title: Homo-succinic acid production by metabolically engineered Mannheimia succiniciproducens.
abstract: Succinic acid (SA) is a four carbon dicarboxylic acid of great industrial interest that can be produced by microbial fermentation. Here we report development of a high-yield homo-SA producing Mannheimia succiniciproducens strain by metabolic engineering. The PALFK strain (ldhA-, pta-, ackA-, fruA-) was developed based on optimization of carbon flux towards SA production while minimizing byproducts formation through the integrated application of in silico genome-scale metabolic flux analysis, omics analyses, and reconstruction of central carbon metabolism. Based on in silico simulation, utilization of sucrose would enhance the SA production and cell growth rates, while consumption of glycerol would reduce the byproduct formation rates. Thus, sucrose and glycerol were selected as dual carbon sources to improve the SA yield and productivity, while deregulation of catabolite-repression was also performed in engineered M. succiniciproducens. Fed-batch fermentations of PALFK with low- and medium-density (OD600of 0.4 and 9.0, respectively) inocula produced 69.2 and 78.4g/L of homo-SA with yields of 1.56 and 1.64mol/mol glucose equivalent and overall volumetric SA productivities of 2.50 and 6.02g/L/h, respectively, using sucrose and glycerol as dual carbon sources. The SA productivity could be further increased to 38.6g/L/h by employing a membrane cell recycle bioreactor system. The systems metabolic engineering strategies employed here for achieving homo-SA production with the highest overall performance indices reported to date will be generally applicable for developing superior industrial microorganisms and competitive processes for the bio-based production of other chemicals as well.
authors.affiliation: Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus program), BioProcess Engineering Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea.
authors.forename: Jeong Wook
authors.initials: JW
authors.lastname: Lee
authors.affiliation: Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus program), BioProcess Engineering Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea.
authors.forename: Jongho
authors.initials: J
authors.lastname: Yi
authors.affiliation: Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus program), BioProcess Engineering Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea.
authors.forename: Tae Yong
authors.initials: TY
authors.lastname: Kim
authors.affiliation: Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus program), BioProcess Engineering Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea; Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea.
authors.forename: Sol
authors.initials: S
authors.lastname: Choi
authors.affiliation: Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus program), BioProcess Engineering Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea; Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea.
authors.forename: Jung Ho
authors.initials: JH
authors.lastname: Ahn
authors.affiliation: Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus program), BioProcess Engineering Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea.
authors.forename: Hyohak
authors.initials: H
authors.lastname: Song
authors.affiliation: Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus program), BioProcess Engineering Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea; Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea.
authors.forename: Moon-Hee
authors.initials: MH
authors.lastname: Lee
authors.affiliation: Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus program), BioProcess Engineering Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea; Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea. Electronic address: leesy@kaist.ac.kr.
authors.forename: Sang Yup
authors.initials: SY
authors.lastname: Lee
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D019149:Bioreactors / Q000382:microbiology; D053898:Biosynthetic Pathways / Q000235:genetics; D024861:Genetic Enhancement / Q000379:methods; D005990:Glycerol / Q000378:metabolism*; D031121:Mannheimia / Q000502:physiology*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019802:Succinic Acid / Q000302:isolation & purification / Q000378:metabolism*; D013395:Sucrose / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013395:Sucrose; D019802:Succinic Acid; D005990:Glycerol
keywords: Byproduct; Carbon catabolite repression; Glycerol; Homo-succinic acid; Mannheimia succiniciproducens; Sucrose