Impact of gene modification of phosphotransferase system on expression of glutamate dehydrogenase protein of Streptococcus suis in Escherichia coli

Abstract

Glutamate dehydrogenase (GDH) protein of Streptococcus suis can be used for detection of S. suis infection and protection of pigs against S. suis infection. Acetate is a primary inhibitory metabolite for cell growth and formation of GDH protein. In this study, the ptsG gene, which encodes the integral membrane permease IICB Glc in the phosphotransferase system, was deleted and the effect of this deletion on the expression of GDH protein was investigated. The plasmids containing glf Z. mobilis (encoding glucose facilitator)-glk E. coli (encoding glucokinase) or galP E. coli (encoding galactose permease)-glk E. coli were transformed into ptsG mutant cells to recover the cell growth and glucose utilization of ptsG mutant. The mutants with deletion of ptsG decreased the accumulation of acetate; and higher cell density and GDH protein concentration were obtained with the ptsG mutants containing glf-glk or galP-glk. When the ptsG mutant containing glf-glk (SSGGFK) was used for expression of GDH protein, the cell density (optical density OD 600 of 2.68) and the concentration of GDH protein (42.34 mg/L) were highest, with an increase by 12.61% and 14.84%, respectively, compared with the parental strain (SSG). The acetate accumulation was reduced to 2.35 g/L, i.e. a 37.33% decrease compared with the SSG strain. High concentration of GDH protein was obtained with reduction of acetate accumulation through gene modification of the phosphotransferase system. This can decrease the production cost of the subunit vaccine of GDH protein and provide theoretical foundation for high-level expression of other recombinant proteins.

Metabolic perturbations in mutants of glucose transporters and their applications in metabolite production in Escherichia coli

“…First, we attempted to take advantage of these effects in the production of recombinant proteins because the host strain produced less acetate but yielded enough amino acids and ATP. Several research groups have successfully reduced acetate production, with a subsequent increase in the production of recombinant proteins, such as DNA vaccines and glutamate dehydrogenase, in ptsG mutants [31,65]. The plasmid containing the EGFP encoding gene under a constitutive promoter, was introduced into ST1, ST2, and ST8 and the resulting strains were named as STE1, STE2, and STE8, respectively.…”

Strategies for efficient production of recombinant proteins in Escherichia coli: alleviating the host burden and enhancing protein activity

“…The GDH expression exhibited a 298-fold increase [35] T7 RNAP activity regulation Single amino acid mutation in T7 RNAP (A102D), which reduced the ability to bind to the P T7 and decreased the RP production rate Seven membrane proteins with varying degrees of production improvement (data not mentioned in the article) [37] T7 RNAP activity regulation The production of pramlintide increased by 40% (protein concentration of 3.09 ± 0.12 g/L) [24] Blocking the phosphotransferase system Blocking the phosphotransferase system (PTS) by knockout ptsG or integration ptsG mutants. can effectively reduce the rate of glucose uptake and decrease the production of acetate A variety of RPs were increased in yield, including eGFP (increased by 282%), vaccines (increased by 3.5-fold) and glutamate dehydrogenase (increased by 14.84%) [61][62][63] Blocking the cellular stress response Transcriptome analysis to identify genes associated with cellular stress response and knockout them. The blocking of CSR alleviates the downregulated expression of a variety of growthessential genes…”

“…The accumulation of acetate is an important factor in the RP production, since it inhibits cell growth and protein synthesis [59]. Blocking the phosphotransferase system (PTS) can effectively reduce the rate of glucose uptake and decrease the production of acetate, which has been applied to increase the production of enhanced GFP (eGFP) [61], vaccines [62], and glutamate dehydrogenase [63]. In addition, knocking out flagellar formation-related genes can reduce energy consumption in E. coli.…”