文献类型：期刊文献

英文题名：A novel methanol-free Pichia pastoris system for recombinant protein expression

作者：Shen, Wei[1];Xue, Ying[1];Liu, Yiqi[1];Kong, Chuixing[1];Wang, Xiaolong[1];Huang, Mengmeng[1];Cai, Menghao[1];Zhou, Xiangshan[1];Zhang, Yuanxing[1,2];Zhou, Mian[1]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, 130 Meilong Rd, Shanghai 200237, Peoples R China;[2]SCICB, Shanghai 200237, Peoples R China

年份：2016

卷号：15

外文期刊名：MICROBIAL CELL FACTORIES

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000385728400001)】；

基金：This work was sponsored by Chinese National High Technology Research and Development Program (2014AA093501), National Special Fund for State Key Laboratory of Bioreactor (2060204), and Shanghai Pujiang Program (15PJ1401600).

语种：英文

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

摘要：Background: As one of the most popular expression systems, recombinant protein expression in Pichia pastoris relies on the AOX1 promoter (P-AOX1) 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 P-AOX1 activation or repression by different carbon sources may be promising targets. Results: We identified two kinase mutants: Delta gut1 and Delta dak, 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:Delta gut1-HpGCY1-glycerol (P-AOX1 induced by glycerol) and Delta dak-DHA (P-AOX1 induced by DHA). By comparing their GFP expression efficiencies, the latter one showed better potential. To further test the Delta dak-DHA system, three more recombinant proteins were expressed as examples. We found that the expression ability of our novel methanol-free Delta dak-DHA system was generally better than the constitutive GAP promoter, and reached 50-60 % of the traditional methanol induced system. Conclusions: We successfully constructed a novel methanol-free expression system Delta dak-DHA. This modified expression platform preserved the favorable regulatable nature of P-AOX1, providing a potential alternative to the traditional system.