title: Pyruvate kinase deletion as an effective phenotype to enhance lysine production in Corynebacterium glutamicum ATCC13032: Redirecting the carbon flow to a precursor metabolite.
abstract: Various attempts have been made to enhance lysine production in Corynebacterium glutamicum. Pyruvate kinase (PYK) defect is one of the strategies used to enhance the supply of oxaloacetic acid (OAA), a precursor metabolite for lysine biosynthesis. However, inconsistent effects of this mutation have been reported: positive effects of PYK defect in mutants having phosphoenolpyruvate carboxylase (PEPC) desensitized to feedback inhibition by aspartic acid, while negative effects in simple PYK gene (pyk) knockout mutants. To address these discrepancies, the effects of pyk deletion on lysine yield were investigated with or without the D299N mutation in ppc rendering PEPC desensitization. C. glutamicum ATCC13032 mutant strain P with a feedback inhibition-desensitized aspartokinase was used as the parent strain, producing 9.36 g/L lysine from 100 g/L glucose in a jar fermentor culture. Under these conditions, while the simple mutant D2 with pyk deletion or R2 with the PEPC-desensitization mutation showed marginally increased lysine yield (∼1.1-fold, not significant), the mutant DR2 strain having both mutations showed synergistically increased lysine productivity (1.38-fold, 12.9 g/L). Therefore, the pyk deletion is effective under a PEPC-desensitized background, which ensures enhanced supply of OAA, thus clarifying the discrepancies. A citrate synthase defective mutation (S252C in gltA) further increased the lysine yield in strain DR2 (1.68-fold, 15.7 g/L). Thus, these three mutations coordinately enhanced the lysine yield. Both the malate:quinone oxidoreductase activity and respiration rate were significantly reduced in strains D2 and DR2. Overall, these results provide valuable knowledge for engineering the anaplerotic reaction to increase lysine yield in C. glutamicum.
authors.affiliation: Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo, Hokkaido 060-8589, Japan. Electronic address: 1134masa@gmail.com.
authors.forename: Masaki
authors.initials: M
authors.lastname: Yanase
authors.affiliation: Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo, Hokkaido 060-8589, Japan. Electronic address: t-aikoh@chem.agr.hokudai.ac.jp.
authors.forename: Tohru
authors.initials: T
authors.lastname: Aikoh
authors.affiliation: Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo, Hokkaido 060-8589, Japan. Electronic address: kazunori.sawada3@gmail.com.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Sawada
authors.affiliation: Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo, Hokkaido 060-8589, Japan. Electronic address: k-ogura@frontier.hokudai.ac.jp.
authors.forename: Kotaro
authors.initials: K
authors.lastname: Ogura
authors.affiliation: Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo, Hokkaido 060-8589, Japan. Electronic address: t-hagiwara@frontier.hokudai.ac.jp.
authors.forename: Takuya
authors.initials: T
authors.lastname: Hagiwara
authors.affiliation: Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo, Hokkaido 060-8589, Japan. Electronic address: keita_imai@showa-sangyo.jp.
authors.forename: Keita
authors.initials: K
authors.lastname: Imai
authors.affiliation: Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo, Hokkaido 060-8589, Japan. Electronic address: wada@chem.agr.hokudai.ac.jp.
authors.forename: Masaru
authors.initials: M
authors.lastname: Wada
authors.affiliation: Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo, Hokkaido 060-8589, Japan. Electronic address: yokota@chem.agr.hokudai.ac.jp.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yokota
mesh_terms: D001224:Aspartic Acid / Q000378:metabolism / Q000494:pharmacology; D016227:Benzoquinones / Q000378:metabolism; D002244:Carbon / Q000378:metabolism*; D002950:Citrate (si)-Synthase / Q000378:metabolism; D048230:Corynebacterium glutamicum / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D025461:Feedback, Physiological / Q000187:drug effects; D017353:Gene Deletion; D008239:Lysine / Q000096:biosynthesis*; D008293:Malates / Q000378:metabolism; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D062907:Oxaloacetic Acid / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D010641:Phenotype; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics / Q000378:metabolism; D011770:Pyruvate Kinase / Q000172:deficiency* / Q000235:genetics*
chemical_list: D016227:Benzoquinones; D008293:Malates; D050505:Mutant Proteins; D062907:Oxaloacetic Acid; D001224:Aspartic Acid; C004532:quinone; D002244:Carbon; C030298:malic acid; D010088:Oxidoreductases; D002950:Citrate (si)-Synthase; D011770:Pyruvate Kinase; D010730:Phosphoenolpyruvate Carboxylase; D008239:Lysine
keywords: Citrate synthase; Corynebacterium glutamicum; Feedback inhibition; Lysine; Phosphoenolpyruvate carboxylase; Pyruvate kinase