基于强化山梨醇同化途径的毕赤酵母非甲醇诱导蛋白表达体系构建

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

摘要:

巴斯德毕赤酵母(Pichia pastoris) AOX1启动子通常依赖甲醇诱导，但甲醇在工业发酵中存在安全性和工艺复杂性等问题。山梨醇因安全性高、对AOX1启动子具有弱阻遏性等特点，成为替代甲醇进行诱导表达的潜力碳源。为降低AOX1启动子体系对甲醇的依赖，本研究以分泌表达软骨素水解酶的工程菌GS-aCe为模型，通过转录调控与碳源代谢协同改造，构建以山梨醇替代甲醇的AOX1启动子表达体系。结果表明，过表达转录激活因子Mit1后，工程菌GS-aCe-Mit1在山梨醇条件下可有效激活AOX1启动子，其酶活达到对照菌GS-aCe在甲醇条件下的91.6%，但生长和碳源利用受限。进一步共表达山梨醇脱氢酶(sorbitol dehydrogenase, SDH)和己糖激酶(hexokinase, HK)强化山梨醇同化代谢，构建的GS-aCe-Mit1-SH显著改善了生长性能。在1 L发酵罐中，其山梨醇消耗量和生物量较GS-aCe-Mit1分别提高82.0%和90.5%，最终生物量较甲醇诱导体系提高10.6%，酶活得率达到1.10×10? U/g，与甲醇诱导体系(1.14×10? U/g)相当，且未观察到山梨醇残留。本研究通过整合Mit1介导的AOX1转录激活与山梨醇同化代谢增强，实现了毕赤酵母在非甲醇条件下的高效外源蛋白表达，为AOX1启动子体系的安全碳源替代提供了实验依据。

Abstract:

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.