过量表达气囊蛋白强化茂原链霉菌合成胰蛋白酶

Enhanced trypsin production in Streptomyces mobaraensis by overexpressing gas vesicle protein

气囊蛋白(gas vesicle protein, Gvp)组成的气体囊泡存在于蓝藻、嗜盐古菌等微生物中,为细胞提供浮力或抗胁迫能力。尽管gvp基因簇广泛存在于链霉菌,但其功能尚不明确。在该研究中,分别在茂原链霉菌(Streptomyces mobaraensis)DSM40587谷氨酰胺转氨酶基因缺失菌株(smY2019Δtg)中表达了内源的gvpA、gvpO以及完整基因簇gvpOAFGJLSK(gvp40587),考察其对胰蛋白酶合成和宿主生理状态的影响。酶活力分析显示,过量表达gvp40587和gvpA使胞外最高胰蛋白酶活力分别提高64.3%和17.0%;过量表达gvpO未明显影响胰蛋白酶活力。SDS-PAGE分析发现,gvp40587和gvpA过表达菌株胞外上清液中的胰蛋白酶条带(26 kDa)较对照菌株明显增粗。同时,过量表达完整基因簇的菌株中8个gvp基因的转录水平均较smY2019Δtg有不同程度的提高。与对照菌相比,gvp40587过表达菌株的胞内ATP含量和NADPH/NADP^(+)分别提高1.4倍和0.4倍。上述结果表明,过量表达内源gvp基因簇能促进S.mobaraensis合成胰蛋白酶,能量代谢和蛋白质前体合成的增强可能是其产量提高的主要原因。研究结果首次报道了S.mobaraensis中gvp基因簇对酶蛋白合成的重要影响,为链霉菌蛋白表达调控提供了新思路。

Gas vesicles,which are composed of gas vesicle protein(Gvp),exist in Cyanobacteria,halophilic Archaea,and other microorganisms,providing buoyancy or stress resistance for cells.Although gvp gene cluster widely exists in Streptomyces strains,its function is still unclear.In this study,the endogenous gvpA,gvpO,and complete gene cluster gvpOAFGJLSK(gvp 40587)were expressed in a transglutaminase gene deleted strain(smY2019Δtg)respectively to investigate their effects on the trypsin synthesis and the physiological status of the host.As indicated by enzyme activity analysis,overexpressing gvp 40587 and gvpA increased the highest extracellular trypsin activity by 64.3%and 17.0%,respectively;the gvpO overexpression had little effect on trypsin activity.SDS-PAGE analysis showed that the trypsin bands(26 kDa)in the extracellular supernatant of gvp 40587 and gvpA overexpression strains were significantly thicker than that of the control strain.In the strain over-expressing the complete gvp gene cluster,the transcriptional levels of the eight gvp genes were increased to different degrees.In addition,the intracellular ATP content and NADPH/NADP^(+)of the gvp 40587-overexpressed strain increased 1.4 and 0.4 times than those of the control strain,respectively.These results showed that the overexpression of endogenous gvp gene cluster could improve the trypsin synthesis by S.mobaraensis,and the increases in energy metabolism and protein precursor synthesis may be the main reason for the increased yield.This is the first report regarding the important role of gvp gene in the enzyme production in S.mobaraensis,providing new ideas for the regulation of protein expression in Streptomyces.