Deletion of rapQNML from the rapamycin gene cluster of Streptomyces hygroscopicus gives production of the 16-O-desmethyl-27-desmethoxy analog

Five contiguous genes in the rapamycin gene cluster, rapQONML, of Streptomy ces hygroscopicus ATCC29253 were replaced with a neomycin resistance marker by double homologous recombination. The resulting strain, if fed pipecolat e, produced the analog 16-O-desmethyl-27-desmethoxyrapamycin instead of rap amycin. This indicates that the P450 hydroxylase encoded by rapN is specifi c for C-27, and that the O-methyltransferases encoded by rapQ and rapM meth ylate the hydroxyl groups on C-16 and C-27. By inference, the remaining P45 0 hydroxylase and methyltransferase genes (rapI and rapJ) are responsible f or hydroxylation of C-9 and methylation of the C-39 hydroxyl, consistent wi th their homology to fkbD and fkbM, respectively, in the FK506 cluster. The relatively high level of 16-O-desmethyl-27-desmethoxyrapamycin produced in dicates that the reactions at C-9 and C-39 do not require previous modifica tion of the macrolactone precursor at either C-16 or C-27.