(19) TZZ __T (11) EP 2 821 494 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 07.01.2015 Bulletin 2015/02 C12P 1/00 (2006.01) C12P 7/18 (2006.01) C12P 7/42 (2006.01) C12P 7/52 (2006.01) (2006.01) (2006.01) (21) Application number: 14178905.7 C12P 17/04 C12N 9/00 C12N 9/02 (2006.01) C12N 9/04 (2006.01) (2006.01) (22) Date of filing: 14.03.2008 C12N 9/88 (84) Designated Contracting States: • Van Dien, Stephen J. AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Encinitas, CA 92024 (US) HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT • Burgard, Anthony RO SE SI SK TR Bellefonte, PA 16825 (US) • Niu, Wei (30) Priority: 16.03.2007 US 918463 P Lincoln, NE 68516 (US) (62) Document number(s) of the earlier application(s) in (74) Representative: Hoffmann Eitle accordance with Art. 76 EPC: Patent- und Rechtsanwälte PartmbB 08732315.0 / 2 137 315 Arabellastraße 30 81925 München (DE) (71) Applicant: Genomatica, Inc. San Diego, CA 92121 (US) Remarks: •This application was filed on 29-07-2014 as a (72) Inventors: divisional application to the application mentioned • Burk, Mark J. under INID code 62. San Diego, CA 92130 (US) •Claims filed after the date of receipt of the divisional application (Rule 68(4) EPC.). (54) Compositions and methods for the biosynthesis of 1,4-butanediol and its precursors (57) The invention provides a non- naturally occurring thetic pathway including at least one exogenous nucleic microbial biocatalyst including a microbial organism hav- acid encoding 4- hydroxybutanoate dehydrogenase, suc- ing a 4-hydroxybutanoic acid (4-HB) biosynthetic path- cinyl-CoA synthetase, CoA-dependent succinic semial- way having at least one exogenous nucleic acid encoding dehyde dehydrogenase or α-ketoglutarate decarboxyla- 4-hydroxybutanoate dehydrogenase, succinyl-CoA syn- se under substantially anaerobic conditions for a suffi- thetase, CoA-dependent succinic semialdehyde dehy- cient period of time to produce monomeric 4-hydroxyb- drogenase, or α-ketoglutarate decarboxylase, wherein utanoic acid (4- HB). Further provided is a method for the the exogenous nucleic acid is expressed in sufficient production of BDO. The method includes culturing a non- amounts to produce monomeric 4- hydroxybutanoic acid naturally occurring microbial biocatalyst, comprising a (4-HB). Also provided is a non- naturally occurring micro- microbial organism having 4-hydroxybutanoic acid (4- bial biocatalyst including a microbial organism having 4- HB) and 1,4-butanediol (BDO) biosynthetic pathways, hydroxybutanoic acid (4-HB) and 1,4-butanediol (BDO) the pathways including at least one exogenous nucleic biosynthetic pathways, the pathways include at least one acid encoding 4- hydroxybutanoate dehydrogenase, suc- exogenous nucleic acid encoding 4-hydroxybutanoate cinyl-CoA synthetase, CoA-dependent succinic semial- dehydrogenase, succinyl-CoA synthetase, CoA-de- dehyde dehydrogenase, 4-hydroxybutyrate:CoA trans- pendent succinic semialdehyde dehydrogenase, 4-hy- ferase, 4-hydroxybutyrate kinase, phosphotranshy- droxybutyrate:CoA transferase, 4- butyrate kinase, phos- droxybutyrylase, α-ketoglutarate decarboxylase, alde- photransbutyrylase, α-ketoglutarate decarboxylase, al- hyde dehydrogenase, alcohol dehydrogenase or an al- dehyde dehydrogenase, alcohol dehydrogenase or an dehyde/alcohol dehydrogenase for a sufficient period of aldehyde/alcohol dehydrogenase, wherein the exoge- time to produce 1,4-butanediol (BDO). The 4-HB and/or nous nucleic acid is expressed in sufficient amounts to BDO products can be secreted into the culture medium. produce 1,4-butanediol (BDO). Additionally provided is a method for the production of 4-HB. The method in- cludes culturing a non-naturally occurring microbial or- ganism having a 4-hydroxybutanoic acid (4-HB) biosyn- EP 2 821 494 A1 Printed by Jouve, 75001 PARIS (FR) EP 2 821 494 A1 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 tetrahydro- furan (Figure 1). These compounds have varied uses as polymer intermediates, solvents, and additives, and have a 20 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 manufacture of the butanediol family of products (DOE Report, "Top Value-Added Chemicals from Biomass", 2004). 25 However, succinic acid is costly to isolate and purify and requires high temperatures and pressures for catalytic reduction to butanediol. [0005] 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 [0006] The invention 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- 35 toglutarate decarboxylase, wherein the exogenous nucleic acid is expressed in sufficient amounts to produce monomeric 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- 40 transbutyrylase, α-ketoglutarate decarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase or an aldehyde/al- 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 45 semialdehyde dehydrogenase or α-ketoglutarate decarboxylase under substantially anaerobic conditions for a sufficient 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 50 succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:CoA transferase, 4-hydroxybutyrate kinase, phosphotran- 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. 55 BRIEF DESCRIPTION OF THE DRAWINGS [0007] 2 EP 2 821 494 A1 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. 5 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.