Directed Evolution Of 4"-Isovalayltransferase By DNA Shuffling

Biotechmycin(4″acylspiramycin),a mixture of spiramycin derivatives, is a new antibiotic produced by a genetic engineering strain constructed by introducing ist gene from Streptomyces thermotolerans into the chromosome of Streptomyces spiramyceticus through homologous double-crossover. The main component of the mixture is 4″ isovaleryl spiramycin, the product of isovaleryltransferase which catalyzes the addition of isovaleryl onto 4″ position of 4″-OH spiramycin. At present, there is still unacylated spiramycin in the fermentation products as well as the acylated spiramycin, and we expect that the percentage of the 4″acylspiramycin will be increased. We choose the method of DNA shuffling to enhance the acylation ability of 4″-isovalayltrasferase in order to advance the percentage of biotechmycin among the fermentation products.DNA shuffling is a type of molecule-level directed evolution. It is based on the principle of sexual PCR, and it is easy to get the expected mutation by the reassembly of DNA fragments with the repeating template exchange.We cloned 6 ist genes from 6 strains with high output of biotechmycin. After shuffled, the finally obtained full-length genes were cloned into pSET152, an integrating Escherichia coli-Streptomyces shuttle plasmid. The 15 recombinant plasmids were obtained and then transferred into Streptomyces spiramyceticus B183. The 15 transformants were fermented after a generation grew on SCVM medium. Five of these strains were selected because their fermentation products contained obviously increased proportion of...