Abstract：
The methylotrophic yeast, Pichia pastoris (P. pastoris; syn. Komagataella spp.), known for its ability to grow to high cell densities, its strong and tightly regulated promoters, and mammalian liked secretion pathway, has been widely used as a robust system to secrete heterologous proteins. The α-mating factor (MF) secretion signal leader from Saccharomyces cerevisiae (S. cerevisiae) is currently the most successfully used secretion signal sequence in the P. pastoris system. In this study, the secretion efficiency mediated by the α-MF secretion signal leaders from Komagataella pastoris (K. pastoris) and Komagataella phaffii (K. phaffii) was assessed using Enhanced Green Fluorescent Protein (EGFP) as a reporter. The results indicated that the secretion efficiency associated with the α-MF secretion signal leaders from K. pastoris and K. phaffii was notably lower in comparison to the α-MF secretion signal leader from S. cerevisiae. Further research indicated that N-linked glycosylation of the α-MF secretion signal leader enhanced the secretion of EGFP. Disruption of calnexin impaired the secretion of EGFP mediated by the N-linked glycosylated α-MF secretion signal leader, without affecting EGFP secretion mediated by the non-N-linked glycosylation α-MF secretion signal leader. The N-linked glycosylated of the α-MF secretion signal leader reduced the unfolded protein response (UPR) in the endoplasmic reticulum (ER). The enhancement of EGFP secretion by the N-linked glycosylated α-MF secretion signal leader might be achieved through the acceleration of proper folding of glycoproteins by the molecular chaperone calnexin. This study enhances the understanding of protein secretion in P. pastoris, specifically highlighting the influence of N-linked glycosylation on secretion efficiency, and could have implications for the production of recombinant proteins in bioengineering and biotechnological applications in P. pastoris.
摘要：
甲基营养酵母，巴斯德毕赤酵母(P.斯帕特里斯;syn.Komagataellaspp.),以其生长到高细胞密度的能力而闻名，它强大且受严格调控的启动子，和哺乳动物喜欢的分泌途径，已被广泛用作分泌异源蛋白的强大系统。酿酒酵母的α-交配因子（MF）分泌信号前导（S.酿酒酵母）是目前在巴斯德毕赤酵母系统中使用最成功的分泌信号序列。在这项研究中,巴斯德科马塔氏菌α-MF分泌信号前导序列介导的分泌效率(K.pastoris)和Komagataellaphafii(K.使用增强的绿色荧光蛋白（EGFP）作为报道分子来评估phafii）。结果表明,与来自酿酒酵母的α-MF分泌信号前导序列相比,与来自巴斯德毕赤酵母和巴斯德毕赤酵母的α-MF分泌信号前导序列相关的分泌效率显著较低。进一步的研究表明，α-MF分泌信号前导序列的N-连接糖基化增强了EGFP的分泌。calnexin的破坏损害了由N连接的糖基化α-MF分泌信号前导介导的EGFP的分泌，不影响由非N连接糖基化α-MF分泌信号前导介导的EGFP分泌。α-MF分泌信号前导的N-连接糖基化降低了内质网（ER）中的未折叠蛋白反应（UPR）。N连接的糖基化α-MF分泌信号前导序列对EGFP分泌的增强可能是通过分子伴侣calnexin加速糖蛋白的正确折叠来实现的。这项研究增强了对巴斯德毕赤酵母蛋白质分泌的理解，特别强调N-连接糖基化对分泌效率的影响，并可能对巴斯德毕赤酵母的生物工程和生物技术应用中重组蛋白的生产产生影响。
