A ketoreductase gene from Streptomyces mycarofaciens 1748 DNA involved in biosynthesis of a spore pigment.
An efficient plasmid transformation system for S. mycarofaciens 1748 has 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 between plasmid-borne MKR gene sequence and the chromosome of S. mycarofaciens 1748 had occurred. A Southern hybridization experiment using a-(32)P-labelled MKR gene as probe indicated that the desired integration event had occurred in the recombinant. The result of gene disruption showed that the alteration of this gene in the chromosome of S. mycarofaciens 1748 made sporulating colonies remain white instead of taking on the typical grey colour of sporulating wild type colonies, suggesting that MKR gene is involved in the biosynthesis of a spore pigment. The recombinant strain was incubated with fermentation medium optimised for midecamycin production. A TLC assay showed that the recombinant strain produced midecamycin in quantities comparable to that of S. mycarofaciens 1748. A pCN8B12 was a clone from genomic library of midecamycin producing strain which contained a 28-kb DNA insert. The 28-kb DNA fragment contained act I-homologous and act III-homologous regions. he PKS (act I-homologous) and MKR (act III-homologous) genes that define spore pigment of midecamycin producing strain were localized by restriction endonuclease digestion with pCN8B12, indicating that they are separated by about 10 kb DNA. The polyketide synthase gene cluster of similar organization has not been reported yet.
Xia
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Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking Union of Medical College, Beijing, China.
Wang
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