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

作　　者：梁晨晨 王立[1,2] 罗秋玲[2] 吴静[1] 

机构地区：[1]江南大学药学院,江苏无锡214122 [2]江南大学食品科学与技术国家重点实验室,江苏无锡214122

出　　处：《生物工程学报》2017年第7期1178-1189,共12页Chinese Journal of Biotechnology

基　　金：江苏省科技支撑计划社会发展项目(No.BE2014652);国家自然科学基金(No.21576117);江苏省自然科学基金(Nos.BE2015307;BK20131103)资助~~

摘　　要：为了提高胰岛素前体(PI)的产量,构建了p PIC9K-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/m L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。To improve the yield of insulin precursor （PI）, we constructed a recombinant expression vector pPIC9K-PI and transformed it into Pichia pastoris GSll5 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 rng/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.

关 键 词：毕赤酵母 胰岛素前体 SNARES 异源表达 

分 类 号：Q78[生物学—分子生物学]