Biochemical Characterization of a Type III Polyketide Biosynthetic Gene Cluster from Streptomyces toxytricini
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A type III polyketide biosynthetic gene cluster has been discovered in the industrially important strain Streptomyces toxytricini NRRL 15443, including four genes stp450-1, stts, stp450-2, and stmo. The stts gene encodes a putative type III polyketide synthase that is homologous to RppA, a 1,3,6,8-tetrahydroxynaphthalene (THN) synthase from Streptomyces griseus. The deduced protein product of stmo resembles the cupin-containing monooxygenase MomA from Streptomyces antibioticus that oxidizes THN into flaviolin. Two cytochrome P450s (CYPs), StP450-1 and StP450-2, are present in the gene cluster. StTS was overexpressed in Escherichia coli BL21(DE3) and identified as a THN synthase. The synthesized THN can be easily oxidized into flaviolin by air. Both CYPs were reconstituted in E. coli BL21(DE3) and can oxidize flaviolin to form oligomers. The k cat/K m values for StP450-1 and StP450-2 were 0.28 and 0.71 min−1 mM−1, respectively. UV irradiation test showed that expression of StTS in E. coli BL21(DE3) significantly protects the cells from UV radiation, and coexpression of StTS and StP450-1 provides even stronger protection.
KeywordsType III polyketide synthase Cytochrome P450 monooxygenases Streptomyces toxytricini UV protection
This work was supported by a Utah State University Research Catalyst Grant. Richard Decker was supported by a Utah State University Engineering Undergraduate Research Program (EURP) Grant. We thank Prof. Lance Seefeldt and Dr. Zhiyong Yang in the Department of Chemistry and Biochemistry at Utah State University for their assistance in the CO difference spectrum assays.
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