Identification Of A Gene Cluster Encoding Phenazine Antibiotic Biosynthesis In Streptomyces Griseoluteus

Streptomyces griseoluteus, a strain which can produce griseolutein, is isolated from soil. It has been proved that griseolutein has strong antibacterial activities. It has very good inhibitory effects against both Gram-positive bacteria and Gram-negative bacteria. The structure of griseolutein is similar to phenazine nucleus. Compared with other phenazine derivatives, such as pyocyanine, chlororaphin and phenazine-1-carboxylic acid, griseolutein has the strongest bacteriostatic effect. These facts suggest that the strong bacteriostatic activity of griseolutein would be dependent on groups such as 1, 2-hydroxyethoxymethyl group or hydroxyacetyl- methyl group at 6-position of phenazine nucleus. But so far, no research on griseolutein biosynthetic genes has been reported.In order to confirm that there are phenazine nucleus synthesis genes and subsequent transformation genes on the chromosome of S. griseoluteus, we constructed Fosmid genomic library of S. griseoluteus. It also lays the foundation for following experiments. By blasting the conserved sequences of phenazine nucleus, we designed specific primers. Then we used several rounds of PCR to screen the Fosmid library, and finally got seven positive clones. Through analyzing the digest map of restriction enzyme, we selected the clone 10D11 which is most likely to contain a complete gene cluster of phenazine synthesis. With shotgun sequencing method, and PCR to complete gaps, we obtained the sequence of 32973bp bases in the end. Using ORF analysis and homology analysis methods, we identified a total of 27 ORFs in 10D11, of which 6 are for phenazine biosynthesis, which are called sghBCDEFG.. ORF21-25 is a gene cluster for following transformation. The final product is griseolutein.