一种增加毕赤酵母生产胰岛素前体的方法
作者:
作者单位:

作者简介:

通讯作者:

中图分类号:

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。


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

Fund Project:

Key Technologies R&D Program of Jiangsu Province (No. BE2014652), National Natural Science Foundation of China (No. 21576117), Natural Science Foundation of Jiangsu Province (Nos. BE2015307, BK20131103).

  • 摘要
  • |
  • 图/表
  • |
  • 访问统计
  • |
  • 参考文献
  • |
  • 相似文献
  • |
  • 引证文献
  • |
  • 资源附件
  • |
  • 文章评论
    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.

    参考文献
    相似文献
    引证文献
引用本文

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

复制
分享
文章指标
  • 点击次数:
  • 下载次数:
  • HTML阅读次数:
  • 引用次数:
历史
  • 收稿日期:2016-01-11
  • 最后修改日期:
  • 录用日期:
  • 在线发布日期: 2017-06-26
  • 出版日期:
您是第位访问者
生物工程学报 ® 2024 版权所有

通信地址:中国科学院微生物研究所    邮编:100101

电话:010-64807509   E-mail:cjb@im.ac.cn

技术支持:北京勤云科技发展有限公司