Cyclic product of the PKS last thioesterase module is then substrate of PimK for transfer of the sugar moiety to the aglycone of pimaricin. Then PimD Cytochrome P-450 monooxygenase formation of the epoxy ring. Natamycin is a polyene amphoteric macrolide antibiotic with antifungal properties. Natamycin exerts its antifungal effects by binding to sterols in the fungal cell membrane thereby increasing membrane permeability. This leads to a leakage and loss of essential cellular constituents. Following ocular application, natamycin is retained in the conjunctival fornices and attains effective concentrations within the corneal stroma where it exerts its effect.
Polyketides serve as rich source of therapeutically relevant drug leads. The manipulation of polyketide synthases (PKSs) for generating derivatives with improved activities usually results in substantially reduced yields. Growing evidence suggests that type I PKS thioesterase (TE) domains are key bottlenecks in the biosynthesis of polyene antibiotics, such as pimaricin and amphotericin, and their unnatural derivatives. Herein, we elucidate the structure of the 26-membered macrolide-complexed TE domain from the pimaricin pathway (Pim TE), which specifies a spacious bifunnel-shaped substrate channel with a highly hydrophobic cleft proximal to the catalytic triad and a hydrophilic loop I region specific for the cyclization of amphiphilic polyene macrolide. Notably, the natural intermediate with C12-COOH is stabilized by a hydrogen-bond network, as well as by interactions between the polyene moiety and the hydrophobic cleft. Moreover, the bottleneck in processing the unnatural intermediate with C12-CH3 is attributed to the unstable and mismatched docking of the curved substrate in the channel. Aided by an in vitro assay with a fully elongated linear polyene intermediate as the substrate, multiple strategies were adopted, herein, to engineer Pim TE, including introducing H-bond donors, enhancing hydrophobic interactions, and modifying the catalytic center. Efficient TE mutations with increased substrate conversion up to 39.2% in vitro were further conducted in vivo, with a titer increase as high as 37.1% for the less toxic decarboxylated pimaricin derivatives with C12-CH3. Our work uncovers the mechanism of TE-catalyzed polyene macrolide formation and highlights TE domains as targets for PKS manipulation for titer increases in engineered unnatural polyketide derivatives.
Title: A complex multienzyme system encoded by five polyketide synthase genes is involved in the biosynthesis of the 26-membered polyene macrolide pimaricin in Streptomyces natalensis Aparicio JF, Fouces R, Mendes MV, Olivera N, Martin JF Ref: Chemical Biology, 7:895, 2000 : PubMed
BACKGROUND: Polyene macrolides are a class of large macrocyclic polyketides that interact with membrane sterols, having antibiotic activity against fungi but not bacteria. Their rings include a chromophore of 3-7 conjugated double bonds which constitute the distinct polyene structure. Pimaricin is an archetype polyene, important in the food industry as a preservative to prevent mould contamination of foods, produced by Streptomyces natalensis. We set out to clone, sequence and analyse the gene cluster responsible for the biosynthesis of this tetraene. RESULTS: A large cluster of 16 open reading frames spanning 84985 bp of the S. natalensis genome has been sequenced and found to encode 13 homologous sets of enzyme activities (modules) of a polyketide synthase (PKS) distributed within five giant multienzyme proteins (PIMS0-PIMS4). The total of 60 constituent active sites, 25 of them on a single enzyme (PIMS2), make this an exceptional multienzyme system. Eleven additional genes appear to govern modification of the polyketide-derived framework and export. Disruption of the genes encoding the PKS abolished pimaricin production. CONCLUSIONS: The overall architecture of the PKS gene cluster responsible for the biosynthesis of the 26-membered polyene macrolide pimaricin has been determined. Eleven additional tailoring genes have been cloned and analysed. The availability of the PKS cluster will facilitate the generation of designer pimaricins by combinatorial biosynthesis approaches. This work represents the extensive description of a second polyene macrolide biosynthetic gene cluster after the one for the antifungal nystatin.
