A novel pyruvate kinase and its application in lactic acid production under oxygen deprivation in Corynebacterium glutamicum

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.

Adaptive laboratory evolution of Corynebacterium glutamicum towards higher growth rates on glucose minimal medium

“…Contrary to the suggested hypothesis, we have not observed an increased glucose uptake rate for strains harboring a less active PK, but for the A271T mutant (Table 3 ). Notably, a simple in-frame deletion of pyk did not result in significant differences in comparison to the wild type regarding growth or glucose consumption as reported by previous studies 42 , 43 . Comparing PK amino acid sequence of C. glutamicum to the published structure of E. coli 44 (sequence identity of 41%) revealed that the two residues T12 and A20 are located in close vicinity to the catalytic center.…”

Metabolic engineering to improve 1,5‐diaminopentane production from cellobiose using β‐glucosidase‐secreting Corynebacterium glutamicum

“…The catalytic activity of Pyk2 was allosterically regulated by fructose 1,6‐bisphosphate activation and ATP inhibition. The mRNA level of pyk2 under oxygen deprivation was higher than that under aerobic conditions (Chai et al, ). However, the effect of its deletion on DAP production has not been reported.…”

“…Here, we deleted pyk2 (cg3218, NCgl2809) and pyk (cg2291, NCgl2008) in the MQN6 strain. The pyk2 gene codes for pyruvate kinase (Chai et al, ). The catalytic activity of Pyk2 was allosterically regulated by fructose 1,6‐bisphosphate activation and ATP inhibition.…”

“…The titer of l ‐lysine produced from 50 g/L cellobiose using the MQN8 strain with only the BGL expression was 12.9 g/L (data not shown), which was higher than that of MQN‐6 strain (9.7 g/L; data now shown). Chai et al () reported that pyk2 and ldhA ( l ‐lactate dehydrogenase) were cotranscribed as a bicistronic mRNA under aerobic condition. In the case of MQN6‐6L6B and MQN8‐6L6B, both strains had the same levels of ldh activity in the crude extract (data not shown), suggesting less effect of pyk2 deletion on ldh activity.…”

Lactic acid production – producing microorganisms and substrates sources-state of art

“…Moreover, recently Chai et al. [ 51 ] found NCgl2809 as another novel pyruvate kinase (Pyk2) in C. glutamicum. These authors grew an engineered C. glutamicum containing Pyk1 or Pyk2 on D-ribose conditions, being the LA production enhanced by overexpression of either Pyk1 or Pyk2, due to the increase of the activity of the Pyk enzyme.…”