title: Boosting Anaplerotic Reactions by Pyruvate Kinase Gene Deletion and Phosphoenolpyruvate Carboxylase Desensitization for Glutamic Acid and Lysine Production in Corynebacterium glutamicum.
abstract: In the 1980s, Shiio and coworkers demonstrated using random mutagenesis that the following three phenotypes were effective for boosting lysine production by Corynebacterium glutamicum: (1) low-activity-level citrate synthase (CSL), (2) phosphoenolpyruvate carboxylase (PEPC) resistant to feedback inhibition by aspartic acid (PEPCR), and (3) pyruvate kinase (PYK) deficiency. Here, we reevaluated these phenotypes and their interrelationship in lysine production using recombinant DNA techniques.The pyk deletion and PEPCR(D299N in ppc) independently showed marginal effects on lysine production, but both phenotypes synergistically increased lysine yield, demonstrating the importance of PEPC as an anaplerotic enzyme in lysine production. Similar effects were also found for glutamic acid production. CSL(S252C in gltA) further increased lysine yield. Thus, using molecular techniques, the combination of these three phenotypes was reconfirmed to be effective for lysine production. However, a simple CSLmutant showed instabilities in growth and lysine yield.Surprisingly, the pyk deletion was found to increase biomass production in wild-type C. glutamicum ATCC13032 under biotin-sufficient conditions. The mutant showed a 37% increase in growth (based on OD660) compared with the ATCC13032 strain in a complex medium containing 100 g/L glucose. Metabolome analysis revealed the intracellular accumulation of excess precursor metabolites. Thus, their conversion into biomass was considered to relieve the metabolic distortion in the pyk-deleted mutant. Detailed physiological studies of various pyk-deleted mutants also suggested that malate:quinone oxidoreductase (MQO) is important to control both the intracellular oxaloacetic acid (OAA) level and respiration rate. These findings may facilitate the rational use of C. glutamicum in fermentation industries.
authors.affiliation: Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo, 060-8589, Japan. yokota@chem.agr.hokudai.ac.jp.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yokota
authors.affiliation: Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo, 060-8589, Japan.
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, 060-8589, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Wada
mesh_terms: D001688:Biological Products / Q000138:chemical synthesis / Q000378:metabolism; D019149:Bioreactors / Q000382:microbiology; D048230:Corynebacterium glutamicum / Q000502:physiology*; D004789:Enzyme Activation; D005285:Fermentation / Q000502:physiology; D017353:Gene Deletion; D024861:Genetic Enhancement / Q000379:methods; D018698:Glutamic Acid / Q000096:biosynthesis* / Q000235:genetics; D008239:Lysine / Q000096:biosynthesis* / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods*; D064688:Metabolic Flux Analysis / Q000379:methods; D053858:Metabolic Networks and Pathways / Q000502:physiology; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics / Q000378:metabolism*; D011770:Pyruvate Kinase / Q000378:metabolism*
chemical_list: D001688:Biological Products; D018698:Glutamic Acid; D011770:Pyruvate Kinase; D010730:Phosphoenolpyruvate Carboxylase; D008239:Lysine
keywords: Anaplerotic pathway; Citrate synthase; Corynebacterium glutamicum; Feedback inhibition; Glutamic acid; Lysine; Malate:quinone oxidoreductase; Phosphoenolpyruvate carboxylase; Pyruvate kinase; Respiration