Patent AU-2013202630-B2 (AU)
Title: Microorganisms and processes for producing terephthalic acid and its salts
Inventor: OSTERHOUT ROBIN E
IPC: C12N15/00
Filed: 2013-01-18
Granted: 2015-10-01
Abstract: (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date W O 2013/109865 A2 25 July 2013 (25.07.2013) W I PO I P CT (51) International Patent Classification: (74) Agents: FRIEBEL, Thomas E. et al.; Jones Day, 222 East C12N 9/02 (2006.01) C12P 7/44 (2006.01) 41st Street, New York, NY 10017-6702 (US). C12N 9/104 (2006.01) C2P 9/00 (2006.01) (81) Designated States (unless otherwise indicated, for every C12N 9/10 (2006.01) C07C 63/04 (2006.01) kind of national protection available): AE, AG, AL, AM, C12N 9/12 (2006.01) C07C 63/26 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, C12N 9/88 (2006.01) C07F 9/109 (2006.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, C12P 7/24 (2006.01) C08G 63/183 (2006.01) DO, DZ, EC, EE, EG, ES, Fl, GB, GD, GE, GH, GM, GT, C12P 7/40 (2006.01) C08L 6 7/02 (2006.01) HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, (21) International Application Number: KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, PCT/US2013/022113 ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, (22) International Filing Date: RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, 18 January 2013 (18.01.2013) TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, (25) Filing Language: English ZM, ZW. (26) Publication Language: English (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (30) Priority Data: GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, 61/589,081 20 January 2012 (20.01.2012) US UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, 61/598,743 14 February 2012 (14.02.2012) US TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, (71) Applicant: GENOMATICA, INC. [US/US]; 10520 Wa- EE, ES, Fl, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, teridge Circle, San Diego, CA 92121 (US). MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, (72) Inventors: OSTERHOUT, Robin E.; 10520 Wateridge ML, MR, NE, SN, TD, TG). Circle, San Diego, CA 92121 (US). BURGARD, Anthony P.; 10520 Wateridge Circle, San Diego, CA 92121 (US). Published: BURK, Mark J.; 10520 Wateridge Circle, San Diego, CA - without international search report and to be republished 92121 (US). upon receipt of that report (Rule 48.2(g)) (54) Title: MICROORGANISMS AND PROCESSES FOR PRODUCING TEREPHTHALIC ACID AND ITS SALTS EryLhrose-4-phosphate 4-PhospheryLhronate 2-xo-3-hydrxy 4- phosphobutanoate - E a Hyaroxy- methyl 2,3,4-Trihydroxy-3-methyi- 2-Acetyi-2,3-dihydroxy 2 -oxo phopho 5- phosphopentanoae 4-phosphobutanoate peota-oLe 2H3M40P FIG. 1 (57) Abstract: The invention provides non-naturally occurring microbial organisms having a (2- hydroxy-3-methyl-4-oxobutoxy) phosphonate (2H3M40P) pathway, p-toluate pathway, and/or terephthalate pathway. The invention additionally provides methods of f4 using such organisms to produce 2H3M40P, p-toluate or terephthalate. Also provided herein are processes for isolating bio-based aromatic carboxylic acid, in particular, p-toluic acid or terephthalic acid, from a culture medium, wherein the processes involve con tacting the culture medium with sufficient carbon dioxide (C02) to lower the pH of the culture medium to produce a precipitate com prised of the aromatic carboxylic acid. WO 2013/109865 PCT/US2013/022113 MICROORGANISMS AND PROCESSES FOR PRODUCING TEREPHTHALIC ACID AND ITS SALTS [0001] This application claims the benefit of priority of United States Provisional application serial No. 61/589,08 1, filed January 20, 2012, and United States Provisional application serial No. 61/598,743, filed February 14, 2012, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD [0002] The present invention relates generally to biosynthetic processes, and more specifically to organisms having (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (2H3M40P), p-toluate or terephthalate biosynthetic capability. Also provided herein are processes for isolating a bio-based aromatic carboxylic acid, for example, p-toluic acid or terephthalic acid, from a culture medium, wherein the processes involve lowering the pH of the culture medium to produce a precipitate comprised of the aromatic carboxylic acid. BACKGROUND OF THE INVENTION [0003] Terephthalate (also known as terephthalic acid and PTA) is the immediate precursor of polyethylene terephthalate (PET), used to make clothing, resins, plastic bottles and even as a poultry feed additive. Nearly all PTA is produced frompara-xylene by oxidation in air in a process known as the Mid Century Process. This oxidation is conducted at high temperature in an acetic acid solvent with a catalyst composed of cobalt and/or manganese salts. Para-xylene is derived from petrochemical sources and is formed by high severity catalytic reforming of naphtha. Xylene is also obtained from the pyrolysis gasoline stream in a naphtha steam cracker and by toluene disproportion. [0004] Cost-effective methods for generating renewable PTA have not yet been developed to date. PTA, toluene and other aromatic precursors are naturally degraded by some bacteria. However, these degradation pathways typically involve monooxygenases that operate irreversibly in the degradative direction. Hence, biosynthetic pathways for PTA are severely limited by the properties of known enzymes to date. [0005] A promising precursor for PTA is p-toluate, also known as p-methylbenzoate. P-Toluate is an intermediate in some industrial processes for the oxidation of p-xylene to PTA. It is also an intermediate for polymer stabilizers, pesticides, light sensitive compounds, animal feed supplements and other organic chemicals. Only slightly soluble WO 2013/109865 2 PCT/US2013/022113 in aqueous solution, p-toluate is a solid at physiological temperatures, with a melting point of 2750 C. Microbial catalysts for synthesizing this compound from sugar feedstocks have not been described to date. [0006] Petrochemical based chemical syntheses for making terephthalic acid are known (see, e.g., U.S. Patent Nos. 2,905,709; 3,023,234; 3,042,717; 3,043,846; 3,064,041; 3,096,366; and 6,441,225). Alternative technologies for the production of terephthalic acid have been made feasible with the advent of molecular recombinant technologies used to modify biosynthetic pathways in microbial organisms. For example, microbial organisms have been described which produce precursors useful for the synthesis of bio-based terephthalic acid. Exemplary indirect semi-synthetic routes of producing bio-based terephthalic acid are described in U.S. Patent Publication No. 2011/0124911 Al. Direct biosynthetic routes, wherein terephthalate is itself biosynthesized in microbial organisms have been described in U.S. Patent Publication No. 2011/0207185 Al. Even with advantages that bio-based production of terephthalic acid offers, improvements and/or additional processes are sought, for example, to improve recovery of terephthalic acid in terms of yields and purity, and to increase efficiency and scalability of the processes including, for instance, reducing the number of manufacturing steps, lowering energy usage, recapturing and recycling materials and reducing environmental discharges. [0007] Thus, there exists a need for alternative methods for effectively producing and isolating commercial quantities of compounds such as p-toluate or terephthalate. The present invention satisfies this need and provides related advantages as well. SUMMARY OF INVENTION [0008] The invention provides non-naturally occurring microbial organisms having a 2H3M40P pathway, p-toluate pathway, and/or terephthalate pathway. The invention additionally provides methods of using such organisms to produce 2H3M40P, p-toluate or terephthalate. [0009] The invention also provides a process for isolating a bio-based aromatic carboxylic acid from a culture medium. In certain embodiments, the process comprises the steps of: (a) culturing a non-naturally occurring microbial organism in a culture medium to produce an aromatic carboxylate anion at a pH sufficient to maintain the aromatic carboxylate anion in soluble form; (b) lowering the pH of the culture medium to produce an aromatic carboxylic acid precipitate from the aromatic carboxylate anion. In certain embodiments, lowering the pH of the culture medium comprises contacting the culture WO 2013/109865 3 PCT/US2013/022113 medium with carbon dioxide (CO2). In certain embodiments, the culture medium is substantially depleted of the aromatic carboxylate anion. In certain embodiments, the process further comprises separating the culture medium from non-soluble materials, for example, cells, cell debris, cellulosic material, feed stock, etc., prior to lowering the pH. In certain embodiments, the process further comprises separating the aromatic carboxylic acid from the culture medium, for example, by centrifugation or membrane filtration, etc. [0010] In certain embodiments of the isolation process, the aromatic carboxylic acid is p-toluic acid. In certain embodiments, the aromatic carboxylic acid is terephthalic acid. [0011] In certain embodiments of the isolation process, a counter ion to the aromatic carboxylate anion in the culture medium is an ammonium, sodium or potassium cation. In certain embodiments, the counter ion is an ammonium cation. [0012] In certain embodiments of the isolation process, the pH sufficient to maintain the aromatic carboxylate anion in soluble form is between about 5.0 - 9.0 pH units. In some embodiments, the pH is about 6.0 to about 8.0 pH units or about 6.2 to about 7.8 pH units. In certain embodiments, the pH sufficient to maintain the aromatic carboxylate anion in soluble form is about 7.0 pH units. In certain embodiments, base is added to the culture medium in the culturing step to maintain the