Analysis of two chromosomal regions adjacent to genes for a type II polyketide synthase involved in the biosynthesis of the antitumor polyketide mithramycin in Streptomyces argillaceus

Mithramycin is an aromatic antitumour polyketide synthesized by Streptomyce s argillaceus. Two chromosomal regions located upstream and downstream of t he locus for the mithramycin type II polyketide synthase were cloned and se quenced. Analysis of the sequence revealed the presence of eight genes enco ding three oxygenases (mtmOI, mtmOII and mtmOIII), three reductases (mtmTI, mtmTII and mtmTIII), a cyclase (mtmY) and an acyl CoA ligase (mtmL). The t hree: oxygenase genes were each inactivated by gene replacement. Inactivati on of one of them (mtmOII) generated a non-producing mutant, while inactiva tion of the other two (mtmOI and mtmOIII) did not affect the biosynthesis o f mithramycin. The mtmOII gene may code for an oxygenase responsible for th e introduction of oxygen atoms at early steps in the biosynthesis of mithra mycin leading to 4-demethylpremithramycinone. One of the reductases may be responsible for reductive cleavage of an intermediate from an enzyme and an other for the reduction of a keto group in the side-chain of the mithramyci n aglycon moiety. A hypothetical biosynthetic pathway showing in particular the involvement of oxygenase MtmOII and of various other gene products in mithramycin biosynthesis is proposed.