Abstract
ABSTRACTGlutamate 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 IICBGlc in the phosphotransferase system, was deleted and the effect of this deletion on the expression of GDH protein was investigated. The plasmids containing glfZ. mobilis (encoding glucose facilitator)–glkE. coli (encoding glucokinase) or galPE. coli (encoding galactose permease)–glkE. 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 OD600 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.
Highlights
The glutamate dehydrogenase (GDH) of Streptococcus suis is conserved and antigenic, as it reacts with serum from animals with S. suis type 2 infection, leading to it being utilized as a serological assay for diagnosis of S. suis infection [1,2]
The accumulation of acetate is due to the combined high rates of glucose uptake and glucose catabolism by the Embden– Meyerhof–Parnas pathway, resulting in a rate of acetylcoenzyme A synthesis surpassing the capacity of the tricarboxylic acid (TCA) cycle to completely consume this metabolite [6,7]
The deletion of ptsG impaired the capacity for cell growth and glucose consumption, which resulted in PARG not being used as a host strain for GDH protein expression [9]
Summary
The glutamate dehydrogenase (GDH) of Streptococcus suis is conserved and antigenic, as it reacts with serum from animals with S. suis type 2 infection, leading to it being utilized as a serological assay for diagnosis of S. suis infection [1,2]. It has been reported that the subunit vaccination based on GDH protein of S. suis is an effective strategy for protecting pigs against S. suis infection [3] but its market is limited because of the high cost. Improvement of the GDH protein by Escherichia coli can decrease the production cost and expand the application market of GDH subunit vaccine, leading to protection of the pig production industry and public health. There are examples where the expression of recombinant protein was significantly improved by decreasing the accumulation of acetate [4]. The accumulation of acetate is due to the combined high rates of glucose uptake and glucose catabolism by the Embden– Meyerhof–Parnas pathway, resulting in a rate of acetylcoenzyme A synthesis surpassing the capacity of the tricarboxylic acid (TCA) cycle to completely consume this metabolite [6,7]. Modification of the glucose transport capacity is a successful approach for decreasing the excretion of acetate under aerobic conditions [7]
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