US 201102.01090A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0201090 A1 Buelter et al. (43) Pub. Date: Aug. 18, 2011 (54) YEAST MICROORGANISMS WITH filed on Feb. 26, 2010, provisional application No. REDUCED BY PRODUCT ACCUMULATION 61/282,641, filed on Mar. 10, 2010, provisional appli FOR IMPROVED PRODUCTION OF FUELS, cation No. 61/352,133, filed on Jun. 7, 2010, provi CHEMICALS, AND AMINO ACIDS sional application No. 61/411.885, filed on Nov. 9, 2010, provisional application No. 61/430,801, filed on (75) Inventors: Thomas Buelter, Denver, CO (US); Jan. 7, 2011. Andrew Hawkins, Parker, CO (US); Stephanie Porter-Scheinman, Conifer, CO Publication Classification (US); Peter Meinhold, Denver, CO (51) Int. Cl. (US); Catherine Asleson Dundon, CI2N I/00 (2006.01) Englewood, CO (US); Aristos Aristidou, Highlands Ranch, CO (52) U.S. Cl. ........................................................ 435/243 (US); Jun Urano, Aurora, CO (US); Doug Lies, Parker, CO (US); Matthew Peters, Highlands Ranch, (57) ABSTRACT CO (US); Melissa Dey, Aurora, CO The present invention relates to recombinant microorganisms (US); Justas Jancauskas, comprising biosynthetic pathways and methods of using said Englewood, CO (US); Kent Evans, recombinant microorganisms to produce various beneficial Denver, CO (US); Julie Kelly, metabolites. In various aspects of the invention, the recombi Denver, CO (US); Ruth Berry, nant microorganisms may further comprise one or more Englewood, CO (US) modifications resulting in the reduction or elimination of 3 keto-acid (e.g., acetolactate and 2-aceto-2-hydroxybutyrate) (73) Assignee: GEVO, INC., Englewood, CO (US) and/or aldehyde-derived by-products. In various embodi (21) Appl. No.: 13/025,801 ments described herein, the recombinant microorganisms may be microorganisms of the Saccharomyces clade, Crab (22) Filed: Feb. 11, 2011 tree-negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD (whole genome duplication) Related U.S. Application Data yeast microorganisms, pre-WGD (whole genome duplica (60) Provisional application No. 61/304,069, filed on Feb. tion) yeast microorganisms, and non-fermenting yeast micro 12, 2010, provisional application No. 61/308,568, organisms. Patent Application Publication Aug. 18, 2011 Sheet 1 of 22 US 2011/02O1090 A1 Licose Glycolysis ?-->2 pyruvate ::: co,-- Als NAP * d KR NAD(P) to Hac. 2, 3-dihydroxy Hac to wiso Yalerate "SoH2-keto-isovalerate (0. -4 KIvo Hach isobutyraldehyde NADP- - isobutanof FGURE 1 Patent Application Publication Aug. 18, 2011 Sheet 2 of 22 US 2011/02O1090 A1 O O NAD(P)H NAD(P) ---OH 3-ketoacid reductase --> 2,3-dihydrox-2-methyl 2-acetolactate butanoate (DH2MB) (AL) O O NAD(P)H NAD(P)" O HO OH — - - O 3-ketoacid reductase OH O 2-ethyl-2,3-dihydroxy-butyrate 2-aceto-2-hydroxy-butyrate OH O O. O. NAD(P)H NAD(P)" -> — - - R R R OH R R 3-ketoacid reductase 3-hydroxyacid 3-ketoacid FIGURE 2 Patent Application Publication Aug. 18, 2011 Sheet 3 of 22 US 2011/02O1090 A1 EC 1.1.1.30 HO 0 O NAD + N- or -- NADH + C + H o O (R)-3-hydroxybutanoate acetoacetate EC 1.1.1.31 O O Hoyo + NAD -- NADH + o + Ht (S)-3-hydroxy-isobutyrate (S)-methylmalonate-semialdehyde EC 1.1.1.103 OH O O -Yo, + NAD" -- --so + NADH + 2 Ht NH3t NH2 L-threonine 2-amino-3-OXobutanoate EC 1.1.1.217 Q OH NADPt cro's benzyl (2r,3s)-2-methyl-3-hydroxybutanoate O NADPH Orar O s + Hit benzyl-2-methyl-3-oxobutanoate FIGURE 3 Patent Application Publication Aug. 18, 2011 Sheet 4 of 22 US 2011/02O1090 A1 EC 1.1.1.298 HO O O r + NADP + NADPH + H 3-hydroxypropionate malonate semialdehyde FIGURE 3 (CONT.) Patent Application Publication Aug. 18, 2011 Sheet 5 of 22 US 2011/02O1090 A1 NAD(P)' NAD(P)H 1-propanal dehydrogenase (ALDH) Chi NAD(P)' NAD(P)H isobutyraldehyde dehydrogenase (ALDH) NAD(P)" NAD(P)H 1-butanal N-2 aldehyde dehydrogenase (ALDH) NAD(P)" NAD(P)H 2-methyl-1-butanal NU aldehyde dehydrogenase (ALDH) 2-methyl 1-butyrate NAD(P)" NAD(P)H 3-methyl-1-butanal Nu' aldehyde dehydrogenase (ALDH) r 3-methyl 1-butyrate FIGURE 4 Patent Application Publication Aug. 18, 2011 Sheet 6 of 22 US 2011/02O1090 A1 y Ewale acetolactate x A synthase (AES) AD(Py -atlacias -i i. --" -- AE. - x -x: x or * Y. Case - MEAEPH- 2,3-dihydrox-2-methyl kit-aid exist- r A isoerase (KARI) heate O-28B) NAD(P) : 2,3-dihydroxyisowalerate. CHIW - “. dihydroxy-acid keysdatase (OHA) 2-keirisowalerate l is KIW) '" A ketasovalerate o, - decarboxylase (KIVE) NAD(P)H isoExityraldehyde a sk PH- all e (AH) dehydrogenase (ADH) isobtstyrate NACP) - is stars l i-BiH) -" ^* FGURE 5 Patent Application Publication Aug. 18, 2011 Sheet 7 of 22 US 2011/02O1090 A1 private x: axeclaxiate E. 1syrittas Ai-S &-acetacitate A.E.) KAEF---, 23-3-hykir-2-retty isratasek&E-xx: recktie- &; bar:3t&ESSE& O-2338E. NADP city oxy-acc cekycratasa is: -kai:33 lease . aretyl-C*. isoppy:Fialate synthase spropy:malise isotreras (x: * 3-sixtylrfalate xietyxixexase 3-rathyl-3-bias -- *: is *-*-** ae EBR- ceigs 8: re-dehydrogenaseE. (AH y-3-is: FGURE 6 Patent Application Publication Aug. 18, 2011 Sheet 9 of 22 US 2011/0201,090 A1 5 s : vvy' - eye KNC0S 08 NHC s 169 - ele190W-Z 098 - ele80W 109 ele,0e s O990 - SNCHO s s s Aug. 18, 2011 Sheet 10 of 22 US 2011/02O1090 A1 ?TOOB l 1992, -92.1KncOS (A ) 19, WHO Patent Application Publication Aug. 18, 2011 Sheet 11 of 22 US 2011/02O1090 A1 Patent Application Publication Aug. 18, 2011 Sheet 12 of 22 US 2011/02O1090 A1 Patent Application Publication Aug. 18, 2011 Sheet 13 of 22 US 2011/02O1090 A1 Patent Application Publication Aug. 18, 2011 Sheet 14 of 22 US 2011/02O1090 A1 O O O 2-5 s D 1. 2 O .9 E O ?t O x O- O en dS s > cy w 5 asE D 2 CD S2 S. O O O d : CD CD s S. 2 O E .9 Ey t O a5 5 o s s Patent Application Publication Aug. 18, 2011 Sheet 15 of 22 US 2011/02O1090 A1 (?Sið Patent Application Publication Aug. 18, 2011 Sheet 16 of 22 US 2011/02O1090 A1 i Patent Application Publication Aug. 18, 2011 Sheet 18 of 22 US 2011/02O1090 A1 Patent Application Publication Aug. 18, 2011 Sheet 19 of 22 US 2011/02O1090 A1 Patent Application Publication Aug. 18, 2011 Sheet 20 of 22 US 2011/02O1090 A1 Patent Application Publication Aug. 18, 2011 Sheet 21 of 22 US 2011/02O1090 A1 | , „s:(~~~~ :•! |- ~?~ Zoo Patent Application Publication Aug. 18, 2011 Sheet 22 of 22 US 2011/02O1090 A1 pyruvate st CH -- NAD(P)HS NAD(P)" - acetyl-CoA illus r O S-s- \-N 1. C} CoA, CO2 H2O y CO2 - CO2 - M -Na1N N-Na' reduced electron acceptor -Na' oxidized electron acceptor NAD(P)HN NADPS(P) . NAD(P)" - NAD(P) c-1\-1N -n- N-N- NAD(P)H - 1-propanol -butano NAD(P)" - N CoA N-Na' NAD(P)H- NAD(P)" - n 1a Oi Y-- Y - 1-butano FIGURE 20 US 2011/020 1 090 A1 Aug. 18, 2011 YEAST MICROORGANISMIS WITH 0006. One of the primary reasons for the sub-optimal per REDUCED BY PRODUCT ACCUMULATION formance observed in many existing microorganisms is the FOR IMPROVED PRODUCTION OF FUELS, undesirable conversion of pathway intermediates to CHEMICALS, AND AMINO ACIDS unwanted by-products. The present inventors have identified various by-products, including 2,3-dihydroxy-2-methylbu CROSS REFERENCE TO RELATED tanoic acid (DH2MB) (CAS #14868-24-7), 2-ethyl-2,3-dihy APPLICATIONS droxybutyrate, 2,3-dihydroxy-2-methyl-butanonate, isobu tyrate, 3-methyl-1-butyrate, 2-methyl-1-butyrate, and 0001. This application claims priority to U.S. Provisional propionate, which are derived from various intermediates of Application Ser. No. 61/304,069, filed Feb. 12, 2010; U.S. biosynthetic pathways used to produce fuels, chemicals, and Provisional Application Ser. No. 61/308,568, filed Feb. 26, amino acids. The accumulation of these by-products nega 2010; U.S. Provisional Application Ser. No. 61/282,641, filed tively impacts the synthesis and yield of desirable metabolites Mar. 10, 2010; U.S. Provisional Application Ser. No. 61/352. in a variety of fermentation reactions. Until now, the enzy 133, filed Jun. 7, 2010; U.S. Provisional Application Ser. No. matic activities responsible for the production of these 61/411,885, filed Nov. 9, 2010; and U.S. Provisional Appli unwanted by-products had not been characterized. More par cation Ser. No. 61/430,801, filed Jan. 7, 2011, each of which ticularly, the present application shows that the activities of a is herein incorporated by reference in its entirety for all pur 3-ketoacid reductase (3-KAR) and an aldehyde dehydroge poses. nase (ALDH) allow for the formation of these by-products ACKNOWLEDGMENT OF GOVERNMENTAL from important biosynthetic pathway intermediates. SUPPORT 0007. The present invention results from the study of these enzymatic activities and shows that the Suppression of the 0002 This invention was made with government support 3-KAR and/or ALDH enzymes considerably reduces or under Contract No. 2009-10006-05919, awarded by the eliminates the formation of unwanted by-products, and con United States Department of Agriculture, and under Contract comitantly improves the yields and titers of beneficial No. W911 NF-09-2-0022, awarded by the United States metabolites. The present application shows moreover, that Army Research Laboratory. The government has certain enhancement of the 3-KAR and/or ALDH enzymatic activi rights in the invention.