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

Abstract

Background: As 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. Results: We identified two kinase mutants: (increment)gut1 and (increment)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: (increment)gut1-HpGCY1-glycerol (PAOX1 induced by glycerol) and (increment)dak-DHA (PAOX1 induced by DHA). By comparing their GFP expression efficiencies, the latter one showed better potential. To further test the (increment)dak-DHA system, three more recombinant proteins were expressed as examples. We found that the expression ability of our novel methanol-free (increment)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 (increment)dak-DHA. This modified expression platform preserved the favorable regulatable nature of PAOX1, providing a potential alternative to the traditional system.