一种增加毕赤酵母生产胰岛素前体的方法

A method to increase the production of insulin precursor in Pichia pastoris

摘要:

为了提高胰岛素前体 (PI) 的产量，构建了pPIC9K-PI表达载体并电转化至毕赤酵母菌株GS115中，在浓度为4.0 mg/mL的G418抗性平板上筛选到了1株拷贝数为12的菌株CL012。将SNAREs (可溶性N-乙基马来酰亚胺敏感因子受体蛋白) 组分中的SNC2和SNC2-SSO2分别转入菌株CL012中，并在摇瓶和5 L发酵罐水平上检测SNAREs对PI产量的影响。结果表明：摇瓶水平上，甲醇诱导96 h后，菌株CL012的PI产量为1.53 mg/L；表达SNC2和SNC2-SSO2的菌株的PI产量分别为1.89 mg/L和2.21 mg/L，分别比菌株CL012提高了23.53%和44.44%。在5 L发酵罐上进行高密度发酵，甲醇诱导96 h后菌株CL012的PI产量为53 mg/L，是摇瓶水平的34.64倍；表达SNC2和SNC2-SSO2的菌株的PI产量分别达到64 mg/L和78 mg/L，分别比菌株CL012提高了20.75%和47.17%。由此得出结论SNAREs可以促进胰岛素前体的分泌，从而提高在毕赤酵母中的异源表达。

Abstract:

To improve the yield of insulin precursor (PI), we constructed a recombinant expression vector pPIC9K-PI and transformed it into Pichia pastoris GS115 using electroporation. After screening, a mutant strain CL012 with 12 copies was obtained on the YPDS plate containing 4.0 mg/mL G418. Then, the components of SNAREs (Soluble N-ethylmaleimide-sensitive factor attachment receptor proteins), SNC2 and SNC2-SSO2, were expressed in the strain CL012 to explore the effect of SNAREs on the yield of PI. In shake flask culture, the strains expressing SNC2 and SNC2-SSO2 yielded PI of 1.89 mg/L and 2.21 mg/L after methanol induction for 96 h, which were improved by 23.53% and 44.44% compared to that of strain CL012 (1.53 mg/L), respectively. Further, in a 5-L bioreactor, the yield of PI with strain CL012 was 53 mg/L for high-density fermentation, after 96 h of methanol induction, which was 34.64-fold higher than that of shake culture. The strains expressing SNC2 and SNC2-SSO2 yielded the PI of 64 mg/L and 78 mg/L, which were respectively increased by 20.75% and 47.17%, compared to that of strain CL012. This work indicated that SNAREs components promoted the secretion of PI to improve its heterologous expression in P. pastoris.