耐盐性谷氨酰胺酶在Bacillus subtilis 168中的整合表达

Over-Expression of Salt-Tolerant Glutaminase by Integration of Mglu Genes in Bacillus subtilis 168

摘要:

微生物质粒过表达外源基因,会由于质粒分离稳定性低、种子培养基中需添加抗生素等原因,限制其在工业生产中的应用。为了得到一株可用于工业生产谷氨酰胺酶的菌株,选择食品安全级Bacillus subtilis 168作为宿主,将来源于Micrococcus luteus K-3的编码耐高浓度盐的谷氨酰胺酶基因（Mglu）插入到其染色体上进行整合表达。选择了2个内源性蛋白酶基因作为整合表达位点,通过lox序列的重组消除抗性基因,得到无抗性基因的重组谷氨酰胺酶表达菌株。遗传稳定性研究表明,在不添加抗生素的条件下,通过连续传代重组菌株并测定发酵酶活,发现重组菌株连续传42代时,酶活基本不变。随后,采用5 L发酵罐对重组菌株进行分批补料发酵,最高酶活达到了41.5 U/mL。本研究对提高谷氨酰胺酶重组菌株遗传稳定性提供了借鉴。

Abstract:

Heterologous over-expression of glutaminase in microorganisms has been extensively studied and its application in industrial production has been limited by its poor separation stability under nonresistant conditions with free plasmids. To obtain a strain used in industrial glutaminase production, glutaminase（from Micrococcus luteus K-3） was successfully over-expressed by integration of the Mglu gene in chromosome of GRAS strain Bacillus subtilis 168. Two genes encoding specific protease were blocked by integration of Mglu gene, and the zeo resistance marker was deleted by Cre recombinase catalyzing the recombination of DNA between lox sequences. After continuous cell culture without adding any antibiotics, the enzyme activity was basically unchanged when the recombinant strain was transmitted for 42 generations. By using 5 L bioreactor for fed-batch fermentation of recombinant glutaminase producing strain, the highest enzyme activity was about 41.5 U/mL. This study provides a reference for improving the genetic stability of glutaminase recombinant strain.