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( 12 ) United States Patent USO10704064B2 (12 ) United States Patent (10 ) Patent No.: US 10,704,064 B2 Park et al. (45 ) Date of Patent : Jul. 7 , 2020 (54 ) RECOMBINANT YEAST PRODUCING (56 ) References Cited 3 - HYDROXYPROPIONIC ACID AND METHOD FOR PRODUCING U.S. PATENT DOCUMENTS 3 - HYDROXYPROPIONIC ACID USING THE 7,655,451 B2 2/2010 Liao et al. SAME 2010/0248233 Al 9/2010 Muller et al . 2012/0135481 A1 * 5/2012 Jessen C12N 1/18 435/141 (71 ) Applicant: SK Innovation Co., Ltd., Seoul (KR ) 2013/0071893 Al 3/2013 Lynch et al . ( 72 ) Inventors: Joong Min Park , Seoul (KR ) ; Jae Yeon Park , Seoul ( KR ); Woo Chan FOREIGN PATENT DOCUMENTS Park , Sejong - si (KR ) ; Sang Min Lee , JP 200835727 A 2/2008 Seoul (KR ) ; Young Bin Seo , Seoul (KR ) ; Merja Oja , Espoo (FI ) ; Outi OTHER PUBLICATIONS Koivistoinen , Espoo (FI ) ; Andrew Conley , Espoo (FI ) ; Marja Ilmen , Alber et al.; “ Malonyl - Coenzyme A Reductase in the Modified Helsinki ( FI) ; Laura Ruohonen , 3 -Hydroxypropionate Cycle for Autotrophic Carbon fixation in Helsinki ( FI) ; Paula Jouhten , Espoo Archaeal Metallosphaera and Sulfolobus spp. ” ; Journal of Bacteri (FI ) ology ; 2006 ; pp . 8551-8559 ; vol . 188 :24 . Berg et al.; “ A 3 -Hydroxypropionate / 4 -Hydroxybutyrate Autotrophic (73 ) Assignee : SK Innovation Co., Ltd. , Seoul (KR ) Carbon Dioxide Assimilation Pathway in Archaea ” ; Science ; 2007 ; pp . 1782-1786 ; vol . 318 . ( * ) Notice : Subject to any disclaimer, the term of this Chen et al.; " Coupled incremental precursor and co - factor supply patent is extended or adjusted under 35 improves 3 -hydroxypropionic acid production in Saccharomyces U.S.C. 154 (b ) by 0 days . cerevisiae ” ; Metabolic Engineering ; 2014 ; pp . 104-109 ; vol . 22 . Diacovich et al .; “ Kinetic and Structural Analysis of a New Group Appl. No.: 15 /506,981 of Acyl- CoA Carboxylases Found in Streptomyces coelicolor A3 ( 2 ) * " ; ( 21) The Journal of Biological Chemistry ; 2002 ; pp . 31228-31236 ; vol. 277 : 34 . ( 22 ) PCT Filed : Aug. 28 , 2015 Hugler et al. , “ Malonyl - Coenzyme A Reductase from Chloroflexus aurantiacus , a Key Enzyme of the 3 -Hydroxypropionate Cycle for ( 86 ) PCT No .: PCT /KR2015 /009061 Autotrophic CO2 Fixation ” ; Journal of Bacteriology ; 2002 ; pp . $ 371 ( c ) ( 1 ) , 2404-2410 ; vol. 184 : 9 . Kockelkorn et al .; “ Malonic Semialdehyde Reductase , Succinic (2 ) Date : Feb. 27 , 2017 Semialdehyde Reductase , and Succinyl- Coenzyme A Reductase from Metallosphaera sedula : Enzymes of the Autotrophic (87 ) PCT Pub . No .: WO2016 /032279 3 - Hydroxypropionate / 4 - Hydroxybutyrate Cycle in Sulfolobales ” ; PCT Pub . Date : Mar. 3 , 2016 Journal of Bacteriology ; 2009 ; pp . 6352-6362 ; vol. 191 : 20 . Kozak et al.; “ Replacement of the Saccharomyces cerevisiae acetyl (65 ) Prior Publication Data COA synthetases by alternative pathways for cytosolic acetyl -CoA synthesis ” ; Metabolic Engineering ; 2014 ; pp . 46-59 ; vol. 21. US 2017/0240932 A1 Aug. 24 , 2017 Kroeger et al.; “ A spectrophotometric assay for measuring acetyl coenzyme A carboxylase ” ; Analytical Biochemistry ; 2011; pp . ( 30 ) Foreign Application Priority Data 100-105 ; vol . 411. (Continued ) Aug. 29 , 2014 (KR ) 10-2014-0114505 (51 ) Int . Cl. Primary Examiner Richard G Hutson C12N 1/16 (2006.01 ) (74 ) Attorney, Agent, or Firm — The Webb Law Firm C12P 7/42 ( 2006.01 ) C12N 9/04 ( 2006.01 ) C12N 9/02 ( 2006.01 (57 ) ABSTRACT C12N 9/00 (2006.01 ) C12N 1/18 ( 2006.01 ) Provided are a recombinant yeast producing 3 -hydroxypro C12N 15/52 ( 2006.01) pionic acid ( 3 -HP ) and a method for producing 3 -HP using (52 ) U.S. CI. the same , more particularly , a recombinant yeast producing CPC C12P 7/42 ( 2013.01) ; C12N 1/16 3 -HP , comprising an exogenous AADH gene ; an endog ( 2013.01) ; C12N 1/18 (2013.01 ) ; C12N enous or exogenous ACC gene ; an exogenous MCR gene ; 9/0006 (2013.01 ) ; C12N 9/0008 ( 2013.01) ; and an exogenous HPDH gene, and producing 3 - HP through C12N 9/93 ( 2013.01 ) ; C12N 15/52 ( 2013.01 ) ; [Pyruvate Acetaldehyde - Acetyl- CoA Malonyl- CoA C12Y 101/01059 ( 2013.01 ) ; C12Y 102/0101 Malonate semialdehyde 3 -HP ) biosynthesis pathway , and a (2013.01 ) ; C12Y 102/01075 (2013.01 ) ; C12Y method for producing 3 - HP using the same. 604/01002 ( 2013.01) (58 ) Field of Classification Search None 4 Claims, 3 Drawing Sheets See application file for complete search history . Specification includes a Sequence Listing . US 10,704,064 B2 Page 2 ( 56 ) References Cited Shi et al.; “ Improving Production of Malonyl Coenzyme A - Derived Metabolites by Abolishing Snf1 -Dependent Regulation of Accl” ; mBio.asm.org ; 2014 ; pp . 1-8 ; vol. 5 : 3 . OTHER PUBLICATIONS Stojiljkovic et al.; “ Ethanolamine Utilization in Salmonella typhimurium : Nucleotide Sequence , Protein Expression , and Mutational Analysis of the cch A cchB eutE eut eutG euth Gene Cluster” ; Journal of Membrillo -Hernandez et al.; “ Evolution of the adhE Gene Product Bacteriology ; 1995 ; pp . 1357-1366 ; vol . 177 : 5 . of Escherichia coli from a Functional Reductase to a Dehydrogenase ” ; Wei et al. , “ Enhanced biofuel production through coupled acetic The Journal of Biological Chemistry ; 2000 ; pp . 33869-33875 ; vol. acid and xylose consumption by engineered yeast” ; Nature Com munications; 2013 ; pp . 1-8 . 275 : 43 . Yao et al.; “ The Catalytic Property of 3 -Hydroxyisobutyrate NCBI; “ 3 -hydroxyisobutyrate dehydrogenase [Pseudomonas aeruginosa Dehydrogenase from Bacillus cereus on 3 -Hydroxypropionate ” ; PAO1] ” ; GenBank accession No. AAG06957.1 ; Jan. 31, 2014 . Appl Biochem Biotechnol; 2010 ; pp . 694-703 ; vol. 160 . NCBI; “ malonyl - CoA reductase (Chloroflexus aurantiacus )” ; GenBank Kildegaard et al ., “ Engineering and systems- level analysis of Sac charomyces cerevisiae for production of 3 -hiydroxypropionic acid accession No. AAS20429.1 ; Feb. 22 , 2004 . via malonyl -CoA reductase -dependent pathway " , Microbial Cell NCBI; malonyl- CoA / succinyl- CoA reductase [ Sulfolobus tokodaii Factories, 2016 , 13 Pages, vol. 15:53 . str . 7 ] ; GenBank accession No. BAB67276.1 ; Oct. 7 , 2016 . NCBI GenBank AGE74568.1 , Jan. 31, 2014 . NCBI; “ ACC1 [Saccharomyces cerevisiae )” ; GenBank accession No. CAA96294.1 ; Jul. 14 , 2016 . * cited by examiner U.S. Patent Jul. 7 , 2020 Sheet 1 of 3 US 10,704,064 B2 [ Fig . 1 ] 12 Glucose NAO : ADP NADA . AIP Pyruvate NAOP POC (Pyruvale decarboxylase ) Acetate U Acetaldehyde MADE COM NA ! .. ( CASHE AADH (Acetylating acetaldehyde de MADHE NADH hydrogenase ) * ... Acetyl -CoA + AT . MCO ACC (Acetyl - CoA carboxylase ) ADPA Mitochondrial PDH route PDH by pass route Natural in yeast Malonyl -COA NAOPIM MCR (Malonyl - CoA reductase ) NAX.p.CA Malonate semialdehyde NAOXOJHO HPDH ( 3.HP dehydrogeanse ) NADP ) COA 3 -Hydroxypropionate [ Fig . 2 ] ACCich < ACCmc < ACCsC5659A < ACCSC < ACCsc51157A ACCSCS659A / $ 1157A ACCke < ACCy ! [ Fig . 3 ] Marker Empty MCRms MCRsa1 MCRst MCRCC MCRca 30 Relativeactivity(%) MCRms MCRms MCRsa1 MCRsa1 MCRst MCRst MCRCC MCRCC MCRca MCRca I (NADPH )wl (NADH ) (NADPH ) (NADH ) (NADPH ) ( NADH ) (NADPH ) (NADH ) (NADPH ) (NADH ) U.S. Patent Jul. 7 , 2020 Sheet 2 of 3 US 10,704,064 B2 [ Fig . 4 ] Sad PRS423(HIS)_FBA1-ENO2TP11PSK085 Gene3 8,767bp SydNot TPUIP 085PSK- Spo Bumit Gene2 22ENO CH ECOR GoneSe Xhol PAMP Sod PT3 Sua Gene3 Nod TEF1-TDH3PDC1(pSK084]PRS426URA) LEP 8,762bp 084-PSK Spol DURAS Bumi Geno Xmas EcoR ???» Gene Xhols U.S. Patent Jul. 7 , 2020 Sheet 3 of 3 US 10,704,064 B2 [ Fig . 5 ) 900 -1( Batch ) 800 2 ( Anaerobic , batch ) 3 (Glucose spiking ) 700 4 (Fed -batch , many tablets ) 5 (Fed -batch , tablet spiking) 600 6 (Galactose spiking ) 500 3-HP(mg/L) 400 300 200 100 0 1 Day 2 3 [ Fig . 6 ] 1400 1200 1000 800 3-HP(mg/L) 600 400 200 0 1 2 Day 3 4 5 US 10,704,064 B2 1 2 RECOMBINANT YEAST PRODUCING base such as sodium hydroxide (NaOH ) and ammonium 3 - HYDROXYPROPIONIC ACID AND hydroxide (NH4OH ) for maintaining a neutral pH . This METHOD FOR PRODUCING causes an increase in the cost of the fermentation process 3 - HYDROXYPROPIONIC ACID USING THE depending on an injected base , makes an extraction and SAME 5 separation process difficult , or significantly increases the cost . CROSS - REFERENCE TO RELATED In a recent case of application of producing organic acid to chemical industry , an organic acid is produced from APPLICATIONS glucose using yeast. Yeast has high resistance against an This application is the United States national phase of 10 organic acid as compared to bacteria , thus the activity of the International Application No. PCT/ KR2015 /009061 filed yeast is not significantly inhibited even at a high acidity (low Aug. 28 , 2015 , and claims priority to Korean Patent Appli pH ) making yeast as more suitable host for producing the cation No. 10-2014-0114505 filed Aug. 29 , 2014 , the dis organic acid . Particularly , yeast has been conventionally closures of which are hereby incorporated in their entirety widely used as an industrial biocatalyst for producing spirits , by reference . 15 industrial ethanol, or the like, and may be mass -cultured and The Sequence Listing associated with this application is may not be contaminated with bacteriophages , such that filed in electronic format via EFS - Web and is hereby incor applicability of the yeast in the chemical industry is more porated by reference into the specification in its entirety . The excellent as compared to bacteria . Yeast has advantages for name of the text file containing the Sequence Listing is producing an organic acid , but there are several disadvan 1701700-2_ST25.txt .
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