title: Multivariate modular engineering of the protein secretory pathway for production of heterologous glucose oxidase in Pichia pastoris.
abstract: Limitations in protein production and secretion have been attributed to the inefficient folding rate of overexpressed proteins and the cellular response to the presence of overexpressed proteins in the endoplasmic reticulum (ER). In this study, we improved the yield of glucose oxidase (GOD) by manipulating genes involved in protein folding machinery and abnormal folding stress responses. First, genes with folding and secretion functions were used to modulate the folding rate of GOD in the ER and its secretion level in the cytoplasm. Next, the potential benefits of the ERAD elements were determined. Cellular resistance to ER derived stress was then strengthened by overexpressing the stress response gene GCN4. Furthermore, a module combination strategy, which co-expressed the SEC53, CNE1 and GCN4 genes, was employed to construct the Pichia pastoris strain S17. This increased the yield of GOD to 21.81g/L, with an activity of 1972.9U/mL, which were 2.53- and 5.11-fold higher, respectively, than the control strain. The work described here improved GOD production significantly, and the strategies employed in this study provide novel information for the large-scale production of heterologous proteins.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Gu
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; School of Biotechnology, Jiangnan University, Wuxi 214122, China. Electronic address: zhangj@jiangnan.edu.cn.
authors.forename: Juan
authors.initials: J
authors.lastname: Zhang
authors.affiliation: The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.affiliation: National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China; School of Biotechnology, Jiangnan University, Wuxi 214122, China. Electronic address: jchen@jiangnan.edu.cn.
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D018274:Electroporation; D060746:Endoplasmic Reticulum-Associated Degradation / Q000235:genetics; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal; D005949:Glucose Oxidase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D017510:Protein Folding; D021381:Protein Transport; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013312:Stress, Physiological / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D005656:Fungal Proteins; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; D005949:Glucose Oxidase
keywords: Glucose oxidase; Heterologous protein secretion; Modular engineering; Pichia pastoris