植酸酶基因中稀有密码子的改造提高其在毕赤酵母中的表达量

Increasing Expression Level of Phytase Gene(phyA) in Pichia pastoris by Changing Rare Codons

对来源于黑曲霉N2 5(AspergillusnigerChinaStrain)的植酸酶基因phyA进行PCR介导的定点突变 ,不改变其所编码氨基酸 ,选用毕赤酵母偏爱的密码子对该基因保守序列中第 81位和第 85位的Arg密码子进行同义突变 .构建了含正确突变的克隆载体pUC18 phyAm 和酵母表达载体pPIC9k phyAm,电击转化毕赤酵母 ,经MM、MD平板筛选和产物的酶活性测定 ,筛选出突变与未突变高酶活酵母转化子各 2株 .这 4株转化子的Southern印迹结果表明 ,phyA基因以单交换方式单拷贝整合到酵母染色体DNA中 .表达产物的SDS PAGE分析表明 ,重组酵母中的植酸酶能有效分泌和表达 ,蛋白质分子大小为 70 15kD .转化子酶活测定结果表明 ,经密码子优化的突变重组酵母酶活力明显高于未进行优化的重组酵母转化子 .经密码子优化的突变重组酵母株PP NPm 8于麦芽汁培养基中诱导 36h后酶活力可达 4 76 0 0U/ml,其活力比未优化重组酵母株PP NP 2 (2 36 6 7U/ml)提高了约 1倍 ,且重组转化子遗传稳定性良好 .

By using long-distance inverse PCR, Arg(cgt) and Arg(cgg) in the expression fragment of phytase phyA gene were mutated synonymously to Arg(aga),which is a bias code of yeast.The mutated gene phyA m was cloned into pUC18 vector, and the two mutated sites were confirmed by sequencing.The expression plasmid pPIC9k-phyA m was constructed and transformed into GS115 strain. By riddling of phenotypes and phytase activities, two high activity strains of mutated and unmutated transformants were selected. Southern blotting analysis to the four yeast transformants showed that phyA gene was intergrated into the chromosome genome through single-crossover events with one copy of phyA gene.SDS-PAGE analysis suggested that the expression product could be secreted and over-expressed, the size of protein was 70.15 kD. The phytase activity of PP-NP m-8 with codons optimized reached 47 600 U/ml in malt wort culture medium after being induced with 36 h, which was the double of the original strain PP-NP-2. Additionally, PP-NP m-8 showed the quite good genetic stability.