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 isomera se (GIG138P, GI1) gene and its natural promoter was inserted into PstI-Bgl/ II linearized Streptomyces vector pIJ702. The ligation mixture was then int roduced into Streptomyces lividans TK54 protoplasts; transformants were ide ntified based on their thiostrepton resistance (Th-R) and insertional inact ivation of the melanin phenotype; and three white colonies, XY-2, 6, and 9, harboring recombinant expression plasmid pYH703, were obtained. Enzyme ass ay and SDS-PAGE analysis indicated that the GI1 gene was expressed, the int racellular GI1 specific activity was 6 U/mg, and GI1 accounted for 20% of t he 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 G I1 expression in S. lividans decreased, and more obviously under non-select ive conditions.