甜蛋白Monellin在毕赤酵母中的表达及响应面法优化发酵培养基

Expression of Monellin in Pichia pastoris and optimization of fermentation medium with response surface methodology

根据毕赤酵母密码子偏好,人工合成了单链甜蛋白Monellin基因,构建了胞内表达载体pPICZA-Mon,重组载体经Sac I线性化后,电击转入毕赤酵母GSll5,转化子经PCR分析鉴定后通过甲醇诱导实现了甜蛋白Monellin在毕赤酵母中的胞内表达。采用Design Expert 7.0软件,利用响应面法对毕赤酵母盐培养基进行优化。首先用Plackett-Burman方法研究发酵培养基各成分对响应值的影响,结果表明酵母提取物、硫酸钙和磷酸缓冲液为3个主要因素;利用最陡爬坡法逼近最大响应区域;最后通过中心组合设计和响应面分析得到最优培养基组成为0.6%酵母提取物,0.03%硫酸钙,0.45%硫酸镁,4%甘油,1.25%硫酸铵,7.7%100mmol/L磷酸缓冲液(pH值为6.0)。在优化培养基条件下,工程菌生物量增加0.5倍,蛋白表达量提高近60%。

According to Pichia pastoris preference of codon usage, single-strand Monellin gene was synthesized and intracellular expression vector pPICZA-Mon was constructed. The recombinant plasmid was linearized with Sacl and transformed into GS115 strain by electroporafion. It was confirmed with PCR that Monellin gene was integrated into the yeast genome and results of SDS-PAGE indicated that Monellin was expressed after induction with methanol. Salt medium for P. pastors was optimized with response surface methodology （RSM） and software Design Expert 7.0. Effect of composition of initial medium was studied with Plackett-Burma design. The results showed that yeast extract, calcium sulfate and potassium phosphate buffer were the main factors. The path of steepest ascent was used to approach the optimal response area. The optimum composition of medium obtained with experimental design of Box-Behnken and RSM were as follows： yeast extract 0.6%, calcium sulfate 0.03%, magnesium sulfate 0.45%, glycerol 4%, ammonium sulfate 1.25%, 100mmol/L potassium phosphate buffer （[oH value 6.0） 7.7%. In the optimum medium, biomass of yeast could increase by 0.5 fold and expression of Monellin could increase by 60%.