Discovery of a Tambjamine Gene Cluster in Streptomyces Suggests Convergent Evolution in Bipyrrole Natural Product Biosynthesis.

While bacterial natural products are a valuable source of therapeutics, the molecules produced by most biosynthetic gene clusters remain unknown. Tambjamine YP1, produced by Pseudoalteromonas tunicata, is partially derived from fatty acids siphoned from primary metabolism. A structurally similar tambjamine produced by Streptomyces, BE-18591, had not been linked to a gene cluster. Using enzymes putatively implicated in the construction of these two tambjamines, we used sequence similarity networks and gene knockout experiments to identify the biosynthetic gene cluster responsible for the production of tambjamine BE-18591 in Streptomyces albus. Despite the structural similarities between YP1 and BE-18591, the biosynthesis of the alkylamine tails of these molecules differs significantly, with the S. albus gene cluster putatively encoding a dedicated system for the construction of the fatty acid precursor to BE-18591. These different pathways in Pseudoalteromonas and Streptomyces suggest that evolutionary convergence is operative, with similar selective pressures leading to the emergence of structurally similar tambjamine natural products using different biosynthetic logic.

Discovery of 3'-O-ß-Glucosyltubercidin and the Nucleoside Specific Glycosyltransferase AvpGT through Genome Mining.

A genome mining approach was used to identify a hybrid tubercidin-nucleocidin biosynthetic gene cluster (BGC) in Streptomyces sp. AVP053U2. Analysis of culture extracts by liquid chromatography-mass spectrometry revealed the presence of a glucosylated tubercidin derivative. A gene, avpGT, was identified within the hybrid cluster that has homology to the glucosyltransferase that is responsible for 3'-O-ß-glucosylation of the fluorinated natural product nucleocidin. AvpGT was heterologously expressed and purified from Escherichia coli for in vitro characterization. AvpGT is active toward UDP-glucose and UDP-galactose as glycosyl donors and several nucleosides as acceptors. Kinetic analysis revealed that AvpGT is most specific for UDP-glucose [kcat/KMapp = (1.1 ± 0.3) × 105 M-1·s-1] as the glycosyl donor and tubercidin [kcat/KMapp = (5.3 ± 1.8) × 104 M-1·s-1] as the glycosyl acceptor. NMR spectroscopic analysis revealed the product of this reaction to be 3'-O-ß-glucopyranosyl tubercidin. A sequence analysis of AvpGT reveals a family of nucleoside-specific GTs, which may be used as markers of BGCs that produce glycosylated nucleosides.

Identification of Genes Essential for Sulfamate and Fluorine Incorporation During Nucleocidin Biosynthesis.

Nucleocidin is an adenosine derivative containing 4'-fluoro and 5'-O-sulfamoyl substituents. In this study, nucleocidin biosynthesis is examined in two newly discovered producers, Streptomyces virens B-24331 and Streptomyces aureorectus B-24301, which produce nucleocidin and related derivatives at titers 30-fold greater than S. calvus. This enabled the identification of two new O-acetylated nucleocidin derivatives, and a potential glycosyl-O-acetyltransferase. Disruption of nucJ, nucG, and nucI, within S. virens B-24331, specifying a radical SAM/Fe-S dependent enzyme, sulfatase, and arylsulfatase, respectively, led to loss of 5'-O-sulfamoyl biosynthesis, but not fluoronucleoside production. Disruption of nucN, nucK, and nucO specifying an amidinotransferase, and two sulfotransferases respectively, led to loss of fluoronucleoside production. Identification of S. virens B-24331 as a genetically tractable and high producing strain sets the stage for understanding nucleocidin biosynthesis and highlights the utility of using 16S-RNA sequences to identify alternative producers of valuable compounds in the absence of genome sequence data.