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
投稿时间:2016-01-11  
DOI:  10.13345/j.cjb.170012
中文关键词:毕赤酵母,胰岛素前体,SNAREs,异源表达
英文关键词:Pichia pastoris, insulin precursor, SNAREs, heterologous expression
基金项目: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。
作者单位E-mail
梁晨晨 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  
王立 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  
罗秋玲 2 江南大学 食品科学与技术国家重点实验室江苏 无锡 214122  
吴静 1 江南大学 药学院江苏 无锡 214122 wujing@jiangnan.edu.cn 
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中文摘要:
      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。
英文摘要:
      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.
梁晨晨,王立,罗秋玲,吴静.一种增加毕赤酵母生产胰岛素前体的方法[J].生物工程学报,2017,33(7):1178~1189
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