Characterization of the Two Methylation Steps Involved in the Biosynthesis of Mycinose in Tylosin

Abstract

The S-adenosyl-L-methionine-dependent O-methyltransferases TylE and TylF catalyze the last two methylation reactions in the tylosin biosynthetic pathway of Streptomyces fradiae. It has long been known that the TylE-catalyzed C2'"-O-methylation of the 6-deoxy-D-allose bound to demethylmacrocin or demethyllactenocin precedes the Ty1F-catalyzed C3"'-O-methylation of the D-javose (C2"'-O-methylated 6-deoxy-D-allose) attached to macrocin or lactenocin. This study reveals the unexpected substrate promiscuity of TylE and TylF responsible for the biosynthesis of D-mycinose (Cr"'-O-methylated D-javose) in tylosin through the identification of a new minor intermediate 2"'-O-demethyldesmycosin (2; 3"'-methyl-demethyllactenocin), which lacks a 2"'-O-methyl group on the mycinose moiety of desmycosin, along with 2"'-O-demethyltylosin (1; 3"'-methyl-demethylmacrocin) that was previously detected from the S. fradiae mutant containing a mutation in the tylE gene. These results unveil the unique substrate flexibility of TylE and TylF and demonstrate their potential for the engineered biosynthesis of novel glycosylated macrolide derivatives.