Hybridizing fragments in the genomic DNA of Streptomyces venezuelae lSP5230, which produces the jadomycin group of angucycline antibiotics, were detected by probing with act/ DNA from Streptomyces coelicolor A3(2). The hybridizing regions were isolated from a 16.5 kb insert of S. venezuelae DNA recovered from a genomic library cloned in a A replacement vector. Subcloning and sequencing of a 4 8 kb segment of the insert, containing regions hybridizing to act/// as well as act/, identified five open reading frames (ORFs). The deduced polypeptide products of the ORFs closely resemble in sequence the components of streptomycete t y p e 4 polyketide synthases (PKSs) : the ORFI product corresponds to the ketoacyl synthase, and the ORF2 product to a polypeptide closely related to the ketoacyl synthase and involved in determining chain length; the ORF3 product matches the acyl carrier protein; ORF4 encodes a bifunctional cyclase/dehydrase; and ORF5 encodes a ketoreductase. Integration into the chromosomal DNA of a plasmid containing a segment of the ORF2-ORF4 region severely depressed jadomycin B biosynthesis; since the integrant showed no change in growth or spore pigmentation, the cloned PKS genes are presumed to encode enzymes in the pathway for jadomycin biosynthesis.

Structurally diverse polyketides provide a rich reservoir of bioactive molecules. Actinorhodin, a model aromatic polyketide, is synthesized by minimal type II polyketide synthase and tailoring enzymes. The ActIII actinorhodin ketoreductase is a key tailoring enzyme in actinorhodin biosynthesis. With purified antibodies against actinorhodin polyketide synthase alpha subunit (KSalpha) and ketoreductase, we conducted systematic localization experiments of the two proteins in Streptomyces coelicolor subproteomes. The results support the membrane location of KSalpha and cell-wall location of ketoreductase. Considering previous evidence that some other tailoring enzymes of actinorhodin biosynthesis may be located outside the cytoplasm, a picture is emerging of an extensive role for extracellular biochemistry in the synthesis of type II polyketide antibiotic.