Transcriptomic analysis of Pichia pastoris (Komagataella phaffii) GS115 during heterologous protein production using a high-cell-density fed-batch cultivation strategy

We investigated gene expression during the bench-scale batch fermentation phase, glycerol feed phase, glycerol-methanol mixture feed (GM) phase, and at different time points following methanol induction using RNA-Seq. We report that the addition of the GM phase may help to alleviate the adverse effects of methanol addition (alone) on P. pastoris cells. Secondly, enhanced upregulation of the mitogen-activated protein kinase (MAPK) signaling pathway was observed in P. pastoris following methanol induction. The MAPK signaling pathway may be related to P. pastoris cell growth, and may regulate the AOX1 promoter via regulatory factors activated by methanol-mediated stimulation. Thirdly, the unfolded protein response (UPR) and ER-associated degradation (ERAD) pathways were not significantly upregulated during the methanol induction period. These results imply that the presence of unfolded or misfolded phytase protein did not represent a serious problem in our study. Finally, the upregulation of the autophagy pathway during the methanol induction phase may be related to the degradation of damaged peroxisomes but not to the production of phytase. This work describes the metabolic characteristics of P. pastoris during heterologous protein production under high-cell-density fed-batch cultivation. We believe that the results of this study will aid further in-depth studies of P. pastoris protein expression, regulation, and secretory mechanisms. Overall design: Investigate the physiological and metabolic characteristics of Pichia pastoris producing exogenous proteins under high-cell-density culture conditions

本研究采用RNA测序（RNA-Seq）技术，对实验室规模分批发酵阶段、甘油补料阶段、甘油-甲醇混合补料（GM）阶段，以及甲醇诱导后的不同时间节点的基因表达情况进行了检测。本研究发现，引入GM补料阶段可有效缓解单独甲醇补料对毕赤酵母（Pichia pastoris，简称P. pastoris）细胞造成的不良影响。其次，甲醇诱导后毕赤酵母中丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）信号通路的上调幅度显著增强；该通路可能与毕赤酵母的细胞生长相关，并可通过甲醇刺激激活的调控因子对醇氧化酶1（AOX1）启动子进行调控。第三，甲醇诱导阶段未折叠蛋白反应（unfolded protein response，UPR）与内质网相关降解（ER-associated degradation，ERAD）通路均未出现显著上调，上述结果表明本研究中未折叠或错误折叠的植酸酶蛋白并未构成严重的表达负担。最后，甲醇诱导阶段自噬通路的上调可能与受损过氧化物酶体的降解相关，而非植酸酶的生产过程。本研究阐明了毕赤酵母在高密度补料分批培养条件下进行异源蛋白生产时的代谢特征，我们认为本研究结果可为后续毕赤酵母蛋白表达、调控及分泌机制的深入研究提供参考。整体实验设计：探究高密度培养条件下产外源蛋白的毕赤酵母的生理与代谢特征。