title: Effect of puuC overexpression and nitrate addition on glycerol metabolism and anaerobic 3-hydroxypropionic acid production in recombinant Klebsiella pneumoniae ΔglpKΔdhaT.
abstract: 3-Hydroxypropionic acid (3-HP), an industrially important platform chemical, is used as a precursor during the production of many commercially important chemicals. Recently, recombinant strains of K. pneumoniae overexpressing an NAD(+)-dependent γ-glutamyl-γ-aminobutyraldehyde dehydrogenase (PuuC) enzyme of K. pneumoniae DSM 2026 were shown to produce 3-HP from glycerol without the addition coenzyme B(12), which is expensive. However, 3-HP production in K. pneumoniae is accompanied with NADH generation, and this always results in large accumulation of 1,3-propanediol (1,3-PDO) and lactic acid. In this study, we investigated the potential use of nitrate as an electron acceptor both to regenerate NAD(+) and to prevent the formation of byproducts during anaerobic production of 3-HP from glycerol. Nitrate addition could improve NAD(+) regeneration, but decreased glycerol flux towards 3-HP production. To divert more glycerol towards 3-HP, a novel recombinant strain K. pneumoniae ΔglpKΔdhaT (puuC) was developed by disrupting the glpK gene, which encodes glycerol kinase, and the dhaT gene, which encodes 1,3-propanediol oxidoreductase. This strain showed improved cellular NAD(+) concentrations and a high carbon flux towards 3-HP production. Through anaerobic cultivation in the presence of nitrate, this recombinant strain produced more than 40±3mM 3-HP with more than 50% yield on glycerol in shake flasks and 250±10mM 3-HP with approximately 30% yield on glycerol in a fed-batch bioreactor.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Pusan National University, San 30, Jangjeon-dong, Geumjeong-gu, Busan 609-735, South Korea.
authors.forename: Somasundar
authors.initials: S
authors.lastname: Ashok
authors.forename: Subramanian
authors.initials: S
authors.lastname: Mohan Raj
authors.forename: Yeounjoo
authors.initials: Y
authors.lastname: Ko
authors.forename: Mugesh
authors.initials: M
authors.lastname: Sankaranarayanan
authors.forename: Shengfang
authors.initials: S
authors.lastname: Zhou
authors.forename: Vinod
authors.initials: V
authors.lastname: Kumar
authors.forename: Sunghoon
authors.initials: S
authors.lastname: Park
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000693:Anaerobiosis / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods*; D005990:Glycerol / Q000378:metabolism*; D007711:Klebsiella pneumoniae / Q000145:classification / Q000502:physiology*; D019344:Lactic Acid / Q000031:analogs & derivatives* / Q000096:biosynthesis; D009566:Nitrates / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics*; D013045:Species Specificity; D015854:Up-Regulation
chemical_list: D009566:Nitrates; D019344:Lactic Acid; C031601:hydracrylic acid; D000445:Aldehyde Oxidoreductases; C019679:aminobutyraldehyde dehydrogenase; D005990:Glycerol