US010246727B2 (12 ) United States Patent ( 10 ) Patent No. : US 10 , 246 ,727 B2 Zanghellini (45 ) Date of Patent: Apr . 2 , 2019 (54 ) FERMENTATION ROUTE FOR THE H05 137568 A 6 / 1993 PRODUCTION OF LEVULINIC ACID , H 05320023 A 12 / 1993 H 06280041 A 10 / 1994 LEVULINATE ESTERS AND H 09190820 A 7 / 1997 VALEROLACTONE AND DERIVATIVES 2009528842 A 8 / 2009 THEREOF WO WO 1995 /022524 AL 8 / 1995 WO WO 1998 / 043684 Al 10 / 1998 WO WO 2004 / 084633 A 10 / 2004 (71 ) Applicant : Arzeda Corp . , Seattle , WA (US ) WO WO 2004 /085048 A2 10 / 2004 WO WO 2004 /085349 A2 10 / 2004 ( 72 ) Inventor : Alexandre Luc Zanghellini, Seattle , wo WO 2004 /085390 A1 10 / 2004 WA (US ) WO WO 2005 / 028529 A2 3 / 2005 WO WO 2005 /063726 AL 7 / 2005 ( 73) Assignee : ARZEDA CORP. , Seattle , WA ( US) WO WO 2005 /097723 A2 10 / 2005 WO WO 2006 /015023 A2 2 / 2006 WO WO 2006 /015024 A1 2 / 2006 ( * ) Notice : Subject to any disclaimer , the term of this WO WO 2006 / 117113 A2 11 /2006 patent is extended or adjusted under 35 WO WO 2007 / 106524 A2 9 / 2007 U . S . C . 154 (b ) by 0 days . WO WO 2009 / 111513 AL 9 / 2009 WO WO 2009 / 142489 A2 11 / 2009 WO WO 2010 /051076 A1 5 / 2010 (21 ) Appl. No. : 15 / 347, 346 WO WO 2010 /065833 A2 6 / 2010 WO WO 2010 /077470 A2 7 / 2010 (22 ) Filed : Nov . 9 , 2016 WO WO 2011 / 066076 Al 6 / 2011 (65 ) Prior Publication Data WO WO 2012 /030860 Al 3 /2012 US 2017 /0275657 A1 Sep . 28 , 2017 OTHER PUBLICATIONS Alonso et al. , “ Catalytic conversion of biomass to biofuels .” Green Related U . S . Application Data Chemistry ( 2010 ) ; 12 ( 9 ) : 1493 - 1513 . (63 ) Continuation of application No . 13 /820 ,028 , filed as Blackwell , N . C ., “ Mechanistic and Structural Investigations of DOG Aldolase ," a thesis submitted for the degree of Doctor of application No. PCT/ US2011 /049788 on Aug . 30 , Philosophy at the University of Leicester, Oct. 2000 . 2011, now Pat . No . 9 , 523, 105 . 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( 56 ) References Cited Ishiyama et al . , “ Structural studies of FlaAl from helicobacter pylori reveal the mechanism for inverting 4 , 6 -dehydratase activity ” , U . S . PATENT DOCUMENTS J . Bio . Chem ., 281 ( 34 ) : 24489 - 24495 ( 2006 ) . 5 ,769 , 929 A 6 / 1998 Gundlach et al . (Continued ) 6 , 965, 058 B1 11 / 2005 Raidel et al. 7 , 153 , 996 B2 12 / 2006 Fagan Primary Examiner — Sharmila G Landau 9 , 523 , 105 B2 12 / 2016 Zanghellini 2008 /0242721 A1 10 / 2008 Selifonov Assistant Examiner — Stephen M Chong 2009 /0191607 A1 7 / 2009 Baker et al. (74 ) Attorney, Agent, or Firm — Cooley LLP 12 / 2013 Zanghellini 2013/ 0330779 Al ( 57 ) ABSTRACT FOREIGN PATENT DOCUMENTS The invention provides processes for the conversion of pyruvate obtained from sugars or other carbon sources , to CN 1643116 A 7 /2005 EP 0496555 A2 7 / 1992 valuable C5 materials such as levulinic acid , levulinate EP 0882745 Al 12 / 1998 esters , valerolactone , and derivatives thereof. EP 1533364 A2 5 /2005 EP 1716131 B1 11 / 2006 6 Claims, 9 Drawing Sheets US 10 ,246 ,727 B2 Page 2 ( 56 ) References Cited PCT/ US2011 / 049788 , International Preliminary Report on Patent ability , dated Mar. 5 , 2013 , 5 pages . Pollard , J . R . et al ., " Substrate selectivity and biochemical proper OTHER PUBLICATIONS ties of 4 -hydroxy - 2 -keto - pentanoic acid aldolase from Escherichia Li, Y ., et al ., “ Biotechnological production of pyruvic acid ” , Applied coli” , Applied and Environmental Microbiology , 64 ( 10 ) : 4093 -4094 Microbiology and Biotechnology , 57 : 451 -459 ( 2001 ) . ( 1998 ) . Lüönd , R . M . , et al ., “ Assessment of the active -site requirements of Serrano -Ruiz et al . , “ Catalytic upgrading of levulinic acid to 5 - aminolaevulinic acid dehydratase : Evaluation of substrate and 5 - nonanone" , Green Chemistry , 12 ( 4 ) : 574 -577 ( 2010 ) . product analogues as competitive inhibitors ” , J . Org . 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Effect of Wang and Seah , “ Purification and biochemical characterization of a remote substituents on p - facial additions to lactam enolates . pyruvate- specific Class II aldolase, Hpal ” , Biochemistry , 44 ( 27 ) : Stereoelectronic and steric effects ” , J . Am . Chem . Soc. , 120 ( 30 ) : 9447 - 9455 ( 2005 ) . 7429 - 7438 ( 1998 ) . Zanghellini, A . et al ., “ New algorithms and an in silico benchmark PCT/ US2011 / 049788 , International Search Report and Written Opin for computational enzyme design ” , Protein Science, 15 ( 12 ) : 2785 ion , dated Jan . 19 , 2012 , 10 pages . 2794 ( 2006 ) . atent Apr. 2 , 2019 Sheet 1 of 9 US 10 ,246 ,727 B2 2-cidroxiperamoiosad substituidowaerersa 50 :*10 ** OXO,2-yarmypantanoirand PA FIG.I atent Apr. 2 , 2019 Sheet 2 of 9 US 10 ,246 ,727 B2 atent Apr. 2 , 2019 Sheet 3 of 9 US 10 ,246 ,727 B2 FIG . 3 . C6 sugars (glucose ) . CS sug?rs mimo www . Lipid metabolism . - . 2NAOH . SZSÁJOOKS . : 2NADH . - . decarboxylation : . ( pyruvate decarboxylase, . aldoi addition . { 8 !dolase ) . NAOH . reduction . EDELS. (dehydrogenase ) . 2. NADH COS oxidation . HOVN ) aseuabookuso{ . 23 derydration . (dehydratase . 4 -valerolacione . FMNH2 NAOH : 9d3lS ee... ZHNW prosta reduction lacionization e ( enoate reductase ) STER ? (lactonase ) v NAO NADH NAOH (dehydrogenaseexcluation ) atent Apr. 2 , 2019 Sheet 4 of 9 US 10 ,246 ,727 B2 FIG . 4 C6 sugars ( glucose ) C5 sugars - - - Lipid metabolism glycolysis 2NAD + 2NADH ??arbaxytation 002 (pyruvate decarboxylase ) aldol addition ( aldolase ) ( NAD oxidation NADH (dehydrogenase ) NADH OM reduction NADH (dehydrogenase ) OH STEPS dehydration (dehydratase ) 4 -valerolactone FMNH2 ?? NADH reduction FMNH2 STEP 7 lactonization NAD + (enoate reductase } (lactonase NADH NAD + OH reduction(dehydrogenase ) atent Apr. 2 , 2019 Sheet 5 of 9 US 10 ,246 ,727 B2 FIG . 5 * . decarboxylation . idol addition . oxidative dersydration valecolactone . :. .. atent Apr. 2 , 2019 Sheet 6 of 9 US 10 ,246 ,727 B2 FIG . 6 C6 sugars (glucose ) C5 sugars im Lipid metabolism glycolysis 2NAD + 2NADH . pyruvate decarboxylase. CO2 . : : : : : : : . other . .. .. .. .. OH . aldolase . COA synthase . hydrolysis . NADA . SCOA NADH lactonization . NAOH . dehydrogenase dehydrogenase . ' ' ' ' ' ' ' ' ' ' ' . .. .. .. .. * dehydrogenase NAD + . NAD + NAD + . OH OH COA synthase OH : hydrolysis . NAD + SCOA thioesterase NAD + OH lactonization dehydrogenase dehydrogenase OH . .. NADH dehydratase NADH . CoA synthase . OH . SCOA thioesterase reductase dehydratase dehydratase CoA synthase g - valerolactone FMNH2 SCOA FMNH2 NADH : thloesterase NADH reductase reductase FMNH2 . FMNH2 . NAD lactonization NAD + . COA synthase . NADA NAD + . thioesterase . dehydrogenase . SCOA OH . U. S . Patent atent Apr. 2 , 2019 Sheet 7 of 9 US 10 ,246 ,727 B2 ANA COAsynthase thioesterase SCOA . : . transferase O-R .. .. HOR withR-El,MetAryl. FIG.7 atent Apr. 2 , 2019