Rational Screening Of Rapamycin-producing Strain Of Sterptomyces Hygroscopicus By Labeling Biosynthetic Gene Cluster And Classical Strain Improvement

High-yield rapamycin-producing strain has industrial and academic application. We are interested in exploring the efficient approach to acquire improved S. hygroscopicus strain, which produces rapamycin cost-competitively. In this study, a novel rational screening method is described as followed.First, to overcome S. hygroscopicus’ complex modification-restriction system, a genetic system, which harnesses interspecies conjugation between Streptomycetes and Escherichia coli, has been established. 40℃heat shock for 20 min to S. hygroscopicus NRRL5491 was the best condition for spore germination. The exconjugant frequency of pSET152 （an integrative plasmid） was 3 to 6 times more efficient than pOJ446 （a self-replicated plasmid）; using overlay concentration of 1μg mL^-1 was 2.4 to 4.6 times more efficient than using 50μg mL^-1 apramycin. The conjugation frequency on MS medium was 3.4 times higher than that on SY agar medium. The conjugation frequency is 2×10^-6 at optimal condition, which is sufficient for genetic manipulation such as gene replacement/disruption.neo gene, which confers its host neomycin and/or kanamycin resistant, as selective reported, is labeled on ORFU, an assumable regulator in rapamycin biosynthesis gene cluster. Combined with Classical Strain Improvement （CSI）, the labeled S. hygroscopicus NRRL5491 strain YM4, was mutagenized with chemical NTG, then grown on appropriate solid medium. Obtained mutants were screened by kanamycin selection. And the mutants possessed higher kanamycin resistant properties were fermented. Rapamycin was isolated from the fermentation broth. HPLC analysis was hired to identify the productivity of rapamycin in these mutants.We attempted to label another structural gene rapB in rapamycin biosynthetic cluster, but failed.