Replacement of streptomyces hygroscopicus genomic segments with in vitro altered DNA sequences

Abstract

We have developed a method for gene replacement in Streptomyces hygroscopicus which permits introduction of an in vitro derived mutation carried on a plasmid into the chromosome. We constructed the plasmid pMSB212 which can replicate in S. hygroscopicus and contains the step 5 gene of the bialaphos biosynthetic pathway which was inactivated by a frame-shift mutation caused by filling in the cohesive ends of the EcoR I site in the structural gene. pMSB212 was introduced into a bialaphos producer strain and by protoplast regeneration of the primary thiostrepton-resistant transformants, non-producing mutants, were obtained. Biochemical and genetical analyses indicated that these mutants were specifically blocked by introduction of the frame-shift mutation in the step 5 gene on the chromosome. This method will enable us to obtain isogenic mutants of known genes and to identify new genes encoded on a cloned fragment. © 1988, JAPAN ANTIBIOTICS RESEARCH ASSOCIATION. All rights reserved.