Biosynthesis of tylosin: Oxidations of 5-O-mycaminosylprtylonolide at C-20 and C-23 with a cell-free extract from Streptomycesfradiae

Cytochrome P450 monooxygenases (P450s) are some of nature’s most ubiquitous and versatile enzymes for performing oxidative metabolic transformations. Their unmatched ability to selectively functionalize inert C-H bonds has led to their increasing employment in academic and industrial settings for the production of fine and commodity chemicals. Many of the most interesting and potentially biocatalytically useful P450s come from microorganisms, where they catalyze key tailoring reactions in natural product biosynthetic pathways. While most of these enzymes act on structurally complex pathway intermediates with high selectivity, they often exhibit narrow substrate scope, thus limiting their broader application. In the present study, we investigated the reactivity of the P450 MycCI from the mycinamicin biosynthetic pathway toward a variety of macrocyclic compounds and discovered that the enzyme exhibits appreciable activity on several 16-membered ring macrolactones independent of their glycosylation state. These results were corroborated by performing equilibrium substrate binding experiments, steady-state kinetics studies, and x-ray crystallographic analysis of MycCI bound to its native substrate mycinamicin VIII. We also characterized TylHI, a homologous P450 from the tylosin pathway, and showed that its substrate scope is severely restricted compared to MycCI. Thus, the ability of the latter to hydroxylate both macrocyclic aglycones and macrolides sets it apart from related biosynthetic P450s and highlights its potential for developing novel P450 biocatalysts with broad substrate scope and high regioselectivity.

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“…While this finding is surprising given the high homology of the two enzymes, it is consistent with the results of previous studies. 66 …”

Section: Resultsmentioning

confidence: 99%

Biochemical and Structural Characterization of MycCI, a Versatile P450 Biocatalyst from the Mycinamicin Biosynthetic Pathway

et al. 2016

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“…Demethyllactenocin might be converted to tylosin by at least three routes: 14 --* 18-*22-> 24; 14 > 18 -> 20 -> 24; or 14 -> 16 --* 20 -4 24. The first two routes do not appear to be normal pathways since neither lactenocin (18) nor desmycosin (22) were converted to tylosin very efficiently. Inefficient uptake of these compounds was ruled out since lactenocin was efficiently converted to desmycosin (69 %), but the endogenously produced desmycosin was not further converted to tylosin.…”

Section: Discussionmentioning

confidence: 99%

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

Baltz

Seno²,

Stonesifer

et al. 1983

J. Antibiot.

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“…The addition of mycaminose to the aglycone is followed by concurrent ring oxidation (to generate the tylonolide moiety) and substitution with mycarose and 6‐deoxyallose. Bis‐ O ‐methylation of the latter generates mycinose and completes the biosynthesis of tylosin (O – mura et al , 1977; 1982a,b; Baltz et al , 1983; Baltz and Seno, 1988; Beckmann et al , 1989). Before the present work, tylR had been characterized as a global activator of the tyl cluster (Bate et al , 1999), but the roles of other regulators had not been addressed.…”

Section: Introductionmentioning

confidence: 99%

Differential roles of two SARP‐encoding regulatory genes during tylosin biosynthesis

Bate

Stratigopoulos

Cundliffe³

2002

Molecular Microbiology

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SummaryThe tylosin biosynthetic gene cluster of Streptomyces fradiae is remarkable in harbouring at least five regulatory genes, two of which (tylS and tylT) encode proteins of the Streptomyces antibiotic regulatory protein (SARP) family. The aim of the present work was to assess the respective contributions of TylS and TylT to tylosin production. A combination of targeted gene disruption, fermentation studies and gene expression analysis via reverse transcriptase-polymerase chain reaction (RT-PCR) suggests that tylS is essential for tylosin production and controls the expression of tylR (previously shown to be a global activator of the biosynthetic pathway) plus at least one other gene involved in polyketide metabolism or regulation thereof. This is the first demonstration of a SARP acting to control another regulatory gene during antibiotic biosynthesis. In contrast, tylT is not essential for tylosin production.

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Biosynthesis of tylosin: Oxidations of 5-O-mycaminosylprtylonolide at C-20 and C-23 with a cell-free extract from Streptomycesfradiae

Cited by 14 publications

References 7 publications

Biochemical and Structural Characterization of MycCI, a Versatile P450 Biocatalyst from the Mycinamicin Biosynthetic Pathway

Biochemical and Structural Characterization of MycCI, a Versatile P450 Biocatalyst from the Mycinamicin Biosynthetic Pathway

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

Differential roles of two SARP‐encoding regulatory genes during tylosin biosynthesis

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