Characterization of 3-phosphoglycerate kinase from Corynebacterium glutamicum and its impact on amino acid production

Abstract

BackgroundCorynebacterium glutamicum cg1790/pgk encodes an enzyme active as a 3-phosphoglycerate kinase (PGK) (EC 2.7.2.3) catalyzing phosphoryl transfer from 1,3-biphosphoglycerate (bPG) to ADP to yield 3-phosphoglycerate (3-PG) and ATP in substrate chain phosphorylation.ResultsC. glutamicum 3-phosphoglycerate kinase was purified to homogeneity from the soluble fraction of recombinant E. coli. PGKHis was found to be active as a homodimer with molecular weight of 104 kDa. The enzyme preferred conditions of pH 7.0 to 7.4 and required Mg2+ for its activity. PGKHis is thermo labile and it has shown maximal activity at 50–65°C. The maximal activity of PGKHis was estimated to be 220 and 150 U mg-1 with KM values of 0.26 and 0.11 mM for 3-phosphoglycerate and ATP, respectively. A 3-phosphoglycerate kinase negative C. glutamicum strain ∆pgk was constructed and shown to lack the ability to grow under glycolytic or gluconeogenic conditions unless PGK was expressed from a plasmid to restore growth. When pgk was overexpressed in L-arginine and L-ornithine production strains the production increased by 8% and by 17.5%, respectively.ConclusionUnlike many bacterial PGKs, C. glutamicum PGK is active as a homodimer. PGK is essential for growth of C. glutamicum with carbon sources requiring glycolysis and gluconeogenesis. Competitive inhibition by ADP reveals the critical role of PGK in gluconeogenesis by energy charge. Pgk overexpression improved the productivity in L-arginine and L-ornithine production strains.

Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

“…3-Phosphoglycerate kinase (3-PGK) uses a special "energy-rich" intermediate, 1,3-bisphosphoglycerate, to produce an additional 1 mol of ATP for a net balance of 2 mol of ATP per mol of glucose (32). Homodimeric 3-PGK in C. glutamicum is tightly regulated by the ADP concentration (K i , Ϸ100 M) and plays a critical role in gluconeogenesis as well as being essential for glycolysis (33). To study the role of the absence of SLP in the glycolysis of C. glutamicum, SLP via 3-PGK was bypassed by replacing endogenous GAPDH with GapN.…”

A RecET-assisted CRISPR–Cas9 genome editing in Corynebacterium glutamicum

“…Downregulation of gap , pgk and gpmA could decrease the split-flow of glyceraldehyde phosphate and drive more carbon fluxes towards 1,2-propanediol synthesis. Among three genes, pgk encoding phosphoglycerate kinase is essential for growth of C. glutamicum on carbon sources requiring glycolysis and gluconeogenesis, and a major control point in the glycolytic pathway during growth on glucose [ 37 ]. The effects of decreased Pgk flux on cell metabolism and 1,2-propanediol synthesis were simulated by i CW773 using FBA.…”

Engineering Corynebacterium glutamicum for fast production of l-lysine and l-pipecolic acid

“…b Means and standard deviations of three replicates are given control by global regulators such as ArcA (Perrenoud and Sauer 2005). Overexpression of genes encoding glycolytic enzymes as applied in E. coli (Xie et al 2014;Seol et al 2015), B. subtilis ), or S. coelicolor (Borodina et al 2008) was also used for C. glutamicum, e.g., to improve the production of high value compounds like amino acids (Yamamoto et al 2012;Reddy and Wendisch 2014), alcohols (Jojima et al 2015;Yamamoto et al 2013), or the organic acid D-lactate (Tsuge et al 2015). Under these conditions or when a repressor gene of glycolysis genes was deleted, i.e., that of the DeoR-type transcriptional regulator SugR, high titers of L-lactate were formed as side effect of faster glucose utilization (Engels and Wendisch 2007;Engels et al 2008;Teramoto et al 2011).…”