CHARACTERIZATION OF METHYLTRANSFERASE AND HYDROXYLASE GENES INVOLVED IN THE BIOSYNTHESIS OF THE IMMUNOSUPPRESSANTS FK506 AND FK520

FK506 and FK520 are 23-membered macrocyclic polyketides with potent im munosuppressive and antifungal activities, The gene encoding 31-O-deme thyl-FK506 methyltransferase, fkbM, was isolated from Streptomyces sp, strains MA6858 and MA6548, two FK506 producers, and Streptomyces hygr oscopicus subsp. ascomyceticus, an FK520 producer. The nucleotide sequ ence of the fkbM gene revealed an open reading frame encoding a polype ptide of 260 amino acids. Disruption of fkbM in Streptomyces sp, strai n MA6518 yielded a mutant that produced 31-O-demethyl-FK506, confirmin g the involvement of the isolated genes in the biosynthesis of FK506 a nd FK520. Heterologous expression of fkbM in Streptomyces lividans est ablished that fkbM encodes an O-methyltransferase catalyzing the methy lation of the C-31 hydroxyl group of 31-O-demethyl-FK506 and FK520. A second open reading frame, fkbD, was found upstream of fkbM in all thr ee aforementioned species and was predicted to encode a protein of 388 residues that showed a strong resemblance to cytochrome P-450 hydroxy lases. Disruption of fkbD had a polar effect on the synthesis of the d ownstream fkbM gene product and resulted in the formation of 9-deoxo-3 1-O-demethyl-FK506. This established the product of fkbD as the cytoch rome P-450 9-deoxo-FK506 hydroxylase, which is responsible for hydroxy lation at position C-9 of the FK506 and FK520 macrolactone ring.