Chalcomycin Biosynthesis Gene Cluster from Streptomyces bikiniensis : Novel Features of an Unusual Ketolide Produced through Expression of the chm Polyketide Synthase in Streptomyces fradiae

Abstract

Chalcomycin, a 16-membered macrolide antibiotic made by the bacterium Streptomyces bikiniensis, contains a 2,3-trans double bond and the neutral sugar D-chalcose in place of the amino sugar mycaminose found in most other 16-membered macrolides. Degenerate polyketide synthase (PKS)-specific primers were used to amplify DNA fragments from S. bikiniensis with very high identity to a unique ketosynthase domain of the tylosin PKS. The resulting amplimers were used to identify two overlapping cosmids encompassing the chm PKS. Sequencing revealed a contiguous segment of >60 kb carrying 25 putative genes for biosynthesis of the polyketide backbone, the two deoxysugars, and enzymes involved in modification of precursors of chalcomycin or resistance to it. The chm PKS lacks the ketoreductase and dehydratase domains in the seventh module expected to produce the 2,3-double bond in chalcomycin. Expression of PKS in the heterologous host Streptomyces fradiae, from which the tyl genes encoding the PKS had been removed, resulted in production of at least one novel compound, characterized as a 3-keto 16-membered macrolactone in equilibrium with its 3-trans enol tautomer and containing the sugar mycaminose at the C-5 position, in agreement with the structure predicted on the basis of the domain organization of the chm PKS. The production of a 3-keto macrolide from the chm PKS indicates that a discrete set of enzymes is responsible for the introduction of the 2,3-trans double bond in chalcomycin. From comparisons of the open reading frames to sequences in databases, a pathway for the synthesis of nucleoside diphosphate-D-chalcose was proposed.

Identification of the Missing trans -Acting Enoyl Reductase Required for Phthiocerol Dimycocerosate and Phenolglycolipid Biosynthesis in Mycobacterium tuberculosis

“…Therefore, these bioinformatic analyses of the Rv2953 protein suggest that it has a Rossmann fold structure and binds NADH or NADPH, consistent with it being an enoyl reductase. To our knowledge, this is the first report of a type I Pks requiring an additional accessory enzyme for polyketide biosynthesis in mycobacteria, although direct interactions between type I Pks and discrete enzymes have been reported for AT, KR, DH, and ER domains in other microorganisms (6,17,27,31). For instance, in Aspergillus terreus, the lovastatin nonaketide synthase, a type I Pks that lacks a functional ER activity, interacts with LovC, a putative ER, for the correct assembly of dihydromonacolin L during lovastatin production (17).…”

Natural‐Product Sugar Biosynthesis and Enzymatic Glycodiversification

“…Tylosin carries both sugars, whereas chalcomycin, dihydrochalcomycin, and mycinamicin contain 38. The biosynthetic gene clusters for these compounds have been sequenced, [46][47][48][49][50] and recent genetic and biochemical studies performed on the tylosin and dihydrochalcomycin systems have fully established the pathways for the formation of these two sugars. [47,[51][52][53][54][55] The key intermediate, TDP-6-deoxy-dallose (37), in the pathway of 38 is synthesized from 21 by C-3 epimerization by the RmlC homologues GerF/TylJ/ChmJ/ MydH and subsequent C-4 ketoreduction by GerKI/TylD/ ChmD/MydI.…”

Structural and Functional Characterization of Three Polyketide Synthase Gene Clusters in Bacillus amyloliquefaciens FZB 42

“…An interesting feature of the difficidin structure is the occurrence of four double bonds with a Z configuration, a geometry that is very rare among polyketides (2,32,41,49). It has been proposed that an aspartate residue in the KR domain plays a crucial role in defining the stereochemistry of the ketoreduction step, which in turn would influence the doublebond configuration after dehydration by the downstream DH domain (38).…”