GENE DISRUPTION AND REPLACEMENT IN THE RAPAMYCIN-PRODUCING STREPTOMYCES-HYGROSCOPICUS STRAIN ATCC-29253

A system for gene disruption and replacement based on a streptomycete temperate phage vector was developed to introduce DNA in the rapamycin -producing Streptomyces hygroscopicus strain ATCC 29253. This will be useful in attempts to produce, through genetic manipulation, novel for ms of the therapeutically important immunosuppressive drug rapamycin. Recombinant phages were constructed from the phi C31 phage derivative KC515 (c(+) attP) carrying a thiostrepton or viomycin resistance gene along with segments of the S. hygroscopicus chromosome. Each of the cl oned segments also contained the aphll neomycin/kanamycin resistance g ene to enable gene replacement by loss of the phage-derived DNA. Speci fic deletion of the entire polyketide synthase (PKS) believed to gover n rapamycin biosynthesis resulted in the loss of rapamycin production. In contrast, disruption or deletion of a region predicted to encode f our PKS open reading frames, or another region predicted to encode ano ther PKS plus a cytochrome P450 hydroxylase and ferredoxin, had no eff ect on the production of rapamycin or nigericin, a polyether antibioti c also produced by S. hygroscopicus. Therefore, S. hygroscopicus may h ave the capacity to produce polyketides additional to rapamycin and ni gericin.