Cloning of large DNA fragments, which hybridize with actinorhodin biosynthesis genes, from kalafungin and nanaomycin A methyl ester producers and identification of genes for kalafungin biosynthesis of the kalafungin producer.
Large actI, III-homologous DNA fragments were isolated from genomic libraries of the strains that produce the benzoisochromanequinone antibiotics kalafungin and nanaomycin A methyl ester, Streptomyces tanashiensis strain Kala and Streptomyces sp. OM-173, respectively. These libraries were prepared in Escherichia coli JM108 by using a novel Streptomyces-E. coli bifunctional cosmid, pKU205, and screened with polyketide synthase genes (actI and III) for actinorhodin biosynthesis from Streptomyces coelicolor A3(2) as probes. The cloned DNA fragments (28 and 42 kb) were analyzed by hybridization with DNA containing actinorhodin biosynthetic genes (actI, II, III, IV, VA, VB, VI and VII). Both fragments hybridized with the actI, III, VA and VI regions, but not with the actII, IV, VB and VII regions. The cloned fragment of S. tanashiensis DNA was analyzed by complementation tests with kalafungin-nonproducing mutants. Seven genes (kalI approximately VII), which correspond to seven steps in kalafungin biosynthesis, were found to be located on a 14 kb continuous DNA fragment. Five of the genes were located on the regions homologous to the genes for actinorhodin biosynthesis, but the other two genes were not. Although kalafungin is an intermediate or shunt product in actinorhodin biosynthesis in S. coelicolor A3(2), the genes for kalafungin biosynthesis in S. tanashiensis are not identical with those in S. coelicolor A3(2).
School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan.
S
S
Kakinuma
Y
Y
Takada
H
H
Ikeda
H
H
Tanaka
S
S
Omura
D A
DA
Hopwood
D000880:Anthraquinones / Q000302:isolation & purification; D000935:Antifungal Agents / Q000096:biosynthesis* / Q000494:pharmacology; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D005798:Genes, Bacterial; D015698:Genomic Library; D006824:Hybridization, Genetic / Q000235:genetics; D009285:Naphthoquinones / Q000302:isolation & purification; D013302:Streptomyces / Q000235:genetics*; D000095622:Benzoisochromanequinones
D000880:Anthraquinones; D000935:Antifungal Agents; D004247:DNA; D009285:Naphthoquinones; C008638:nanaomycin A; D000095622:Benzoisochromanequinones; C013079:kalafungin; C013390:actinorhodin