The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 is a potential host for heterologous protein production. However, overproduction of heterologous protein can induce cellular stress and limit the level of its secretion. To improve the secretion of heterologous protein, we identified the candidate proteins with altered production during production of heterologous protein in O. thermomethanolica by using a label‐free comparative proteomic approach. Four hundred sixty‐four proteins with various biological functions showed differential abundance between O. thermomethanolica expressing fungal xylanase (OT + Xyl) and a control strain. The induction of proteins in transport and proteasomal proteolysis was prominently observed. Eight candidate proteins involved in cell wall biosynthesis (Chs3, Gas4), chaperone (Sgt2, Pex19), glycan metabolism (Csf1), protein transport (Ypt35), and vacuole and protein sorting (Cof1, Npr2) were mutated by a CRISPR/Cas9 approach. An Sgt2 mutant showed higher phytase and xylanase activity compared with the control strain (13%–20%), whereas mutants of other genes including Cof1, Pex19, Gas4, and Ypt35 showed lower xylanase activity compared with the control strain (15%–25%). In addition, an Npr2 mutant showed defective growth, while overproduction of Npr2 enhanced xylanase activity. These results reveal genes that can be mutated to modulate heterologous protein production and growth of O. thermomethanolica TBRC656.

中文翻译：

---

在耐热甲基营养酵母 Ogataea thermomethanolica TBRC656 中鉴定对异源蛋白质生产有反应的蛋白质

耐热甲基营养酵母Ogataea thermomethanolica TBRC656 是异源蛋白质生产的潜在宿主。然而，异源蛋白的过量产生会引起细胞应激并限制其分泌水平。为了改善异源蛋白质的分泌，我们通过使用无标记的比较蛋白质组学方法鉴定了在O.thermomethanolica中生产异源蛋白质期间产生改变的候选蛋白质。四百64蛋白具有多种生物学功能表现之间的差异丰O. thermomethanolica表达真菌木聚糖酶 (OT + Xyl) 和对照菌株。显着地观察到蛋白质在运输和蛋白酶体蛋白水解中的诱导。涉及细胞壁生物合成（Chs3、Gas4）、分子伴侣（Sgt2、Pex19）、聚糖代谢（Csf1）、蛋白质转运（Ypt35）以及液泡和蛋白质分选（Cof1、Npr2）的八种候选蛋白质被 CRISPR/Cas9 突变方法。与对照菌株相比，Sgt2 突变体显示出更高的植酸酶和木聚糖酶活性（13%–20%），而其他基因的突变体，包括 Cof1、Pex19、Gas4 和 Ypt35，与对照菌株相比（15%–25 %）。此外，Npr2 突变体表现出生长缺陷，而 Npr2 的过度生产增强了木聚糖酶活性。这些结果揭示了可以突变以调节异源蛋白质产生和生长的基因O. thermomethanolica TBRC656。