A novel methanol-free Pichia pastoris system for recombinant protein expression. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: Shen, Wei; Xue, Ying; Liu, Yiqi; Kong, Chuixing; Wang, Xiaolong; Huang, Mengmeng; Cai, Menghao; Zhou, Xiangshan; Zhang, Yuanxing; Zhou, Mian
• Is Part Of: Microbial cell factories. Volume 15:Issue 1(2016)
• Page Start: 1
• Page End: 11
• Publication Date: 2016-12
• Subjects: Recombination protein expression -- AOX1 promoter -- Dihydroxyacetone -- GUT1 -- DAK -- Pichia pastoris; Microbial biotechnology -- Periodicals; Recombinant proteins -- Synthesis -- Periodicals; 660.62
• DOI: 10.1186/s12934-016-0578-4
• Languages: English
• ISSNs: 1475-2859

Abstract

Abstract Background As one of the most popular expression systems, recombinant protein expression inPichia pastoris relies on theAOX1 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. Results We 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 constitutiveGAP promoter, and reached 50–60 % of the traditional methanol induced system. Conclusions We 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.