[Construction of a methanol-free protein expression system in Pichia pastoris based on the enhanced sorbitol...

The AOX1 promoter in Pichia pastoris is conventionally induced by methanol. However, methanol-based processes arouse safety concerns and operational complexity in industrial fermentation. Sorbitol has emerged as a promising alternative carbon source due to its favorable safety profile and minimal repressive effect on AOX1. To eliminate methanol dependence in the conventional AOX1-based expression system, in this study, the engineered strain GS-aCe, capable of secreting chondroitin hydrolase, was employed as a model to establish a methanol-free AOX1 expression system through coordinated engineering of transcriptional regulation and carbon metabolism. Overexpression of the transcriptional activator Mit1 in strain GS-aCe-Mit1 effectively activated the AOX1 promoter under sorbitol conditions, achieving 91.6% of the enzyme activity observed in the methanol-induced parental strain. Further co-expression of sorbitol dehydrogenase and hexokinase to enhance sorbitol assimilation significantly improved the growth performance of the resulting strain GS-aCe-Mit1-SH. In 1-L fermenters, the sorbitol consumption and biomass of GS-aCe-Mit1-SH increased by 82.0% and 90.5%, respectively, compared with those of GS-aCe-Mit1. The final biomass was 10.6% higher than that achieved in the methanol system, and the enzyme yield reached 1.10×10⁶ U/g, which was comparable to that of the methanol-induced system (1.14×10⁶ U/g), with no detectable accumulation of residual sorbitol. Collectively, integration of Mit1-mediated AOX1 transcriptional activation with reinforced sorbitol assimilation enabled efficient heterologous protein expression in P. pastoris under methanol-free conditions. This work provides a practical strategy for developing safe carbon source alternatives for AOX1-driven expression systems.