title: Site-specific integration of the double-mutation glucose isomerase (GIG138PG247D) gene in Streptomyces lividans and its stable expression.
abstract: A recombinant expression plasmid pYH12, containing the double-mutation glucose isomerase (GIG138PG247D, GI2) coding gene and its natural regulatory sequence, was constructed for site-specific integration in Streptomyces. The resulting plasmid was introduced into Streptomyces lividans TK54 by protoplast transformation and two apramycin-resistance (AmR) transformants, designated GY2 and BY7, respectively, were obtained further based on enzyme assays. These results for polymerase chain reaction (PCR), Dot blot, and recovery of cloned fragments from the transformant chromosome indicated that the GI2 gene was integrated into the S. lividans chromosome by site-specific recombination, and which was further verified by Southern blot. We found that the free form of plasmid pYH12 co-existing with the integrated form was present in S. lividans. SDS-PAGE analysis showed that the GI2 gene was expressed in S. lividans. The intracellular GI2 specific activity was 1.15 U/mg. The stability of integrants demonstrated that the cloned GI2 gene was stably integrated and expressed even in the absence of selective pressure.
authors.affiliation: Department of Molecular and Cell Biology, School of Life Sciences, University of Science and Technology of China, Chinese Academy of Sciences, Hefei, Anhui 230026, P. R. China.
authors.forename: Yonghui
authors.initials: Y
authors.lastname: Yang
authors.forename: Chong
authors.initials: C
authors.lastname: Xu
authors.forename: Feng
authors.initials: F
authors.lastname: Ge
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Lu
authors.forename: Guoping
authors.initials: G
authors.lastname: Zhu
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.forename: Jun
authors.initials: J
authors.lastname: Liao
authors.forename: Liwen
authors.initials: L
authors.lastname: Niu
authors.forename: Yuzhen
authors.initials: Y
authors.lastname: Wang
authors.forename: Mian
authors.initials: M
authors.lastname: Wu
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000235:genetics*; D000818:Animals; D015870:Gene Expression; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D013302:Streptomyces / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase