Heterologous expression of the single-mutation glucose isomerase (GIG138P) gene in Streptomyces lividans and its genetic instability.
A 1.3-kb PstI-BamHI fragment containing the single-mutation glucose isomerase (GIG138P, GI1) gene and its natural promoter was inserted into PstI-BglII linearized Streptomyces vector pIJ702. The ligation mixture was then introduced into Streptomyces lividans TK54 protoplasts; transformants were identified based on their thiostrepton resistance (ThR) and insertional inactivation of the melanin phenotype; and three white colonies, XY-2, 6, and 9, harboring recombinant expression plasmid pYH703, were obtained. Enzyme assay and SDS-PAGE analysis indicated that the GI1 gene was expressed, the intracellular GI1 specific activity was 6 U/mg, and GI1 accounted for 20% of the soluble proteins in S. lividans. Restriction analysis and Southern blot of pYH703 showed the existence of plasmid deletion, presumably owing to the interaction between the mel and GI1 sequences. Continuous liquid cultures of the recombinant strain demonstrated that the GI1 specific activity and GI1 expression in S. lividans decreased, and more obviously under non-selective conditions.
Yang
Y
Y
Department of Molecular and Cell Biology, School of Life Sciences, University of Science and Technology of China, The Chinese Academy of Sciences, Hefei, Anhui 230026, PR China.
Xu
C
C
Ge
F
F
Lu
Z
Z
Zhu
G
G
Li
H
H
Liao
J
J
Teng
M
M
Niu
L
L
Wang
Y
Y
D019747:Aldose-Ketose Isomerases / Q000096:biosynthesis* / Q000235:genetics*; D015139:Blotting, Southern; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D009693:Nucleic Acid Hybridization; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase