title: Improved Production of Active Streptomyces griseus Trypsin with a Novel Auto-Catalyzed Strategy.
abstract: N-terminal sequences play crucial roles in regulating expression, translation, activation and enzymatic properties of proteins. To reduce cell toxicity of intracellular trypsin and increase secretory expression, we developed a novel auto-catalyzed strategy to produce recombinant trypsin by engineering the N-terminus of mature Streptomyces griseus trypsin (SGT). The engineered N-terminal peptide of SGT was composed of the thioredoxin, glycine-serine linker, His6-tag and the partial bovine trypsinogen pro-peptide (DDDDK). Furthermore, we constructed a variant TLEI with insertion of the artificial peptide at N-terminus and site-directed mutagenesis of the autolysis residue R145. In fed-batch fermentation, the production of extracellular trypsin activity was significantly improved to 47.4 ± 1.2 U·ml(-1) (amidase activity, 8532 ± 142.2 U·ml(-1) BAEE activity) with a productivity of 0.49 U·ml(-1)·h(-1), which was 329% greater than that of parent strain Pichia pastoris GS115-SGT. This work has significant potential to be scaled-up for microbial production of SGT. In addition, the N-terminal peptide engineering strategy can be extended to improve heterologous expression of other toxic enzymes.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.|Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.|The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
authors.forename: Zhenmin
authors.initials: Z
authors.lastname: Ling
authors.affiliation: Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.|The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.|Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.|Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.|The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Kang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001329:Autolysis; D061249:Batch Cell Culture Techniques; D055162:Biocatalysis; D002417:Cattle; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000378:metabolism; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013305:Streptomyces griseus / Q000201:enzymology*; D014357:Trypsin / Q000235:genetics / Q000378:metabolism*; D014362:Trypsinogen / Q000737:chemistry
chemical_list: D010455:Peptides; D011993:Recombinant Fusion Proteins; D014362:Trypsinogen; D014357:Trypsin