title: High-level overproduction of Thermobifida enzyme in Streptomyces lividans using a novel expression vector.
abstract: In this study, we constructed a novel Streptomyces-E.coli shuttle vector pZRJ362 combining the xylose isomerase promoter and amylase terminator. A gene encoding the endoglucanase Cel6A in Thermobifida fusca was amplified by PCR, cloned into Streptomyces lividans host strain using the novel expression vector and Pichia pastoris GS115 host strain using the vector pPICZα-C, respectively. Afterwards, the expression pattern and the maximum expression level were comparatively studied in both expression systems. The maximum enzyme activity of Cel6A-(His)₆ secreted in S. lividans supernatant after 84-h of cultivation amounted to 5.56 U/mL, which was dramatically higher than that secreted in P. pastoris about 1.4 U/mL after 96-h of cultivation. The maximum expression level of Cel6A-(His)₆ in S. lividans supernatant reached up to 173 mg/L after 84-h of cultivation. The endoglucanase activity staining SDS-PAGE showed that there were some minor proteins in S. lividans supernatant which may be the Cel6A derivant by proteolytic degradation, while there was no proteolytic product detected in supernatant of P. pastoris.
authors.affiliation: State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China.
authors.forename: Jun-Xia
authors.initials: JX
authors.lastname: Li
authors.forename: Long-Mei
authors.initials: LM
authors.lastname: Zhao
authors.forename: Ru-Juan
authors.initials: RJ
authors.lastname: Wu
authors.forename: Zhao-Jun
authors.initials: ZJ
authors.lastname: Zheng
authors.forename: Ri-Jun
authors.initials: RJ
authors.lastname: Zhang
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000235:genetics / Q000378:metabolism; D002480:Cellulase / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D048170:Streptomyces lividans / Q000201:enzymology* / Q000235:genetics
chemical_list: D002480:Cellulase; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase
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