密码子优化的植酸酶基因appA-P在毕赤酵母中的高效表达

High expression of codon optimized phytase-encoding gene appA-P in Pichia Pastoris

根据毕赤酵母（Pichia pastoris）密码子使用偏爱性,对Escherichia coli来源的高比活植酸酶基因（appA）全面地进行密码子优化,人工合成了植酸酶基因（appA-P）,并将该基因克隆到毕赤酵母诱导型分泌表达载体pHBM905A上,获得重组质粒pHBM905A-appA-P,通过PEG1000转化法将线性化的重组质粒转化毕赤酵母GS115菌株,筛选得到一个高效表达植酸酶的重组菌株GS115-appA-P,在25℃摇瓶培养168 h后酶活力达到422 U/mL,该植酸酶最适反应温度为60℃,最适反应pH为4.5,在20-50℃、pH1.0-6.0范围内较稳定.

According to the bias in codon choice of the high expression gene in Pichia pastoris, the gene encoding the mature portion of Escherchia coli phytase（AppA） with high specific activity was optimized and artificially synthesized without changing the amino acid sequence, and the modified gene was inserted into the Pichia pastoris expression vector pHBM905A, then the linearized recombinant plasmid, pHBM905A-appA-P, was introduced into Pichia pastoris GS115 by the PEG1000 transformation method. A high expression recombinant Pichia pastoris GS115-appA-P was obtained. After induced by 1%（V/V） methanol for 168 h at 25℃, the activity of phytase achieved 422 U/mL in the supernatant of the 100 mL shaking flask culture, which was among the highest of those recombinant strains reported now. The phytase expressed by GS115-appA-P has a temperature optimum of 60℃ and a pH optimum of 4. 5, and are active at temperature ranged from 20℃ to 50℃ and pH ranged from 1.0 to 6. 0.