A KETOREDUCTASE GENE FROM STREPTOMYCES-MYCAROFACIENS-1748 DNA INVOLVED IN BIOSYNTHESIS OF A SPORE PIGMENT

An efficient plasmid transformation system for S. mycarofaciens 1748 h as been established. In order to determine the function of MKR gene in S. mycarofaciens 1748, the gene disruption experiment was carried out . For this purpose the plasmid pKC1139 was used. A recombinant strain with white spore appeared, in contrast to the grey-colour spore of S. mycarofaciens 1748. This suggested that homologous recombination betwe en plasmid-borne MKR gene sequence and the chromosome of S. mycarofaci ens 1748 had occurred. A Southern hybridization experiment using alpha -P-32-labelled MKR gene as probe indicated that the desired integratio n event had occurred in the recombinant. The result of gene disruption showed that the alteration of this gene in the chromosome of S. mycar ofaciens 1748 made sporulating colonies remain white instead of taking on the typical grey colour of sporulating wild type colonies, suggest ing that MKR gene is involved in the biosynthesis of a spore pigment. The recombinant strain was incubated with fermentation medium optimise d for midecamycin production. A TLC assay showed that the recombinant strain produced midecamycin in quantities comparable to that of S. myc arofaciens 1748. A pCN8B12 was a clone from genomic library of midecam ycin producing strain which contained a 28-kb DNA insert. The 28-kb DN A fragment contained act I-homologous and act III-homologous regions. The PKS (act I-homologous) and MKR (act III-homologous) genes that def ine spore pigment of midecamycin producing strain were localized by re striction endonuclease digestion with pCN8B12, indicating that they ar e separated by about 10 kb DNA. The polyketide synthase gene cluster o f similar organization has not been reported yet.