picromycin and neomethymycin

A biocatalytic platform that employs the final two monomodular type I polyketide synthases of the pikromycin pathway in vitro followed by direct appendage of D-desosamine and final C-H oxidation(s) in vivo was developed and applied toward the synthesis of a suite of 12- and 14-membered ring macrolide natural products. This methodology delivered both compound classes in 13 steps (longest linear sequence) from commercially available (R)-Roche ester in >10% overall yields.

Topics: Biocatalysis; Biotransformation; Lactones; Macrolides; Polyketide Synthases

The in vitro characterization of the catalytic activity of DesVII, the glycosyltransferase involved in the biosynthesis of the macrolide antibiotics methymycin, neomethymycin, narbomycin, and pikromycin in Streptomyces venezuelae, is described. DesVII is unique among glycosyltransferases in that it requires an additional protein component, DesVIII, for activity. Characterization of the metabolites produced by a S. venezuelae mutant lacking the desVIII gene confirmed that desVIII is important for the biosynthesis of glycosylated macrolides but can be replaced by at least one of the homologous genes from other pathways. The addition of recombinant DesVIII protein significantly improves the glycosylation efficiency of DesVII in the in vitro assay. When affinity-tagged DesVII and DesVIII proteins were coproduced in Escherichia coli, they formed a tight (αβ)(3) complex that is at least 10(3)-fold more active than DesVII alone. The formation of the DesVII/DesVIII complex requires coexpression of both genes in vivo and cannot be fully achieved by mixing the individual protein components in vitro. The ability of the DesVII/DesVIII system to catalyze the reverse reaction with the formation of TDP-desosamine was also demonstrated in a transglycosylation experiment. Taken together, our data suggest that DesVIII assists the folding of DesVII during protein production and remains tightly bound during catalysis. This requirement must be taken into consideration in the design of combinatorial biosynthetic experiments with new glycosylated macrolides.

Topics: Amino Sugars; Anti-Bacterial Agents; Biosynthetic Pathways; Escherichia coli; Glycosylation; Glycosyltransferases; Macrolides; Proteins; Recombinant Proteins; Streptomyces

In Streptomyces venezuelae, four polyketide synthase (PKS) polypeptides encoded by pikAI-pikAIV are used to generate 10 and 12-membered macrocyclic structures, narbonolide and 10-deoxymethynolide. Sequence analysis suggests these genes are translationally coupled with downstream genes, pikAV (encoding a type II thioesterase), desVIII-desVI (encoding enzymes responsible for production of the final glycosylated products pikromycin, narbomycin, methymycin and neomethymycin) and desR (a resistance gene). Type II thioesterases have been suggested to have an editing function in polyketide biosynthesis and deletion of the corresponding genes often leads to decreased levels of polyketide production. Surprisingly an in-frame deletion of 687 bp of the 843 bp pikAV ORF led to a strain SC1022 that produced normal yields of polyketide products, but only in the aglycone form. Plasmid-based expression of the desVIII-VI and desR in the SC1022 strain completely restored production of glycosylated products, despite the absence of a functional pikAV gene product. Under these conditions the PikAV TEII therefore does not play an important role in polyketide biosynthesis, and its function remains an enigma. These observations also demonstrate that the region of pikAV DNA deleted in strain SC1022 contains a transcription unit essential for expression of the des genes. A sequence alignment of PikAV with members of the highly conserved type II thioesterases revealed a short divergent region at the carboxy terminus, suggesting a region of pikAV that might contain such a transcription unit. DNA containing this region of pikAV was shown to be able to increase plasmid-based expression of both crotonyl CoA reductase gene (ccr) and the erythromycin resistance gene (ermE) in S. venezuelae.

Topics: Acyl-CoA Dehydrogenases; Amino Acid Sequence; Anti-Bacterial Agents; Bacterial Proteins; Cosmids; DNA, Recombinant; Fatty Acid Synthases; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Genetic Complementation Test; Glycosylation; Lactones; Macrolides; Molecular Sequence Data; Mutation; Operon; Oxidoreductases; Sequence Deletion; Sequence Homology, Amino Acid; Streptomyces; Thiolester Hydrolases; Time Factors; Transcription, Genetic

The final step in the biosynthesis of methymycin, neomethymycin, and picromycin is an hydroxylation, shown to be carried out by the cytochrome P-450 monooxygenase, PicK. Direct comparison of the relative Kcat/K(m) values for the two substrates, YC-17 and narbomycin, showed a threefold rate preference of picK for narbomycin.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Cytochrome P-450 Enzyme System; Hydroxylation; Macrolides; Mixed Function Oxygenases; Streptomyces