Biosynthesis of the macrolide antibiotic tylosin. A preferred pathway from tylactone to tylosin

The efficiencies of bioconversion of twenty-three potential intermediates in the biosynthesis of tylosin were determined with a mutant strain blocked only in tylactone biosynthesis. The results indicated that tylactone, the first intermediate excreted by Streptomyces fradiae, is converted to tylosin by a preferred sequence of reactions which include: (1) addition of mycaminose to the C-5 hydroxyl position of the lactone; (2) hydroxylation of the C-20 methyl group to a hydroxymethyl group; (3) dehydrogenation of the C-20 hydroxymethyl group to a formyl group; (4) hydroxylation of the C-23 methyl group to a hydroxymethyl; (5) addition of 6-deoxy-D-allose to the C-23 hydroxymethyl group; (6) addition of mycarose to the 4'-hydroxyl group of mycaminose; (7) addition of a methyl group to the 2"'-hydroxyl position of demethylmacrocin, and (8) addition of a methyl group to the 3"'-hydroxyl position of macrocin to produce tylosin. The intermediates which lacked both neutral sugars (mycarose and 6-deoxy-D-allose) were biologically unstable, and substantial quantities of these compounds were degraded during standard bioconversion experiments. However, the amount of one such intermediate (O-mycaminosyltylonolide) degraded was substantially reduced when low concentrations of the compound were used for bioconversion, and under these conditions, much higher efficiencies of bioconversion to tylosin were obtained. We have shown that a mutant blocked in hydroxylation of the C-20 methyl group is also blocked in the further dehydrogenation of the C-20 hydroxymethyl group to a formyl group, and have confirmed in in vitro studies that the 2"'-O-methylation of demethylmacrocin must proceed the 3"'-O-methylation of macrocin to produce tylosin.