增加植酸酶基因appA-m的拷贝提高其在巴斯德毕赤酵母的表达量

Improving Phytase Expression by Increasing the Gene Copy Number of appA-m in Pichia pastoris

为了进一步提高植酸酶的发酵效价,降低植酸酶生产成本,对毕赤酵母表达载体pGAPZα-A进行了改造。将表达载体pPIC9的AOX1启动子序列引入pGAPZα-A,使之成为甲醇可诱导型表达载体pAOXZα,插入植酸酶基因appA-m后得到重组载体pAOXZα-appA-m。以染色体上带有一个拷贝的appA-m基因、发酵效价可达到7.5×106IU/mL发酵液的重组酵母菌株74#为受体菌进行转化,在该重组菌株的染色体上的另一位点整合含有植酸酶基因的表达盒,经筛选到高表达植酸酶的重组子。通过PCR进行验证,植酸酶基因被整合到重组酵母的染色体上,且受体菌中原有的植酸酶基因结构未改变。重组菌在5L发酵罐经甲醇诱导120h植酸酶蛋白表达量达到4mg/mL发酵液,酶活性(发酵效价)达到1.2×107IU/mL发酵液以上,较含单拷贝植酸酶基因的受体菌株表达量有较大程度提高。PCR检测及表达量分析证明改良的菌株具有很好的遗传稳定性和表达稳定性。

In order to improve the fermentation potency of phytase in recombinant host and decrease the production cost, the pichia expression vector pGAPZα-A was modified by introduction of an AOX1 promoter from vector pPIC9 and the resulted vector pAOXZα is an methanol induced vector. After that, a phytase gene appA-m was cloned into pAOXZα to construct the recombinant vector pAOXZα-appA-m. The recombinant Pichia pastoris 74^# , which already contains one copy of appA-m and its fermentation potency exceeded 7.5 ×10^6IU/mL, was used as the host strain for the transformation of pAOXZα-appA-m. The Pichia pastoris transformants were gained by electroporation. PCR results indicated that the appA-m expression box has integrated into the genome of Pichia pastoris and the original construction of phytase gene has not changed. SDS-PAGE analysis revealed that phytase was overexpressed and secreted into the medium supornatant. Recombinants with high expression level were screened and used for fermentation. In 5L fermentor, the expression level of phytase protein achieved 4mg/mL and the phytase activity （fermentation potency） exceeded 1.2 ×10^7IU/mL, which was about 1.6-fold compared with that of the host strain 74^# . Moreover, the improved recombinant Pichia pastoris is excellent at expression stability and heredity stability.