Integrative Gene Cloning and Expression System forStreptomycessp. US 24 andStreptomycessp. TN 58 Bioactive Molecule Producing Strains

Abstract

Streptomycessp. US 24 andStreptomycessp. TN 58, two strains producing interesting bioactive molecules, were successfully transformed usingE. coliET12567 (pUZ8002), as a conjugal donor, carrying the integrative plasmid pSET152. For theStreptomycessp. US 24 strain, two copies of this plasmid were tandemly integrated in the chromosome, whereas forStreptomycessp. TN 58, the integration was in single copy at theattBsite. Plasmid pSET152 was inherited every time for all analysedStreptomycessp. US 24 andStreptomycessp. TN 58 exconjugants under nonselective conditions. The growth, morphological differentiation, and active molecules production of all studied pSET152 integrated exconjugants were identical to those of wild type strains. Consequently, conjugal transfer using pSET152 integration system is a suitable means of genes transfer and expression for both studied strains. To validate the above gene transfer system, the glucose isomerase gene (xylA) fromStreptomycessp. SK was expressed in strainStreptomycessp. TN 58. Obtained results indicated that heterologous glucose isomerase could be expressed and folded effectively. Glucose isomerase activity of the constructed TN 58 recombinant strain is of about eighteenfold higher than that of theStreptomycessp. SK strain. Such results are certainly of importance due to the potential use of improved strains in biotechnological process for the production of high-fructose syrup from starch.