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Ep 2821494 B1 (19) TZZ __T (11) EP 2 821 494 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12P 1/00 (2006.01) C12P 7/18 (2006.01) 08.03.2017 Bulletin 2017/10 C12P 7/42 (2006.01) C12P 7/52 (2006.01) C12P 17/04 (2006.01) C12N 9/00 (2006.01) (2006.01) (2006.01) (21) Application number: 14178905.7 C12N 9/02 C12N 9/04 C12N 9/88 (2006.01) (22) Date of filing: 14.03.2008 (54) Compositions and methods for the biosynthesis of 1,4-butanediol and its precursors Zusammensetzungen und Verfahren für die Biosynthese von 1,4-Butandiol und dessen Vorläufern Compositions et procédés pour la biosynthèse de 1,4-butanediol et de ses précurseurs (84) Designated Contracting States: WO-A2-2009/031766 KR-A- 20070 096 348 AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT • JEWELL J B ET AL: "BIOCONVERSION OF RO SE SI SK TR PROPIONIC-ACID VALERIC-ACID AND 4 HYDROXYBUTYRIC-ACID INTO THE (30) Priority: 16.03.2007 US 918463 P CORRESPONDING ALCOHOLS BY CLOSTRIDIUM-ACETOBUTYLICUM NRRL-527", (43) Date of publication of application: CURRENT MICROBIOLOGY, vol. 13, no. 4, 1986, 07.01.2015 Bulletin 2015/02 pages 215-220, XP009132372, ISSN: 0343-8651 • "PERP Program, New Report Alert (60) Divisional application: 1,4-Butanediol/THF (02/03-7)", INTERNET 17151965.5 CITATION, 2004, pages 1-5, XP008138255, Retrieved from the Internet: (62) Document number(s) of the earlier application(s) in URL:http://www.chemsystems.com/reports/sea accordance with Art. 76 EPC: rch/docs/abstracts/0203-7-abs.pdf [retrieved on 08732315.0 / 2 137 315 2011-06-22] • SOEHLING BRIGITTE ET AL: "Molecular analysis (73) Proprietor: Genomatica, Inc. of the anaerobic succinate degradation pathway San Diego, CA 92121 (US) in Clostridium kluyveri", JOURNAL OF BACTERIOLOGY, vol. 178, no. 3, 1996, pages (72) Inventors: 871-880, XP002578594, ISSN: 0021-9193 • Burk, Mark J. • VALENTIN H E ET AL: "Production of San Diego, CA 92130 (US) poly(3-hydroxybutyrate-co-4-hydroxybutyrat e) • Van Dien, Stephen J. in recombinant Escherichia coli grown on Encinitas, CA 92024 (US) glucose", JOURNAL OF BIOTECHNOLOGY, • Burgard, Anthony ELSEVIER SCIENCE PUBLISHERS, Bellefonte, PA 16825 (US) AMSTERDAM, NL LNKD- • Niu, Wei DOI:10.1016/S0168-1656(97)00127-2, vol. 58, no. Lincoln, NE 68516 (US) 1, 2 October 1997 (1997-10-02), pages 33-38, XP004126101, ISSN: 0168-1656 (74) Representative: Hoffmann Eitle Patent- und Rechtsanwälte PartmbB Arabellastraße 30 81925 München (DE) (56) References cited: WO-A1-2009/023493 WO-A2-99/14313 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 821 494 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 821 494 B1 • HARRY YIM ET AL: "Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol", NATURE CHEMICAL BIOLOGY, NATURE PUB. GROUP, [Online] vol. 7, no. 7, 22 May 2011 (2011-05-22), pages 445-452, XP002706396, ISSN: 1552-4450, DOI: 10.1038/NCHEMBIO.580 Retrieved from the Internet: URL:http://www.nature.com/nchembio/journal /v7/n7/full/nchembio.580.html> [retrieved on 2011-05-22] 2 EP 2 821 494 B1 Description BACKGROUND OF THE INVENTION 5 [0001] This invention relates generally to in silico design of organisms and, more particularly to organisms having 1,4- butanediol biosynthesis capability. [0002] The compound 4-hydroxybutanoic acid (4-hydroxybutanoate, 4-hydroxybutyrate, 4-HB) is a 4-carbon carboxylic acid that has industrial potential as a building block for various commodity and specialty chemicals. In particular, 4-HB has the potential to serve as a new entry point into the 1,4-butanediol family of chemicals, which includes solvents, 10 resins, polymer precursors, and specialty chemicals. 1,4-Butanediol (BDO) is a polymer intermediate and industrial solvent with a global market of about 3 billion lb/year. BDO is currently produced from petrochemical precursors, primarily acetylene, maleic anhydride, and propylene oxide. [0003] For example, acetylene is reacted with 2 molecules of formaldehyde in the Reppe synthesis reaction (Kroschwitz and Grant, Encyclopedia of Chem. Tech., John Wiley and Sons, Inc., New York (1999)), followed by catalytic hydrogen- 15 ation to form 1,4-butanediol. It has been estimated that 90% of the acetylene produced in the U.S. is consumed for butanediol production. Alternatively, it can be formed by esterification and catalytic hydrogenation of maleic anhydride, which is derived from butane. Downstream, butanediol can be further transformed; for example, by oxidation hto- butyrolactone, which can be further converted to pyrrolidone and N-methyl-pyrrolidone, or hydrogenolysis to tetrahydrofuran (Figure 20 1). These compounds have varied uses as polymer intermediates, solvents, and additives, and have a combined market of nearly 2 billion lb/year. [0004] It is desirable to develop a method for production of these chemicals by alternative means that not only substitute renewable for petroleum-based feedstocks, and also use less energy- and capital-intensive processes. The Department of Energy has proposed 1,4-diacids, and particularly succinic acid, as key biologically-produced intermediates for the 25 manufacture of the butanediol family of products (DOE Report, "Top Value-Added [0005] Chemicals from Biomass", 2004). However, succinic acid is costly to isolate and purify and requires high temperatures and pressures for catalytic reduction to butanediol. [0006] Thus, there exists a need for alternative means for effectively producing commercial quantities of 1,4-butanediol and its chemical precursors. The present invention satisfies this need and provides related advantages as well. 30 SUMMARY OF THE INVENTION [0007] The invention is defined by the subject matter of the claims. [0008] KR 20070096348 discloses the production of 1,4-BDO, converted from 4-HB, in a recombinant microorganism 35 expressing heterologous enzymes. [0009] The description provides a non-naturally occurring microbial biocatalyst including a microbial organism having a 4-hydroxybutanoic acid (4-HB) biosynthetic pathway having at least one exogenous nucleic acid encoding 4-hydroxyb- utanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, or α-ke- toglutarate decarboxylase, wherein the exogenous nucleic acid is expressed in sufficient amounts to produce monomeric 40 4-hydroxybutanoic acid (4-HB). Also provided is a non-naturally occurring microbial biocatalyst including a microbial organism having 4-hydroxybutanoic acid (4-HB) and 1,4-butanediol (BDO) biosynthetic pathways, the pathways include at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA- dependent succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:CoA transferase, 4-butyrate kinase, phospho- transbutyrylase, α-ketoglutarate decarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase or an aldehyde/al- 45 cohol dehydrogenase, wherein the exogenous nucleic acid is expressed in sufficient amounts to produce 1,4-butanediol (BDO). Additionally provided is a method for the production of 4-HB. The method includes culturing a non-naturally occurring microbial organism having a 4-hydroxybutanoic acid (4-HB) biosynthetic pathway including at least one ex- ogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase or α-ketoglutarate decarboxylase under substantially anaerobic conditions for a sufficient 50 period of time to produce monomeric 4-hydroxybutanoic acid (4-HB). Further provided is a method for the production of BDO. The method includes culturing a non-naturally occurring microbial biocatalyst, comprising a microbial organism having 4-hydroxybutanoic acid (4-HB) and 1,4-butanediol (BDO) biosynthetic pathways, the pathways including at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:CoA transferase, 4-hydroxybutyrate kinase, phosphotran- 55 shydroxybutyrylase, α-ketoglutarate decarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase or an alde- hyde/alcohol dehydrogenase for a sufficient period of time to produce 1,4-butanediol (BDO). The 4-HB and/or BDO products can be secreted into the culture medium. 3 EP 2 821 494 B1 BRIEF DESCRIPTION OF THE DRAWINGS [0010] 5 Figure 1 is a schematic diagram showing an entry point of 4-hydroxybutanoic acid (4-HB) into the product pipeline of the 1,4-butanediol (BDO) family of chemicals, and comparison with chemical synthesis routes from petrochemical feedstocks. Solid black arrows show chemical synthesis routes; dashed blue arrows show a biosynthetic route to 4-HB and subsequent conversion steps to BDO family chemicals. 10 Figure 2 is a schematic diagram showing biochemical pathways to 4-hydroxybutyurate (4-HB) and to 1,4-butanediol production. The first 5 steps are endogenous toE. coli, while the remainder can be expressed heterologously. Enzymes catalyzing the biosynthetic reactions are: (1) succinyl-CoA synthetase; (2) CoA-independent succinic semialdehyde dehydrogenase; (3) α-ketoglutarate dehydrogenase;
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