Zheng Xueyun, Ye Zhifang, Gao Jiao, Hao Yuechuo, Li Cheng, Xie Hongsen, Lin Ying, Liang Shuli
Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei University of Technology, Wuhan, 430068, China.
Guangdong Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology, Guangzhou, 510006, China.
Arch Microbiol. 2025 May 9;207(6):140. doi: 10.1007/s00203-025-04339-7.
The methylotrophic yeast Pichia pastoris (P. pastoris) exhibits remarkable capability for methanol-driven protein biosynthesis, positioning it as an attractive platform for carbon-neutral biomanufacturing utilizing methanol as a renewable feedstock. However, challenges arising from methanol metabolism, particularly the accumulation of toxic formaldehyde intermediates, significantly hinder efficient methanol biotransformation. To address this limitation, we implemented a metabolic engineering strategy involving dual knockout of alcohol oxidase genes (aox1 and aox2) combined with glycerol co-substrate supplementation. Using enhanced green fluorescent protein (EGFP) as a model heterologous product, we demonstrated that the ΔAOX1/2 strain achieved superior protein productivity in glycerol-methanol co-feeding cultures. Under optimized conditions (0.5% methanol + 0.4% glycerol), the engineered strain attained a biomass density of 38.5 (OD) and EGFP fluorescence intensity of 494,723 units, representing improvements of 32.8% and 53.6%, respectively, compared to the wild-type (WT) strain cultivated with 1% methanol alone. Transcriptome profiling revealed that the observed enhancement in protein synthesis originated from optimized methanol utilization through coordinated upregulation of both assimilatory and dissimilatory metabolic modules. This study demonstrates that alcohol oxidase suppression coupled with glycerol co-metabolism constitutes an effective strategy to alleviate methanol-derived metabolic stress while enhancing heterologous protein yields in P. pastoris.
甲基营养型酵母巴斯德毕赤酵母（P. pastoris）在甲醇驱动的蛋白质生物合成方面表现出卓越能力，使其成为利用甲醇作为可再生原料进行碳中和生物制造的有吸引力的平台。然而，甲醇代谢产生的挑战，特别是有毒甲醛中间体的积累，严重阻碍了高效的甲醇生物转化。为了解决这一限制，我们实施了一种代谢工程策略，包括双重敲除醇氧化酶基因（aox1和aox2）并补充甘油共底物。以增强型绿色荧光蛋白（EGFP）作为模型异源产物，我们证明了ΔAOX1/2菌株在甘油 - 甲醇共进料培养中实现了更高的蛋白质生产力。在优化条件下（0.5%甲醇 + 0.4%甘油），工程菌株的生物量密度达到38.5（OD），EGFP荧光强度达到494,723单位，与仅用1%甲醇培养的野生型（WT）菌株相比，分别提高了32.8%和53.6%。转录组分析表明，观察到的蛋白质合成增强源于通过同化和异化代谢模块的协同上调实现的甲醇利用优化。这项研究表明，抑制醇氧化酶并结合甘油共代谢是一种有效的策略，可减轻甲醇衍生的代谢应激，同时提高巴斯德毕赤酵母中异源蛋白质的产量。
