Identification of proteins responsive to heterologous protein production in thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656

• Published in: Yeast (Chichester, England) Vol. 38; no. 5; pp. 316 - 325
• Main Authors: Phithakrotchanakoon, Chitwadee, Puseenam, Aekkachai, Kruasuwan, Worarat, Likhitrattanapisal, Somsak, Phaonakrop, Narumon, Roytrakul, Sittiruk, Ingsriswang, Supawadee, Tanapongpipat, Sutipa, Roongsawang, Niran
• Format: Journal Article
• Language: English
• Published: England Wiley Subscription Services, Inc 01.05.2021
• Subjects: Cell walls; Cellular stress response; comparative proteomic analysis; CRISPR; heterologous expression; Mutants; Ogataea thermomethanolica; Proteasomes; Protein transport; Protein turnover; Proteins; Proteolysis; Secretion; thermotolerant methylotrophic yeast; xylanase; heterologous expression; Ogataea thermomethanolica; thermotolerant methylotrophic yeast; comparative proteomic analysis; xylanase
• ISSN: 0749-503X; 1097-0061
• DOI: 10.1002/yea.3548

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. Eight candidate proteins identified by label‐free comparative proteomic analysis were mutated by CRISPR/Cas9 approach to reveal the proteins responsive to heterologous protein production in thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656.