A novel methanol-free Pichia pastoris system for recombinant protein expression

Microbial Cell Factories

Research

Menghao Cai¹  Ying Xue¹  Chuixing Kong¹  Xiangshan Zhou¹  Mengmeng Huang¹  Xiaolong Wang¹  Yiqi Liu¹  Mian Zhou¹  Wei Shen¹  Yuanxing Zhang² 

[1] State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, 200237, Shanghai, China;State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, 200237, Shanghai, China;Shanghai Collaborative Innovation Center for Biomanufacturing (SCICB), 200237, Shanghai, China;

关键词: Recombination protein expression;    AOX1;    Dihydroxyacetone;    GUT1;    DAK;    Pichia pastoris;   

DOI  :  10.1186/s12934-016-0578-4

 received in 2016-05-23, accepted in 2016-10-13,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundAs one of the most popular expression systems, recombinant protein expression in Pichia pastoris relies on the AOX1 promoter (PAOX1) which is strongly induced by methanol. However, the toxic and inflammatory nature of methanol restricts its application, especially in edible and medical products. Therefore, constructing a novel methanol-free system becomes necessary. The kinases involved in PAOX1 activation or repression by different carbon sources may be promising targets.ResultsWe identified two kinase mutants: Δgut1 and Δdak, both of which showed strong alcohol oxidase activity under non-methanol carbon sources. Based on these two kinases, we constructed two methanol-free expression systems: Δgut1-HpGCY1-glycerol (PAOX1 induced by glycerol) and Δdak-DHA (PAOX1 induced by DHA). By comparing their GFP expression efficiencies, the latter one showed better potential. To further test the Δdak-DHA system, three more recombinant proteins were expressed as examples. We found that the expression ability of our novel methanol-free Δdak-DHA system was generally better than the constitutive GAP promoter, and reached 50–60 % of the traditional methanol induced system.ConclusionsWe successfully constructed a novel methanol-free expression system Δdak-DHA. This modified expression platform preserved the favorable regulatable nature of PAOX1, providing a potential alternative to the traditional system.

【 授权许可】

CC BY   
© The Author(s) 2016