富含蛋氨酸醇溶蛋白在产朊假丝酵母中的表达研究

为构建表达富含蛋氨酸醇溶蛋白的产朊假丝酵母工程菌株，利用基因工程技术，将玉米δ—10kD-醇溶蛋白基因（zein），酵母的GAP启动子（GAP—P）、终止子（GAP—t）拼接合成表达框，以18S rDNA片段为整合介导区．用放线菌酮（CYH）抗性基因作为筛选标记，结合pBR322质粒构建了同源整合表达载体．把zein基因同源重组整合到产朊假丝酵母染色体上，构建表达zein基因的产朊假丝酵母工程菌株。蛋氨酸含量测定结果显示，重组菌株的蛋氨酸表达量比起始菌株提高了17．14％。该产朊假丝酵母工程菌的构建为下一步提高其蛋氨酸产量的研究和应用打下了良好的基础。

Expression of Methionine-rich Zein Protein in Candida utilis

The present study was designed to construct a genetically engineered strain of Candida utilis that expresses methionine-rich zein protein. By using genetic engineering method, a methionine-rich δ-10kD-zein gene of maize endosperm, the promoter （GA P-p） gene and the terminator （GAP-t） gene from yeast were spliced into an expression cassette; subsequently, a homologous recombinant expression vector was constructed with a 18S rDNA fragment （serving as an integrated mediated region）, a cycloheximide （CYH） resistance gene （serving as a selection marker） and the pBR322 plasmid; the zein gene was integrated into the chromosome of Candida utilis by using homologous recombination. The determination results of methionine content by HPLC demonstrated that the expression of methionine in the recombinant strain was increased by 17.14% than the wild one. The constructed genetically engineered strain of Candida utilis provided technological foundation for further study on increasing its production of methionine.