Abstract
BackgroundPyruvate 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.ResultsHere, 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.ConclusionsPyk2 functioned as a pyruvate kinase and contributed to the increased level of Pyk activity under oxygen deprivation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0313-6) contains supplementary material, which is available to authorized users.
Highlights
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
Enzymatic properties of Pyk2 To detect the activity of Pyk2 in vitro, the pyk2 gene of C. glutamicum was expressed in E. coli BL21 (DE3) using the pET-28a expression system
In the present study, we identified that Pyk2 is a novel pyruvate kinase in C. glutamicum and has significantly different biochemical properties than Pyk1
Summary
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. In S. cerevisiae, FBP-activated Pyk1p was the major enzyme that catalyzed the conversion of PEP to pyruvate, whereas Pyk2p with relatively low enzymatic activity was insensitive to FBP and subject to glucose repression, which suggested that Pyk2p played a major role under conditions of low glycolytic flux [8]. Little is known about other Pyk isoenzymes and their physiological function in other bacteria
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