米黑根毛霉脂肪酶基因的酵母重组表达和Kex2位点改造

The Recombinant Expression and Kex2 Cleavage Site Modification of Rhizomucor miehei Lipase in Pichia pastoris

米黑根毛霉脂肪酶(RML)具有较高的合成活性、热稳定性、溶剂耐受性以及sn-1,3位置专一性等一系列适合工业化推广的特点。本研究通过密码子优化改造RML编码基因,并将其克隆、转化到毕赤酵母GS115菌株,经诱导发酵156 h,酶活达到3.77 U/mL。为了进一步改善RML脂肪酶的重组表达,通过定点突变的方式去掉潜在的Kex2酶切位点,结果显示:RML突变体表达量均显著低于野生型的RML脂肪酶,表明"-Arg196-Arg197-"序列并不影响RML的重组表达。通过对RML蛋白结构分析,Kex2酶切位点的肽键被包埋在蛋白内部,这表明该位点区域的折叠在进入分泌途径时已基本完成。本研究对异源蛋白的酵母重组表达具有一定参考价值。

Rhizomucor miehei lipase(RML) has many advantages like high synthetic activity, thermostability, solvent tolarence and sn-1,3 specificity, which make itself very suitable for industrial usage. In this study, the modified RML coding sequence through codon optimization has been cloned and transformed into Pichia pastoris GS115 strain. After 156 h fermentation in flask, the lipase activity of recombinant RML strain reached 3.77 U/mL in the supernatant. To improve the RML expression in P. pastoris, a potential Kex2 cleavage sequence Arg196-Arg197 was modified by site-directed mutatgenesis. However, the result showed that the variants displayed lower expression levels than wild type, indicating that the potential Kex2 site would not be cleaved in the secretary pathway, which might be attributed to the deep location of the Kex2 site inside the folding structure of RML. This implies that the folding process of RML has basically completed before it enters the secretary pathway of P. pastoris. This study has a certain valuable reference for recombinant expression of other heterologous proteins in P. pastoris.