title: Metabolic engineering of Corynebacterium glutamicum for enhanced production of 5-aminovaleric acid.
abstract: BACKGROUND
  5-Aminovaleric acid (5AVA) is an important five-carbon platform chemical that can be used for the synthesis of polymers and other chemicals of industrial interest. Enzymatic conversion of L-lysine to 5AVA has been achieved by employing lysine 2-monooxygenase encoded by the davB gene and 5-aminovaleramidase encoded by the davA gene. Additionally, a recombinant Escherichia coli strain expressing the davB and davA genes has been developed for bioconversion of L-lysine to 5AVA. To use glucose and xylose derived from lignocellulosic biomass as substrates, rather than L-lysine as a substrate, we previously examined direct fermentative production of 5AVA from glucose by metabolically engineered E. coli strains. However, the yield and productivity of 5AVA achieved by recombinant E. coli strains remain very low. Thus, Corynebacterium glutamicum, a highly efficient L-lysine producing microorganism, should be useful in the development of direct fermentative production of 5AVA using L-lysine as a precursor for 5AVA. Here, we report the development of metabolically engineered C. glutamicum strains for enhanced fermentative production of 5AVA from glucose.
  
  
  RESULTS
  Various expression vectors containing different promoters and origins of replication were examined for optimal expression of Pseudomonas putida davB and davA genes encoding lysine 2-monooxygenase and delta-aminovaleramidase, respectively. Among them, expression of the C. glutamicum codon-optimized davA gene fused with His6-Tag at its N-Terminal and the davB gene as an operon under a strong synthetic H36promoter (plasmid p36davAB3) in C. glutamicum enabled the most efficient production of 5AVA. Flask culture and fed-batch culture of this strain produced 6.9 and 19.7 g/L (together with 11.9 g/L glutaric acid as major byproduct) of 5AVA, respectively. Homology modeling suggested that endogenous gamma-aminobutyrate aminotransferase encoded by the gabT gene might be responsible for the conversion of 5AVA to glutaric acid in recombinant C. glutamicum. Fed-batch culture of a C. glutamicum gabT mutant-harboring p36davAB3 produced 33.1 g/L 5AVA with much reduced (2.0 g/L) production of glutaric acid.
  
  
  CONCLUSIONS
  Corynebacterium glutamicum was successfully engineered to produce 5AVA from glucose by optimizing the expression of two key enzymes, lysine 2-monooxygenase and delta-aminovaleramidase. In addition, production of glutaric acid, a major byproduct, was significantly reduced by employing C. glutamicum gabT mutant as a host strain. The metabolically engineered C. glutamicum strains developed in this study should be useful for enhanced fermentative production of the novel C5 platform chemical 5AVA from renewable resources.
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21 Plus program), Institute for the BioCentury, Center for Systems and Synthetic Biotechnology, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.|Metabolic Engineering National Research Laboratory and BioProcess Engineering Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.
authors.forename: Jae Ho
authors.initials: JH
authors.lastname: Shin
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21 Plus program), Institute for the BioCentury, Center for Systems and Synthetic Biotechnology, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.|Metabolic Engineering National Research Laboratory and BioProcess Engineering Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.
authors.forename: Seok Hyun
authors.initials: SH
authors.lastname: Park
authors.affiliation: Division of Convergence Chemistry, Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology, P.O. Box 107, 141 Gajeong-ro, Yuseong-gu, Daejeon, 34602, Republic of Korea.
authors.forename: Young Hoon
authors.initials: YH
authors.lastname: Oh
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21 Plus program), Institute for the BioCentury, Center for Systems and Synthetic Biotechnology, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.
authors.forename: Jae Woong
authors.initials: JW
authors.lastname: Choi
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21 Plus program), Institute for the BioCentury, Center for Systems and Synthetic Biotechnology, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.|Metabolic Engineering National Research Laboratory and BioProcess Engineering Research Center, 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), Institute for the BioCentury, Center for Systems and Synthetic Biotechnology, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.|Metabolic Engineering National Research Laboratory and BioProcess Engineering Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.
authors.forename: Jae Sung
authors.initials: JS
authors.lastname: Cho
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21 Plus program), Institute for the BioCentury, Center for Systems and Synthetic Biotechnology, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.
authors.forename: Ki Jun
authors.initials: KJ
authors.lastname: Jeong
authors.affiliation: Division of Convergence Chemistry, Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology, P.O. Box 107, 141 Gajeong-ro, Yuseong-gu, Daejeon, 34602, Republic of Korea.
authors.forename: Jeong Chan
authors.initials: JC
authors.lastname: Joo
authors.affiliation: Metabolic Engineering National Research Laboratory and BioProcess Engineering Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.
authors.forename: James
authors.initials: J
authors.lastname: Yu
authors.affiliation: Department of Environmental Engineering and Energy, Myongji University, 116 Myongji-ro, Cheoin-gu, Yongin, Gyeonggido, 17058, Republic of Korea. parksj93@mju.ac.kr.
authors.forename: Si Jae
authors.initials: SJ
authors.lastname: Park
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21 Plus program), Institute for the BioCentury, Center for Systems and Synthetic Biotechnology, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea. leesy@kaist.ac.kr.|Metabolic Engineering National Research Laboratory and BioProcess Engineering Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea. leesy@kaist.ac.kr.|Bioinformatics Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea. leesy@kaist.ac.kr.
authors.forename: Sang Yup
authors.identifier: http://orcid.org/0000-0003-0599-3091
authors.initials: SY
authors.lastname: Lee
mesh_terms: D000581:Amidohydrolases / Q000235:genetics; D021542:Amino Acids, Neutral / Q000096:biosynthesis*; D061249:Batch Cell Culture Techniques; D048230:Corynebacterium glutamicum / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005285:Fermentation; D005947:Glucose / Q000378:metabolism*; D005977:Glutarates / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D006899:Mixed Function Oxygenases / Q000235:genetics; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics
chemical_list: D021542:Amino Acids, Neutral; D005977:Glutarates; C013809:5-aminovaleric acid; D006899:Mixed Function Oxygenases; C100031:lysine monooxygenase; D000581:Amidohydrolases; C019694:5-aminovaleramidase; C035736:glutaric acid; D005947:Glucose; D008239:Lysine
keywords: 5-Aminovaleric acid; Corynebacterium glutamicum; Glutaric acid; L-Lysine; Metabolic engineering