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

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.