MAIMOTOMONTANT US009828618B2 DE LLU TO MO NA MINI (12 ) United States Patent ( 10 ) Patent No. : US 9 ,828 ,618 B2 Hidesaki et al. (45 ) Date of Patent: *Nov . 28 , 2017 (54 ) MICROORGANISM HAVING CARBON EP 2 738 247 Al 6 / 2014 DIOXIDE FIXATION CYCLE INTRODUCED TW 201311889 3 / 2013 THEREINTO WO WO - 2009 /046929 A2 4 / 2009 WO WO - 2009 /094485 A1 7 / 2009 WO WO - 2010 /071697 A1 6 / 2010 ( 71 ) Applicant: Mitsui Chemicals , Inc . , Tokyo ( JP ) WO WO - 2011 /099006 A2 8 / 2011 ( 72 ) Inventors: Tomonori Hidesaki, Singapore (SG ) ; WO WO 2013 /018734 A12 / 2013 Ryota Fujii, Chiba ( JP ); Yoshiko Matsumoto , Mobara ( JP ) ; Anjali OTHER PUBLICATIONS Madhavan , Singapore ( SG ) ; Su Sun Wendisch et al. , Current Opinion in Microbiology 2006, vol . 9 , p . Chong, Singapore ( SG ) 268 - 274 . * Chistoserdova et al. , Journal of Bacteriology , 1994 , vol. 176 , No . ( 73 ) Assignee : MITSUI CHEMICALS , INC . , Tokyo 23 , p . 7398 - 7404. * ( JP ) Schneider et al. , The Journal of Biological Chemistry , 2012 , vol . 287 , No. 1 , p . 757 - 766 Only . * ( * ) Notice : Subject to any disclaimer , the term of this Office Action issued in Chinese Patent Application No . patent is extended or adjusted under 35 201480002416 . 5 dated Jun . 23 , 2016 . U . S .C . 154 (b ) by 0 days . Erb et al. , “ The Apparent Malate Synthase Activity of Rhodobacter sphaeroides is Due to Two Paralogous Enzymes , (3S ) -Malyl - Co This patent is subject to a terminal dis enzyme A (COA ) B -Methylmalyl - CoA Lyase and (3S ) -Malyl -CoA claimer . Thioesterase ” , J . Bacteriol, Sep . 2009 , pp . 1249 - 1258 , vol. 192 , No . (21 ) Appl. No. : 14 /428 , 928 Yim et al. , “ Metabolic engineering of Escherichia coli for direct production of 1 , 4 -butanediol ,” Nature Chemical Biology , vol. 7 , ( 22 ) PCT Filed : Jan . 23, 2014 Jul. 2011, pp . 445 -452 . Atomi, Haruyuki, “ Review Microbial Enzymes Involved in Carbon ( 86 ) PCT No. : PCT / JP2014 / 051402 Dioxide Fixation, ” Journal of Bioscience and Bioengineering , vol. $ 371 ( c ) ( 1 ) , 94 , No . 6 , 2002 , pp . 497 - 505 . ( 2 ) Date : Mar. 17, 2015 Berg , Ivan A . , “Minireview Ecological Aspects of the Distribution of Different Autotrophic CO2 Fixation Pathways, ” Applied and (87 ) PCT Pub . No .: WO2014 / 115815 Environmental Microbiology , vol. 77 , No . 6 , Mar. 2011, pp . 1925 1936 . PCT Pub . Date : Jul. 31, 2014 Atsumi et al ., Non -fermentative pathways for synthesis of branched - chain higher alcohols as biofuels , Nature vol . 451 ( 3 ) Jan . (65 ) Prior Publication Data 2008, pp . 86 - 89 . US 2015 /0232903 A1 Aug. 20 , 2015 International Search Report dated Apr . 8 , 2014 issued in Application No. PCT/ JP2014 /051402 . ( 30 ) Foreign Application Priority Data ( Continued ) Jan . 24 , 2013 ( JP ) .. .. .. .. .. .. .. .. 2013 -011536 Jan . 24 , 2013 ( JP ) . .. .. .. .. 2013 - 011538 Primary Examiner — Kade Ariani (51 ) Int. CI. ( 74 ) Attorney , Agent, or Firm — Foley & Lardner LLP C12P 19 /32 ( 2006 .01 ) C12P 7 / 04 ( 2006 .01 ) ABSTRACT C12P 7 / 28 ( 2006 .01 ) (57 ) C12P 13 / 14 ( 2006 .01 ) An acetyl -CoA producing microorganism obtained by C12P 7748 ( 2006 .01 ) imparting at least one enzymatic activity selected from the C12P 7 /46 ( 2006 . 01 ) group consisting of malate thiokinase , malyl- CoA lyase , C12P 7 /62 ( 2006 . 01) glyoxylate carboligase , 2 -hydroxy - 3 - oxopropionate reduc (52 ) U . S . CI. tase , and hydroxypyruvate reductase , to a microorganism CPC .. ... C12P 19 /32 (2013 .01 ) ; C12P 7 / 04 that does not have any of the following (a ), ( b ), ( c ) , ( d ) or ( 2013 .01 ) ; C12P 7 /28 ( 2013 . 01 ) ; C12P 7 /46 ( C ): ( a ) a carbon dioxide fixation cycle including an enzy (2013 .01 ) ; C12P 7748 ( 2013 .01 ) ; C12P 77625 matic reaction from malonyl- CoA to malonate semialdehyde (2013 . 01 ) ; C12P 13 / 14 ( 2013 . 01 ) or 3 - hydroxypropionate ; (b ) a carbon dioxide fixation cycle ( 58 ) Field of Classification Search including an enzymatic reaction from acetyl - CoA and CO2 None to pyruvate ; ( c ) a carbon dioxide fixation cycle including an See application file for complete search history. enzymatic reaction from crotonyl - CoA and Co , to ethylma lonyl- CoA or glutaconyl -CoA ; ( d ) a carbon dioxide fixation (56 ) References Cited cycle including an enzymatic reaction from CO2 to formate ; or ( e ) at least one selected from the group consisting of FOREIGN PATENT DOCUMENTS malate thiokinase and malyl -CoA lyase . DE 10 2007 047 206 AL 4 / 2009 DE 1020070 59 248 A1 6 /2009 10 Claims, 2 Drawing Sheets US 9 ,828 ,618 B2 Page 2 ( 56 ) References Cited OTHER PUBLICATIONS Notice of Submission of Opinion issued in Korean Patent Appli cation No . 10 - 2015 -7006510 dated Mar . 28, 2016 . Chatsurachai et al. , “ An in silico platform for the design of heterologous pathways in nonnative metabolite production ,” BMC Bioinformatics 13 :93 ( 2012 ) . Melzer et al. , “ Flux Design : In silico design of cell factories based on correlation of pathway fluxes to desired properties, ” BMC Systems Biology 3 : 120 ( 2009 ) . Extended European Search Report issued in European Patent Appli cation No . 14743436 . 9 dated Sep . 7 , 2016 . Office Action dated Mar. 15 , 2017 in corresponding Chinese Patent Application No . 201480002416 . 5 . Office Action issued in Taiwan patent application No . 103102745 dated Aug . 28 , 2017 . * cited by examiner U . S . Patent Nov. 28 , 2017 Sheet 1 of 2 US 9 ,828 ,618 B2 * * * * * * * * W * * W * * WWW om c * : WW * W * M WOW X W o M now W . Ww * M W X W X WWW on 2 wW K W noonn.ex:* mci *Wwwww ww 1000 Malyl-CoA hyi w OOOOXXOOXXOOOOSSSOOS 0 deincomemoltinSuccinate 2-Hydroxy3 w Glyoxylate Hydroxypyruvate w CO2eminoch frd,sdh cano w Oba 0tonexMe* oxopropionate WU* mik *w * OMMIGRANKENONCONCHAKOMAX CIXR ycdw white oano wwww #44 Acetyl-CoA w w o 0 Fumarate ?{} Glyceratewhether w w w :*X OxaloacetatemannensomMalate * ** NK#KKMKM* * * CO glxx * 1.FIG ** gark nnoono *DO306 W wwwwww * x o D * wwww o 200 xW Ond.209200 W MAW Acetyl-CoAproductioncycle ensta XX MOM SK x* Yeew'vols ?{“????1} ond UX*w * *om 70% w ,ppc w oncuts w o eno wd6 w ock VVXboxopok w n w 3-Phosphoglycerate w in 2-Phosphoglycerate w ? ? ? ? ? ? ? ? ??? ??? ??? ?? w * * wuuu ? ? ? ? ? ? ? ? ? ? o ACCOUKKO (glucose,etc.) pood wwwwwwww mePhosphoenolpyruvate TxRd Pyruvate Acetyl-CoA 200 Intendedproduct(IPA ,glutamicacid,etc.) * * * Carbonsourcematerial * wwwpuukautuuwwe * * www www mmmmmwwwwwwwwww U . S . Patent Nov. 28 , 2017 Sheet 2 of 2 US 9 ,828 ,618 B2 * * * * * * * * * * * * * * * * * * * * * * * * A DO C O M O Malyl-COA Glyoxylate Glycine (03) Hydroxypyruvate ppopo 93)( o pogoon (13) (3) Acetyl-CoA CO2 GOOOD HOMWAKIERONDHATRONIKKOKOHAMEDHONINGCHAWI ERACEEANCAK DOOLERO Glycerateoffre Glycinepathway OxaloacetatereserverenenbesoMalate(C3) (C3) Serine (53) " FIG.2 HONCERNASONONO O (h3) (€9) 99004 2-Phosphoglycerate (m3) N3 A 2 4 www * w 4 LR 4 - AR * w w K w w (glucose,etc.)Opony careajeansdousoydsoudwo w cocco como W om kkxxi Acetyl-CoA Pyruvate * Phosphoglycerate3-)etc,aciditaconic,acidcitric( O w 00 Intendedproduct w Carbonsourcematerial w ARAKARRAGAWARABARAW w +w #" ttab xHxa > * A * * * * * * #* US 9 , 828 ,618 B2 MICROORGANISM HAVING CARBON by formate dehydrogenase , CO dehydrogenase , etc . , and , DIOXIDE FIXATION CYCLE INTRODUCED ultimately converted into acetyl- CoA . The 3 -hydroxypropi THEREINTO onate cycle is found in Chloroflexus bacteria and the like , and comprises 13 enzymes. In the 3 -hydroxypropionate CROSS REFERENCE TO RELATED 5 cycle , CO2 is fixed by the action of acetyl- CoA (propionyl APPLICATIONS COA ) carboxylase , and acetyl- CoA is produced via malonyl COA and the like . The 4 -hydroxybutyrate cycle exists in This application is the National Phase of PCT/ JP2014 ) archaebacteria and the like . In the 4 -hydroxybutyrate cycle , 051402 , filed Jan . 23 , 2014 , which claims priority to Japa CO , is fixed by the actions of pyruvate synthase , acetyl -CoA nese Application No . 2013 -011536 , filed Jan . 24 , 2013 and 10 (propionyl -CoA ) carboxylase , and phosphoenolpyruvate Japanese Application No . 2013 -011538 , filed Jan . 24 , 2013 . carboxylase , whereby acetyl - CoA is produced via 4 -hy droxybutyryl CoA and the like . TECHNICAL FIELD Several approaches of producing a useful substance by introducing a carbon dioxide fixation pathway to a micro The present invention relates to a microorganism having 15 organism that produces a useful compound have been pro a carbon dioxide fixation cycle introduced thereinto and a posed . For example , International Publication Nos. WO method of producing a substance using the microorganism . 2009 /094485 and WO 2010 / 071697 disclose approaches for producing acetyl- CoA from CO2 by using a microorganism BACKGROUND ART to which a pathway similar to the Wood - Ljungdahl pathway 20 of acetic acid bacteria has been introduced . WO 2009/ Acetyl- CoA is one of significantly important intermedi- 046929 discloses an approach for producing lactic acid from ates in metabolic pathways of microorganisms. Various CO2 by using a microorganism to which hydrogenase and metabolites are produced via acetyl- CoA . Well -known tetrahydrofolate lyase have been introduced . WO 2011/ examples of such substances produced via acetyl- CoA 099006 proposes a cycle in which CO , is fixed via a carbon include amino acids such as L - glutamic acid , L - glutamine, 25 dioxide fixation reaction into acetyl- CoA or a malonyl- CoA L - proline, L -arginine , L - leucine , and L - isoleucine ; organic reduction reaction . German Patent Application Laid -