关键词: γ-多聚谷氨酸, 合成基因, SEFA-PCR, 异源表达, 金属离子

Abstract: Poly-γ-glutamic acid（γ-PGA）, a new type of biodegradable material with high molecular weight, is synthesized by many microbes; the construction of genetic engineering strain will be highly valuable to the research of synthesis mechanism and production of γ-PGA. The gene cluster, pgsBCAE, involving in the biosynthesis of γ-PGA in Bacilus amyloliqueficiens C1 were amplified by SEFA-PCR, which were then ligated in expression vector pET29a（+）and transformed into E. coli BL21（DE3）host cells. The recombinant clone, E. coli BL21（pET29a-pgsBCAE）induced by IPTG obtained the ability to synthesize γ-PGA by fermentation in shake flask, and the yield of γ-PGA was 0.14 g/L. Both Mn²⁺ and Zn²⁺ increased γ-PGA production of E. coli BL21（pET29a-pgsBCAE）. With the increase of the concentration of Mn²⁺ and Zn²⁺, this promotion became much more significantly. On the other hand, Mg²⁺ in low concentration（1 mmol/L）also promoted γ-PGA production, the promotion decreased with the increase of Mg²⁺ concentration, while high concentration of Mg²⁺（4 mmol/L）inhibited γ-PGA production of the recombinant clone significantly. The above results suggested that Bacilus amyloliqueficiens C1 contained the intact gene cluster pgsBCAE for the synthesis of γ-PGA, which can be cloned and expressed in E. coli BL21. The production of γ-PGA in the genetic engineering strain was affected by several metal ions.

Key words: poly-γ-glutamic acid, synthesis gene, SEFA-PCR, heterologous expression, metal ions