Engineering Biology Research Center, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.
Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.
Microb Biotechnol. 2022 Sep;15(9):2364-2378. doi: 10.1111/1751-7915.14061. Epub 2022 Jun 3.
In our previous study, we serendipitously discovered that protein secretion in the methylotrophic yeast Pichia pastoris is enhanced by a mutation (V50A) in the mating factor alpha (MFα) prepro-leader signal derived from Saccharomyces cerevisiae. In the present study, we investigated 20 single-amino-acid substitutions, including V50A, located within the MFα signal peptide, indicating that V50A and several single mutations alone provided significant increase in production of the secreted proteins. In addition to hydrophobicity index analysis, both an unfolded protein response (UPR) biosensor analysis and a microscopic observation showed a clear difference on the levels of UPR induction and mis-sorting of secretory protein into vacuoles among the wild-type and mutated MFα signal peptides. This work demonstrates the importance of avoiding entry of secretory proteins into the intracellular protein degradation pathways, an observation that is expected to contribute to the engineering of strains with increased production of recombinant secreted proteins.
在我们之前的研究中，我们偶然发现，源自酿酒酵母的交配因子 α（MFα）前导肽中的一个突变（V50A）增强了甲醇营养型酵母巴斯德毕赤酵母中的蛋白分泌。在本研究中，我们研究了 20 个位于 MFα 信号肽内的单个氨基酸取代，包括 V50A，结果表明 V50A 和几个单独的突变单独提供了分泌蛋白产量的显著增加。除了疏水性指数分析外，未折叠蛋白反应（UPR）生物传感器分析和显微镜观察都表明，在野生型和突变 MFα 信号肽之间，UPR 诱导和错误分拣到液泡中的分泌蛋白水平有明显差异。这项工作表明了避免分泌蛋白进入细胞内蛋白降解途径的重要性，这一观察结果有望有助于提高重组分泌蛋白产量的工程菌株的设计。
