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 TylF-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 (C3‴-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.

Deciphering Nature’s Intricate Way of N,S-Dimethylating l-Cysteine: Sequential Action of Two Bifunctional Adenylation Domains

“…As methylation is an important and prevalent modification of natural products, it has attracted a great deal of attention in the natural product community. Because methylation may beneficially alter the activity of natural products, engineered methyltransferases or ones (naturally occurring or engineered) with promiscuous activity − have been investigated as biocatalysts for the synthesis of novel bioactive compounds. However, although very important, M domains, which are commonly found embedded in A domains of NRPSs, remain poorly understood .…”

Discovery of a highly efficient TylF methyltransferase via random mutagenesis for improving tylosin production

“…Enzyme activity assays were carried out as described previously with some modifications [30] . The optimized enzyme reaction was conducted in 1 ml reaction buffer (50 mM Tris-HCl, 10 mM MgCl 2 , 1 mM phenylmethanesulfonyl fluoride (PMSF), 6 mM 2-mercaptoethanol, pH 7.6), and initiated by adding protein, SAM (0.4 mM final), and macrocin (0.2 mM final).…”

Exploration of Streptomyces fradiae J1–021 as a Potential Host for the Heterologous Production of Spinosad

“…fradiae genome and includes at least 43 open reading frames (ORF). Tylosin PKS includes one loading module and seven extension modules, which are encoded by five biosynthetic genes ( tylG1 , tylG2 , tylG3 , tylG4 , and tylG5 ) sequentially transcribed in the same direction. , As shown in Figure , stepwise addition of three deoxy sugar moieties ( d -mycaminose, 5′-deoxy- d -allose, and l -mycarose) to tylactone affords tylosin …”

“…Tylosin PKS includes one loading module and seven extension modules, which are encoded by five biosynthetic genes (tylG1, tylG2, tylG3, tylG4, and tylG5) sequentially transcribed in the same direction. 11,12 As shown in Figure 1, stepwise addition of three deoxy sugar moieties (Dmycaminose, 5′-deoxy-D-allose, and L-mycarose) to tylactone affords tylosin. 13 The commercial tylosin-producer S. fradiae can produce abundant common polyketide precursors (methylmalonyl-CoA, malonyl-CoA, and propionyl-CoA), which is essential for spinosad and tylosin polyketide syntheses (Figure 1).…”

“…Tylosin comprises tylosin A, B, C, and D 9 and is a 16membered polyketide macrocyclic compound derived from S. fradiae and utilized as a growth-promoting additive and a veterinary antibiotic. 10,11 The tylosin BGC comprises an 86 kb region of the S. f radiae genome and includes at least 43 open reading frames (ORF). Tylosin PKS includes one loading module and seven extension modules, which are encoded by five biosynthetic genes (tylG1, tylG2, tylG3, tylG4, and tylG5) sequentially transcribed in the same direction.…”