CLONING AND INSERTIONAL INACTIVATION OF STREPTOMYCES-ARGILLACEUS GENES INVOLVED IN THE EARLIEST STEPS OF BIOSYNTHESIS OF THE SUGAR MOIETIESOF THE ANTITUMOR POLYKETIDE MITHRAMYCIN

Two genes (mtmD and mtmE) were cloned and sequenced from the mithramyc in producer Streptomyces argillaceus. Comparison with proteins ire dat abases and enzymatic assays after expression in Escherichia coli showe d that they encode a glucose-1-phosphate:TTP thymidylyl transferase an d a TDP-D-glucose 4,6-dehydratase, respectively. The mtmD gene was ina ctivated by gene replacement, generating a nonproducing mutant that ac cumulates a tetracyclic compound designated premithramyicinone. The id entification of premithramycinone reveals new aspects of the mithramyc in biosynthetic pathway and suggests that at least some glycosylations occur before breakage of the fourth ring.