title: Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032.
abstract: Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0 g/L from 100 g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0 g/L (1.19-fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8 g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9 g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production 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: 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: 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: shimono-yuta@frontier.hokudai.ac.jp.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Shimono
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: Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan. Electronic address: masakazu_sugimoto@ajinomoto.com.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Sugimoto
authors.affiliation: Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan. Electronic address: akiko_oonuki@ajinomoto.com.
authors.forename: Akiko
authors.initials: A
authors.lastname: Onuki
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; D019149:Bioreactors; D001710:Biotin / Q000378:metabolism; D002952:Citric Acid Cycle; D048230:Corynebacterium glutamicum / Q000201:enzymology / Q000378:metabolism*; D025461:Feedback, Physiological; D005947:Glucose / Q000378:metabolism; D018698:Glutamic Acid / Q000096:biosynthesis* / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism / Q000494:pharmacology; D010730:Phosphoenolpyruvate Carboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011770:Pyruvate Kinase / Q000172:deficiency / Q000235:genetics
chemical_list: D007656:Ketoglutaric Acids; D001224:Aspartic Acid; D018698:Glutamic Acid; D001710:Biotin; D011770:Pyruvate Kinase; D010730:Phosphoenolpyruvate Carboxylase; D005947:Glucose
keywords: Anaplerotic pathway; Aspartic acid; Corynebacterium glutamicum; Feed-back inhibition; Glutamic acid; Phosphoenolpyruvate carboxylase; Pyruvate kinase