来源于Selenomonas ruminantium的高比活植酸酶基因在毕赤酵母中的高效表达

Overexpression of Selenomonas ruminantium Phytase with High Specific Activity in Pichia pastoris

应用酶解效率更高的高比活植酸酶是进一步提高植酸酶发酵效价、降低植酸酶生产成本的有效途径。根据毕赤酵母对于密码子的选择偏向,对来源于原核瘤胃微生物Selenomonasruminantium的高比活植酸酶基因phyS进行了密码子优化,将优化后的植酸酶基因phyS(m)与未优化的phyS插入到毕赤酵母转移载体pPIC9中,转化毕赤酵母得到重组子,在摇瓶水平上,phyS(m)的表达水平比phyS高10倍。在5L发酵罐中,优化后的植酸酶基因phyS(m)其蛋白表达量达到4mgml-1发酵液,效价达到1.6×106IUml-1,高于目前报道的各种植酸酶基因工程菌株的发酵效价。

Utilization of the phytase with high specific activity is a useful way to improve the fermentation potency ofphytase in recombinant host and decrease the production cost. The gene phyS encoding phytase with high specific activityfrom ruminal bacteria Selenomonas ruminantium was modified for the optimal expression according to bias in codon choiceof high expression gene in Pichia pastoris. Further, the modified phytae gene phyS(m) and the origin phytase gene phySwithout origin signal peptide sequence were cloned into the plasmid pPIC9 under the control of AOX1 promoter andintroduced into the host Pichia pastoris by electroporation, respectively. The concentration of the phytase expressed byP. pastoris with modified gene phyS(m) was 10 times higher than that of recombinant P. pastoris with non-modified genephyS in shake culture. In 5-liter fermentor in fed-batch fermentation, the expression level of phytase in recombinant P.pastoris with modified gene phyS(m) was increased to 4mgml-1 and 1.6106IUml-1, which was the highest in fermentationpotency among all of the reported recombinant strain.