USOO8420375B2 (12) United States Patent (10) Patent No.: US 8.420,375 B2 Osterhout et al. (45) Date of Patent: Apr. 16, 2013 (54) MICROORGANISMS AND METHODS FOR 5,487.987 A 1/1996 Frost et al. CARBON-EFFICIENT BOSYNTHESIS OF 559: A : 3: St. MEK AND 2-BUTANOL 5,573.931 A 11/1996 Guettler et al. 5,616,496 A 4/1997 Frost et al. (75) Inventors: Robin E. Osterhout, San Diego, CA 5,686,276 A 11/1997 Lafendet al. (US); Wei Niu, San Diego, CA (US); 5,700.934. A 12/1997 Wolters et al. Anthony P. Burgard, Bellefonte, PA 5,770.435 A 6/1998 Donnelly et al. (US) 336 A 3-86 SE,ghiem et al. 5,908,924 A 6/1999 Burdette et al. (73) Assignee: Genomatica, Inc., San Diego, CA (US) 5,958,745 A 9/1999 Gruys et al. 6,117,658 A 9, 2000 Dennis et al. (*) Notice: Subject to any disclaimer, the term of this 6,133,014 A 10/2000 Mukouyama et al. patent is extended or adjusted under 35 & S. 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Primary Examiner — Anand Desai (56) Ref Cited Assistant Examiner — Iqbal H Chowdhury eerees e (74) Attorney, Agent, or Firm — Jones Day U.S. PATENT DOCUMENTS (57) ABSTRACT 3,513,209 A 5, 1970 Clement A non-naturally occurring microbial organism has at least one 3,912,586 A 10/1975 Kaneyuki et al. exogenous nucleic acid encoding a MEK pathway enzyme 3,965, 182 A 6, 1976 Worrel expressed in a sufficient amount to produce MEK. The MEK 4,048,196 A 9, 1977 Broecker et al. pathway includes an enzyme selected from an acetoacetyl 4,082,788 A 4, 1978 Mims 4,190.495 A 2f1980 Curtiss CoA dehydrogenase (bifunctional), an acetoacetyl-CoA 4,301,077 A 11, 1981 Pesa et al. aldehyde dehydrogenase, a 3-oxobutyraldehyde reductase, a 4,624,920 A 11, 1986 Inoue 3-oxobutanol dehydratase, an MEK oxidoreductase, a 3-ox 4,652,685 A 3, 1987 Cawse et al. obutyraldehyde aminotransferase, a 4-aminobutan-2-one 4,871,667 A 10, 1989 Imada et al. deaminase, a 2-amino-4-ketopentanoate (AKP) thiolase, an 5,079,143 A 1/1992 Klein et al. AKP aminotransferase, a 2,4-dioxopentanoate decarboxy 5,143,833. A 9, 1992 Datta lase, an AKP deaminase, an acetylacrylate decarboxylase, an 5,143,834 A 9, 1992 Glassner et al. 5,168,055 A 12/1992 Datta et al. AKP decarboxylase, a glutamate dehydrogenase, a 3-oxobu 5,173,429 A 12/1992 Gaddy et al. tyraldehyde oxidoreductase (aminating) and an AKP oxi 5, 182,199 A 1/1993 Hartley doreductase (aminating). A 2-butanol pathway further 5, 192,673 A 3, 1993 Jain et al. includes an MEK reductase. A method for producing MEKor 5,403,721 A 4, 1995 Ward, Jr. et al. 2-butanol includes culturing these organisms under condi 5,413,922 A 5/1995 Matsuyama et al. tions and for a sufficient period of time to produce MEK or 5,416,020 A 5, 1995 Severson et al. 2-butanol. 5,457,040 A 10/1995 Jarry et al. 5,478,952 A 12/1995 Schwartz 26 Claims, 4 Drawing Sheets US 8,420,375 B2 Page 2 U.S. PATENT DOCUMENTS 2007/0292927 A1 12/2007 Donaldson et al. 2008/O13887.0 A1 6/2008 Bramucci et al. S. 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