title: Highly selective production of succinic acid by metabolically engineered Mannheimia succiniciproducens and its efficient purification.
abstract: Succinic acid (SA) is one of the fermentative products of anaerobic metabolism, and an important industrial chemical that has been much studied for its bio-based production. The key to the economically viable bio-based SA production is to develop an SA producer capable of producing SA with high yield and productivity without byproducts. Mannheimia succiniciproducens is a capnophilic rumen bacterium capable of efficiently producing SA. In this study, in silico genome-scale metabolic simulations were performed to identify gene targets to be engineered, and the PALK strain (ΔldhA and Δpta-ackA) was constructed. Fed-batch culture of PALK on glucose and glycerol as carbon sources resulted in the production of 66.14 g/L of SA with the yield and overall productivity of 1.34 mol/mol glucose equivalent and 3.39 g/L/h, respectively. SA production could be further increased to 90.68 g/L with the yield and overall productivity of 1.15 mol/mol glucose equivalent and 3.49 g/L/h, respectively, by utilizing a mixture of magnesium hydroxide and ammonia solution as a pH controlling solution. Furthermore, formation of byproducts was drastically reduced, resulting in almost homo-fermentative SA production. This allowed the recovery and purification of SA to a high purity (99.997%) with a high recovery yield (74.65%) through simple downstream processes composed of decolorization, vacuum distillation, and crystallization. The SA producer and processes developed in this study will allow economical production of SA in an industrial-scale. Biotechnol. Bioeng. 2016;113: 2168-2177. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Metabolic and Biomolecular Engineering National Research Laboratory, BioProcess Engineering Research Center, and 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: Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Metabolic and Biomolecular Engineering National Research Laboratory, BioProcess Engineering Research Center, and Center for Systems and Synthetic Biotechnology, Institute for the BioCentury; KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea.
authors.forename: Hyohak
authors.initials: H
authors.lastname: Song
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Metabolic and Biomolecular Engineering National Research Laboratory, BioProcess Engineering Research Center, and Center for Systems and Synthetic Biotechnology, Institute for the BioCentury; KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea.
authors.forename: Sung Won
authors.initials: SW
authors.lastname: Lim
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Metabolic and Biomolecular Engineering National Research Laboratory, BioProcess Engineering Research Center, and Center for Systems and Synthetic Biotechnology, Institute for the BioCentury; KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea.
authors.forename: Tae Yong
authors.initials: TY
authors.lastname: Kim
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Metabolic and Biomolecular Engineering National Research Laboratory, BioProcess Engineering Research Center, and 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: Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Metabolic and Biomolecular Engineering National Research Laboratory, BioProcess Engineering Research Center, and Center for Systems and Synthetic Biotechnology, Institute for the BioCentury; KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea.
authors.forename: Jeong Wook
authors.initials: JW
authors.lastname: Lee
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Metabolic and Biomolecular Engineering National Research Laboratory, BioProcess Engineering Research Center, and 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: Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Metabolic and Biomolecular Engineering National Research Laboratory, BioProcess Engineering Research Center, and Center for Systems and Synthetic Biotechnology, Institute for the BioCentury; KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea. leesy@kaist.ac.kr.
authors.forename: Sang Yup
authors.initials: SY
authors.lastname: Lee
mesh_terms: D003198:Computer Simulation; D024861:Genetic Enhancement / Q000379:methods*; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D031121:Mannheimia / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D064688:Metabolic Flux Analysis; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D019802:Succinic Acid / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D009097:Multienzyme Complexes; D019802:Succinic Acid; D005947:Glucose; D005990:Glycerol
keywords: Mannheimia succiniciproducens; byproducts; in silico metabolic flux analysis; metabolic engineering; succinic acid