title: A novel pyruvate kinase and its application in lactic acid production under oxygen deprivation in Corynebacterium glutamicum.
abstract: Pyruvate kinase (Pyk) catalyzes the generation of pyruvate and ATP in glycolysis and functions as a key switch in the regulation of carbon flux distribution. Both the substrates and products of Pyk are involved in the tricarboxylic acid cycle, anaplerosis and energy anabolism, which places Pyk at a primary metabolic intersection. Pyks are highly conserved in most bacteria and lower eukaryotes. Corynebacterium glutamicum is an industrial workhorse for the production of various amino acids and organic acids. Although C. glutamicum was assumed to possess only one Pyk (pyk1, NCgl2008), NCgl2809 was annotated as a pyruvate kinase with an unknown role.
  
  
  
  Here, we identified that NCgl2809 was a novel pyruvate kinase (pyk2) in C. glutamicum. Complementation of the WTΔpyk1Δpyk2 strain with the pyk2 gene restored its growth on D-ribose, which demonstrated that Pyk2 could substitute for Pyk1 in vivo. Pyk2 was co-dependent on Mn2+and K+and had a higher affinity for ADP than phosphoenolpyruvate (PEP). The catalytic activity of Pyk2 was allosterically regulated by fructose 1,6-bisphosphate (FBP) activation and ATP inhibition. Furthermore, pyk2 and ldhA, which encodes L-lactate dehydrogenase, were co-transcribed as a bicistronic mRNA under aerobic conditions and pyk2 deficiency had a slight effect on the intracellular activity of Pyk. However, the mRNA level of pyk2 in the wild-type strain under oxygen deprivation was 14.24-fold higher than that under aerobic conditions. Under oxygen deprivation, pyk1 or pyk2 deficiency decreased the generation of lactic acid, and the overexpression of either pyk1 or pyk2 increased the production of lactic acid as the activity of Pyk increased. Fed-batch fermentation of the pyk2-overexpressing WTΔpyk1 strain produced 60.27 ± 1.40 g/L of lactic acid, which was a 47% increase compared to the parent strain under oxygen deprivation.
  
  
  
  Pyk2 functioned as a pyruvate kinase and contributed to the increased level of Pyk activity under oxygen deprivation.
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China.|University of Chinese Academy of Sciences, Beijing, 100049, People's Republic of China.
authors.forename: Xin
authors.initials: X
authors.lastname: Chai
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China.
authors.forename: Xiuling
authors.initials: X
authors.lastname: Shang
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China.|University of Chinese Academy of Sciences, Beijing, 100049, People's Republic of China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China.
authors.forename: Shuwen
authors.initials: S
authors.lastname: Liu
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Liang
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China. zhangyun@im.ac.cn.
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China. wenty@im.ac.cn.|Savaid Medical School, University of Chinese Academy of Sciences, Beijing, 100049, People's Republic of China. wenty@im.ac.cn.
authors.forename: Tingyi
authors.initials: T
authors.lastname: Wen
mesh_terms: D048230:Corynebacterium glutamicum / Q000378:metabolism*; D004789:Enzyme Activation; D051418:Focal Adhesion Kinase 2 / Q000378:metabolism*; D019344:Lactic Acid / Q000096:biosynthesis*; D010100:Oxygen / Q000378:metabolism*
chemical_list: D019344:Lactic Acid; D051418:Focal Adhesion Kinase 2; D010100:Oxygen
keywords: Corynebacterium glutamicum; Fructose 1,6-bisphosphate; Lactate dehydrogenase; Oxygen deprivation; Pyruvate kinase