The Pichia pastoris transmembrane protein GT1 is a glycerol transporter and relieves the repression of glycerol on AOX1 expression

摘要

Promoter of alcohol oxidase I (PAOX1) is the most efficient promoter involved in the regulation of recombinant protein expression in Pichia pastoris (P. pastoris). PAOX1 is tightly repressed by the presence of glycerol in the culture medium; thus, glycerol must be exhausted before methanol can be taken up by P. pastoris and the expression of the heterologous protein can be induced. In this study, a candidate glycerol transporter (GT1, GeneID: 8197545) was identified, and its role was confirmed by further studies (e. g. bioinformatics analysis, heterologous complementation in Schizosaccharomyces pombe (S. pombe)). When GT1 is co-expressed with enhanced green fluorescent protein (EGFP), it localizes to the membrane and S. pombe carrying gt1 but not the wild-type strain can grow on medium containing glycerol as the sole carbon source. The present study is the first to report that AOX1 in the X-33 Delta gt1 mutant can achieve constitutive expression in medium containing glycerol; thus, knocking down gt1 can eliminate the glycerol repression of P-AOX1 in P. pastoris. These results suggest that the glycerol transporter may participate in the process of P-AOX1 inhibition in glycerol medium.