Cloning and characterization of actinorhodin biosynthetic gene clusters fromStrep to my ces lividansTK24

Abstract

Actinorhodin antibiotics produced by Streptomyces lividans TK24 are blue pigments with a weak antibiotic activity, derived from one acetyl‐CoA and 15 malonyl‐CoA units via a typical ployketide pathway. In an attempt to clone polyketide biosynthetic genes of S. lividans TK24, hybridizing fragments in the genomic DNA of S. lividans TK24 were detected by use of actl and act III polyketide synthase gene probes. Since typical aromatic polyketide biosynthetic gene clusters are roughly 22–34 Kb long, we constructed in E. coli XL‐Blue MR using the Streptomyces‐E. coli bifunctional shuttle cosmid vector (pOJ446). Then, about 5,000 individual E. coli colonies were thoroughly screened with actI‐ORFI and actIII probes. From these cosmid libraries, 12 positive clones were identified. Restriction analysis and southern hybridization showed two polyketide biosynthetic gene clusters in this organism. These cosmid clones can be transformed into Streptomyces parvulus 12434 for expression test that identify product of actino...