (US); Jun Sun, San from the group consisting of a beta-ketothiolase, a beta-ke Diego, CA (US) toadipyl-CoA hydrolase, a beta-ketoadipyl-CoA transferase, a beta-ketoadipyl-CoA ligase, a 2-fumarylacetate reductase, (21) Appl. No.: 12/851,478 a 2-fumarylacetate dehydrogenase, a trans-3-hydroxy-4-hex endioate dehydratase, a 2-fumarylacetate aminotransferase, a (22) Filed: Aug. 5, 2010 2-fumarylacetate aminating oxidoreductase, a trans-3- amino-4-hexenoate deaminase, a beta-ketoadipate enol-lac Related U.S. Application Data tone hydrolase, a muconolactone isomerase, a muconate cycloisomerase, a beta-ketoadipyl-CoA dehydrogenase, a (60) Provisional application No. 61/231,637, filed on Aug. 3-hydroxyadipyl-CoA dehydratase, a 2.3-dehydroadipyl 5, 2009. CoA transferase, a 2.3-dehydroadipyl-CoA hydrolase, a 2.3- dehydroadipyl-CoA ligase, a muconate reductase, a 2-maley Publication Classification lacetate reductase, a 2-maleylacetate dehydrogenase, a cis-3- hydroxy-4-hexendioate dehydratase, a 2-maleylacetate (51) Int. C. aminoatransferase, a 2-maleylacetate aminating oxidoreduc CD7C 63/26 (2006.01) tase, a cis-3-amino-4-hexendioate deaminase, and a mucon CI2N I/2 (2006.01) ate cis/trans isomerase. Other muconate pathway enzymes CI2P 7/44 (2006.01) also are provided. Additionally provided are methods of pro (52) U.S. Cl. ..................... 562/480:435/252.33: 435/142 ducing muconate. Patent Application Publication May 26, 2011 Sheet 1 of 6 US 2011/O124911A1 O O 2 s S o D al O 1 O n O O O a. Vo O O 92 L t c5 5.2 $2J. : -- C - O 2 cs c s > Patent Application Publication US 2011/O124911A1 Patent Application Publication US 2011/O124911A1 US 2011/O124911 A1 May 26, 2011 SEM-SYNTHETICTEREPHTHALIC ACID pathways for producing trans,trans-muconate and cis, trans VLAMCROORGANISMIS THAT PRODUCE muconate would be useful because these isomers can serve as MUCONCACID direct synthetic intermediates for producing renewable PTA via the inverse electron demand Diels-Alder reaction with acetylene. The present invention satisfies this need and pro 0001. This application claims the benefit of priority of vides related advantages as well. U.S. Provisional Application No. 61/231,637, filed Aug. 5, 2009, the entire contents of which are incorporated herein by this reference. SUMMARY OF INVENTION BACKGROUND OF THE INVENTION 0008. The invention provides a non-naturally occurring microbial organism having a muconate pathway having at 0002 The present disclosure relates generally to the least one exogenous nucleic acid encoding a muconate path design of engineered organisms and, more specifically to way enzyme expressed in a sufficient amount to produce organisms having selected genotypes for the production of muconate. The muconate pathway including an enzyme muconic acid. selected from the group consisting of a beta-ketothiolase, a 0003 Terephthalate (also known as terephthalic acid and beta-ketoadipyl-CoA hydrolase, a beta-ketoadipyl-CoA PTA) is the immediate precursor of polyethylene terephtha transferase, a beta-ketoadipyl-CoA ligase, a 2-fumarylacetate late (PET), used to make clothing, resins, plastic bottles and reductase, a 2-fumarylacetate dehydrogenase, a trans-3-hy even as a poultry feed additive. Nearly all PTA is produced droxy-4-hexendioate dehydratase, a 2-fumarylacetate ami from para-Xylene by the oxidation in airina process known as notransferase, a 2-fumarylacetate aminating oxidoreductase, the Mid Century Process (Roffia et al., Ind. Eng. Chem. Prod. a trans-3-amino-4-hexenoate deaminase, a beta-ketoadipate Res. Dev. 23:629-634 (1984)). This oxidation is conducted at enol-lactone hydrolase, a muconolactone isomerase, a high temperature in an acetic acid solvent with a catalyst muconate cycloisomerase, a beta-ketoadipyl-CoA dehydro composed of cobalt and/or manganese salts. Para-Xylene is genase, a 3-hydroxyadipyl-CoA dehydratase, a 2.3-dehy derived from petrochemical sources, and is formed by high droadipyl-CoA transferase, a 2,3-dehydroadipyl-CoA hydro severity catalytic reforming of naphtha. Xylene is also lase, a 2.3-dehydroadipyl-CoA ligase, a muconate reductase, obtained from the pyrolysis gasoline stream in a naphtha a 2-maleylacetate reductase, a 2-maleylacetate dehydroge steam cracker and by toluene disproportion. nase, a cis-3-hydroxy-4-hexendioate dehydratase, a 2-maley 0004 PTA, toluene and other aromatic precursors are lacetate aminoatransferase, a 2-maleylacetate aminating oxi naturally degraded by some bacteria. However, these degra doreductase, a cis-3-amino-4-hexendioate deaminase, and a dation pathways typically involve monooxygenases that muconate cis/trans isomerase. Other muconate pathway operate irreversibly in the degradative direction. Hence, bio enzymes also are provided. Additionally provided are meth synthetic pathways for PTA are severely limited by the prop ods of producing muconate. erties of known enzymes to date. 0005 Muconate (also known as muconic acid, MA) is an unsaturated dicarboxylic acid used as a raw material for res BRIEF DESCRIPTION OF THE DRAWINGS ins, pharmaceuticals and agrochemicals. Muconate can be converted to adipic acid, a precursor of Nylon-6,6, via hydro 0009 FIG. 1 shows the synthesis of terepthalate from genation (Draths and Frost, J. Am. Chem. Soc. 116:399-400 muconate and acetylene via Diels-Alder chemistry. P1 is (1994)). Alternately, muconate can be hydrogenated using cyclohexa-2,5-diene-1,4-dicarboxylate. biometallic nanocatalysts (Thomas et al., Chem. Commun. 0010 FIG. 2 shows pathways to trans-trans muconate 10:1126-1127 (2003)). from succinyl CoA. Enzymes are A) beta-ketothiolase, B) 0006 Muconate is a common degradation product of beta-ketoadipyl-CoA hydrolase, transferase and/or ligase, C) diverse aromatic compounds in microbes. Several biocata 2-fumarylacetate reductase, D) 2-fumarylacetate dehydroge lytic strategies for making cis,cis-muconate have been devel nase, E) trans-3-hydroxy-4-hexendioate dehydratase, F) oped. Engineered E. coli strains producing muconate from 2-fumarylacetate aminotransferase and/or 2-fumarylacetate glucose via shikimate pathway enzymes have been developed aminating oxidoreductase, G) trans-3-amino-4-hexenoate in the Frost lab (U.S. Pat. No. 5,487.987 (1996): Niu et al., deaminase, H) beta-ketoadipate enol-lactone hydrolase, I) Biotechnol Prog., 18:201-211 (2002)). These strains are able muconolactone isomerase, J) muconate cycloisomerase, K) to produce 36.8 g/L of cis,cis-muconate after 48 hours of beta-ketoadipyl-CoA dehydrogenase, L) 3-hydroxyadipyl culturing under fed-batch fermenter conditions (22% of the CoA dehydratase, M) 2.3-dehydroadipyl-CoA transferase, maximum theoretical yield from glucose). Muconate has also hydrolase or ligase, N) muconate reductase, O) 2-maleylac been produced biocatalytically from aromatic starting mate etate reductase, P) 2-maleylacetate dehydrogenase, Q) cis-3- rials such as toluene, benzoic acid and catechol. Strains pro hydroxy-4-hexendioate dehydratase, R) 2-maleylacetate ducing muconate from benzoate achieved titers of 13.5 g/L aminotransferase and/or 2-maleylacetate aminating oxi and productivity of 5.5 g/L/hr (Choi et al., J. Ferment. Bioeng. doreductase, S) cis-3-amino-4-hexendioate deaminase, T) 84:70-76 (1997)). Muconate has also been generated from the muconate cis/trans isomerase, W) muconate cis/trans effluents of a styrene monomer production plant (Wu et al., isomerase. Enzyme and Microbiology Technology 35:598-604 (2004)). 0011 FIG. 3 shows pathways to muconate from pyruvate 0007 All biocatalytic pathways identified to date proceed and malonate semialdehyde. Enzymes are A) 4-hydroxy-2- through enzymes in the shikimate pathway, or degradation ketovalerate aldolase, B) 2-oxopentenoate hydratase, C) enzymes from catechol. Consequently, they are limited to 4-oxalocrotonate dehydrogenase, D) 2-hydroxy-4-hexene producing the cis, cis isomer of muconate, since these path dioate dehydratase, E) 4-hydroxy-2-oxohexanedioate oxi ways involve ring-opening chemistry. The development of doreductase, F) 2,4-dihydroxyadipate dehydratase (acting on US 2011/O124911 A1 May 26, 2011 2-hydroxy), G) 2,4-dihydroxyadipate dehydratase (acting on 0019. As used herein, the term “non-naturally occurring 4-hydroxyl group) and H) 3-hydroxy-4-hexenedioate dehy when used in reference to a microbial organism or microor dratase. ganism of the invention is intended to mean that the microbial 0012 FIG. 4 shows pathways to muconate from pyruvate organism has at least one genetic alteration not normally and succinic semialdehyde. Enzymes are A) HODHaldolase, foundina naturally occurring Strain of the referenced species, B) OHED hydratase, C) OHED decarboxylase, D) HODH including wild-type strains of the referenced species. Genetic formate-lyase and/or HODH dehydrogenase, E) OHED for alterations include, for example, modifications introducing mate-lyase and/or OHED dehydrogenase, F) 6-OHE dehy expressible nucleic acids encoding metabolic polypeptides,
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