Liang Chenchen, Wang Li, Luo Qiuling, Wu Jing
College of Pharmacy, Jiangnan University, Wuxi 214122, Jiangsu, China.
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2017 Jul 25;33(7):1178-1189. doi: 10.13345/j.cjb.170012.
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.
为提高胰岛素前体（PI）的产量，我们构建了重组表达载体pPIC9K-PI，并通过电穿孔法将其转化到毕赤酵母GS115中。筛选后，在含有4.0 mg/mL G418的YPDS平板上获得了具有12个拷贝的突变菌株CL012。然后，在菌株CL012中表达可溶性N-乙基马来酰亚胺敏感因子附着受体蛋白（SNAREs）的组分SNC2和SNC2-SSO2，以探究SNAREs对PI产量的影响。在摇瓶培养中，表达SNC2和SNC2-SSO2的菌株在甲醇诱导96小时后，PI产量分别为1.89 mg/L和2.21 mg/L，与菌株CL012（1.53 mg/L）相比，分别提高了23.53%和44.44%。此外，在5-L生物反应器中，菌株CL012进行高密度发酵，甲醇诱导96小时后PI产量为53 mg/L，比摇瓶培养高34.64倍。表达SNC2和SNC2-SSO2的菌株PI产量分别为64 mg/L和78 mg/L，与菌株CL012相比，分别提高了20.75%和47.17%。这项工作表明，SNAREs组分促进了PI的分泌，从而提高了其在毕赤酵母中的异源表达。
