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(12) United States Patent (10) Patent No.: US 9,012,189 B2 Bastian Et Al US0090 12189B2 (12) United States Patent (10) Patent No.: US 9,012,189 B2 Bastian et al. (45) Date of Patent: Apr. 21, 2015 (54) MODIFIED ALCOHOL DEHYDROGENASES “Request for Inter Partes Reexamination of U.S. Patent No. FOR THE PRODUCTION OF FUELS AND 8,158.404 Under 35 U.S.C. S 311 and 37 C.F.R.S 1913.”2583 pages, CHEMICALS U.S. Reexamination Control No. 95/002, 177 (filed Sep. 11, 2012). “Request for Inter Partes Reexamination of U.S. Patent No. (75) Inventors: Sabine Bastian, Pasadena, CA (US); 8,153,415 Under 35 U.S.C. S 311 and 37 C.F.R.S 1913.”3262 pages, Frances Arnold, La Cañada, CA (US); U.S. Reexamination Control No. 95/002,174 (filed Sep. 10, 2012). Peter Meinhold, Denver, CO (US) Overkamp et al., “Metabolic Engineering of Glycerol Production in Saccaromyces cerevisiae," AEM 68(6):2814-2821, American Soci (73) Assignees: Gevo, Inc., Englewood, CO (US); The ety for Microbiology, United States (2002). California Institute of Technology, Van Maris et al., “Directed Evolution of Pyruvate Decarboxylase Pasadena, CA (US) Negative Saccharomyces cerevisiae, Yielding a C-Independent, Glucose-Tolerant, and P-yruvate-Hyperpproducing Yeast. Appl. (*) Notice: Subject to any disclaimer, the term of this Environ. Microbiol. 70(1): 159-166, American Society for Microbiol patent is extended or adjusted under 35 ogy, United States (2004). U.S.C. 154(b) by 597 days. Würdig et al., “Vorkommen, Nachweis und Bestimmung von 2-und 3-methyl-2,3-dihydroxybuttersä und 2-hydroxyglutarav im Wein.” (21) Appl. No.: 13/025,805 Vitis 8:216-230, Bundesanstalt fur Zuechtungsforschung an (22) Filed: Feb. 11, 2011 Kulturpflanzen, Germany (1969). “Order Granting Inter Partes Reexamination of U.S. Patent No. (65) Prior Publication Data 8,133,715.” 35 pages, U.S. Reexamination Control No. 95/002,159 (mailed Dec. 3, 2012). US 2011 FO2O1072 A1 Aug. 18, 2011 “Order Granting Inter Partes Reexamination of U.S. Patent No. Related U.S. Application Data 8,158.404” 20 pages, U.S. Reexamination Control No. 95/002,177 (mailed Dec. 10, 2012). (60) Provisional application No. 61/304,069, filed on Feb. “Order Granting Inter Partes Reexamination of U.S. Patent No. 12, 2010, provisional application No. 61/308,568, 8,153,415.” 38 pages, U.S. Reexamination Control No. 95/002,174 filed on Feb. 26, 2010, provisional application No. (mailed Dec. 7, 2012). 61/282,641, filed on Mar. 10, 2010, provisional Written Opinion for International Application No. PCT/US2011/ application No. 61/352,133, filed on Jun. 7, 2010, 024482, 10 pages (mailed Apr. 9, 2012). provisional application No. 61/411.885, filed on Nov. Scannell et al., “Independent sorting-out of thousands of duplicated 9, 2010, provisional application No. 61/430,801, filed gene pairs in two yeast species descended from a whole-genome on Jan. 7, 2011. duplication.” PNAS 2007, Vo. 104(20): 8397-8402. “Office Action in Inter Partes Reexamination of U.S. Patent No. (51) Int. Cl. 8,133,715.” 46 pages, U.S. Reexamination Control No. 95/002,159 CI2P 7/16 (2006.01) (mailed Jun. 14, 2013). CI2N 15/8 (2006.01) “Office Action in Inter Partes Reexamination of U.S. Patent No. CI2N 9/04 (2006.01) 8,158.404” 28 pages, U.S. Reexamination Control No. 95/002,177 (52) U.S. Cl. (mailed Jun. 14, 2013). CPC .............. CI2N 15/81 (2013.01); C12N 9/0006 “Office Action in Inter Partes Reexamination of U.S. Patent No. (2013.01); CI2P 7/16 (2013.01); C12Y 8,153,415.” 47 pages, U.S. Reexamination Control No. 95/002,174 102/01005 (2013.01); C12Y 101/01 (2013.01); (mailed Jun. 14, 2013). Y02E 50/10 (2013.01) (58) Field of Classification Search * cited by examiner None Primary Examiner — Hope Robinson See application file for complete search history. (74) Attorney, Agent, or Firm — Cooley LLP (56) References Cited (57) ABSTRACT U.S. PATENT DOCUMENTS The present invention relates to recombinant microorganisms 6,753,314 B1* 6/2004 Giot et al. .................... 424,169 comprising biosynthetic pathways and methods of using said 8,017,376 B2 9, 2011 Dundon et al. recombinant microorganisms to produce various beneficial 8,133,715 B2 * 3/2012 Buelter et al. ........... 435.254.11 8,153,415 B2 * 4/2012 Buelter et al. .. ... 435,254.11 metabolites. In various aspects of the invention, the recombi 8, 158.404 B2 * 4/2012 Lies et al. .................. 435/254.2 nant microorganisms may further comprise one or more 2007/00929.57 A1 4/2007 Donaldson et al. modifications resulting in the reduction or elimination of 3 2011/0076733 A1 3/2011 Urano et al. keto-acid (e.g., acetolactate and 2-aceto-2-hydroxybutyrate) and/or aldehyde-derived by-products. In various embodi FOREIGN PATENT DOCUMENTS ments described herein, the recombinant microorganisms may be microorganisms of the Saccharomyces clade, Crab WO WO 2009/086423 A2 T 2009 tree-negative yeast microorganisms, Crabtree-positive yeast WO WO 2012/O27642 A1 3, 2012 microorganisms, post-WGD (whole genome duplication) OTHER PUBLICATIONS yeast microorganisms, pre-WGD (whole genome duplica Bastian et al. tion) yeast microorganisms, and non-fermenting yeast micro “Request for Inter Partes Reexamination of U.S. Patent No. organisms. 8,133,715 Under 35 U.S.C.S 311 and 37 C.F.R.S 1913.”3131 pages, U.S. Reexamination Control No. 95/002,159 (filed Sep. 7, 2012). 10 Claims, 22 Drawing Sheets U.S. Patent Apr. 21, 2015 Sheet 1 of 22 US 9,012,189 B2 Glucose 2 NAD' -- s 2 NADH Glycolysis - 2 ATP 2?e-se pyruvate co,--- Als Hs H 2-aceto-lactate HcoH NAD(P)HNAD(P)' - d. KAR Hac 2,3-dihydroxy H3C O -isowaterate DHAD s o 2-keto-sowaterate co, KVD c H3 isobutyraldehyde NAD(P)HNAD(P) . O ADH c s isobutara of FIGURE 1 U.S. Patent Apr. 21, 2015 Sheet 2 of 22 US 9,012,189 B2 O O NAD(P)H NAD(P) O -- OH 3-ketoacid -reductase - -> OH OH 2,3-dihydrox-2-methyl 2-acetolactate butanoate (DH2MB) (AL) O O NAD(P)H NAD(P)" O HO OH — — . OH 3-ketoacid reductase OH O 2-ethyl-2,3-dihydroxy-butyrate 2-aceto-2-hydroxy-butyrate O O O. O. NAD(P)H NAD(P)" R OH R R R. R 3-ketoacid-k reductase 3-hydroxyacid 3-ketoacid FIGURE 2 U.S. Patent Apr. 21, 2015 Sheet 3 of 22 US 9,012,189 B2 Ark orra ^i:SS: & -'. r . is -3-hycisas seiss oxy & is . a. a . S-8ietysaicrate-se: iaisiehysie -- Y - & a S S ;, x Nii 3-8: Yi (3-3-3X3:333s :seizy S (2,3s-2-retiyiis s: iris'xylisatioaie FIGURE 3 U.S. Patent Apr. 21, 2015 Sheet 4 of 22 US 9,012,189 B2 s NADP" 5 NAP 3-hydroxypropionate alonate serialdehyde FIGURE 3 (CONT.) U.S. Patent Apr. 21, 2015 Sheet 5 of 22 US 9,012,189 B2 NAD(P)" NAD(P)H 1-propanal aldehyde -N-4' dehydrogenase (ALDH) propionat e Chis NAD(P)' NAD(P)H Cis isobutyraldehyde Nu aldehyde -- dehydrogenase (ALDH) r isobutyrat e NAD(P)" NAD(P)H 1-butana N aldehyde dehydrogenase (ALDH) - butyrate NAD(P)' NAD(P)H 2-methyl-1-butanal Nu aldehyde dehydrogenase (ALDH) s 2-methyl 1-butyrate NAD(P)' NAD(P)H 3-methyl-1-butanal N aldehyde dehydrogenase (ALDH) rC 3-methyl 1-butyrate FIGURE 4 U.S. Patent Apr. 21, 2015 Sheet 6 of 22 US 9,012,189 B2 pyruvate - a Cetolactate co, - synthase (ALS) - - cases 3i 2,3-dihydrox-2-methyl butanoate (DH2MB) ii dihydroxy-acid dehydratase (DHAD) 2-ketoisowalerate (KIV) ^ 3. ketoisowalerate o, - decarboxylase (KVD) his 33. isobutyraldehyde -- x- -- i (P)H all A dehydrogenase (ADH) isobutyrate NAD(P)- isitar (BuOH) --- FIGURE 5 U.S. Patent Apr. 21, 2015 Sheet 7 of 22 US 9,012,189 B2 2,3-dihydro-2-methy butariate (DH2MB) 3-methyl-1-butarial r *...* FIGURE 6 U.S. Patent Apr. 21, 2015 Sheet 8 of 22 US 9,012,189 B2 " acetohydroxybutanoate . NADPy" 2-aceto-2-hydroxy-butyrate s-X"* us six's: r / r y NPN-: keto-acid reducto 2-ethyl-2,3-dihydroxy-butyrate A isomerase (KARI) NAD(P) 2,3-dihydroxy-3-methyvalerate -eH : dihydroxy-acid dehydratase (DAD) 2-keto-3-methylwaterate ---s A kitselaerate - decarboxylase (KIMD) 2-methy--butara -- as - NAD(P)H- s NAD(P)- de ydrogenase (ADH)A. 2-methyl-1-butyrate 2-methyl-1-butano sus FIGURE 7 U.S. Patent Apr. 21, 2015 Sheet 9 of 22 US 9,012,189 B2 33 83 U.S. Patent Apr. 21, 2015 Sheet 10 of 22 US 9,012,189 B2 xxxxx s it ig 338ARides Seiics 88,38 sis. f f s st i C RS is . & wer is \ was is is try S. is ... x X -- six is v a x\\\ a rr a U.S. Patent Apr. 21, 2015 Sheet 11 of 22 US 9,012,189 B2 {udd T. st L. c. ur C. L. c. ge is " U.S. Patent Apr. 21, 2015 Sheet 13 of 22 US 9,012,189 B2 &ssssssssssss U.S. Patent Apr. 21, 2015 Sheet 16 of 22 US 9,012,189 B2 i. U.S. Patent Apr. 21, 2015 Sheet 17 of 22 US 9,012,189 B2 r) |e?OLHOng!-e- (1/3) uoeulueouoo U.S. Patent Apr. 21, 2015 Sheet 18 of 22 US 9,012,189 B2 U.S. Patent Apr. 21, 2015 Sheet 19 of 22 US 9,012,189 B2 U.S.
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