Genome Mining of Streptomyces sp. Tü 6176: Characterization of the Nataxazole Biosynthesis Pathway

Abstract

Streptomyces sp. Tü 6176 produces the cytotoxic benzoxazole nataxazole. Bioinformatic analysis of the genome of this organism predicts the presence of 38 putative secondary-metabolite biosynthesis gene clusters, including those involved in the biosynthesis of AJI9561 and its derivative nataxazole, the antibiotic hygromycin B, and ionophores enterobactin and coelibactin. The nataxazole biosynthesis gene cluster was identified and characterized: it lacks the O-methyltransferase gene required to convert AJI9561 into nataxazole. This O-methyltransferase activity might act as a resistance mechanism, as AJI9561 shows antibiotic activity whereas nataxazole is inactive. Moreover, heterologous expression of the nataxazole biosynthesis gene cluster in S. lividans JT46 resulted in the production of AJI9561. Nataxazole biosynthesis requires the shikimate pathway to generate 3-hydroxyanthranilate and an iterative type I PKS to generate 6-methylsalicylate. Production of nataxazole was improved up to fourfold by disrupting one regulatory gene in the cluster. An additional benzoxazole, 5-hydroxynataxazole is produced by Streptomyces sp. Tü 6176. 5-Hydroxynataxazole derives from nataxazole by the activity of an as yet unidentified oxygenase; this implies cross-talk between the nataxazole biosynthesis pathway and an unknown pathway.

High diversity and suggested endemicity of culturable Actinobacteria in an extremely oligotrophic desert oasis

“…While genome mining represents a major paradigm shift for exploration of rare taxa (Cano-Prieto et al, 2015; Tang et al, 2015; Smanski, Schlatter & Kinkel, 2016), recent studies from genome mining for secondary metabolites gene clusters of unculturable Actinobacteria support the culturable approach for natural product discovery targeting “gifted microbes”, obtaining samples from unexplored habitats. In particular, untapped marine sediments are recommended when searching for cultivable potentially bioactive natural products from Actinobacteria (Baltz, 2016).…”

Elucidation of the glycosylation steps during biosynthesis of antitumor macrolides PM100117 and PM100118 and engineering for novel derivatives

“…caniferus GUA-06-05-006A wild type, or the corresponding mutant strains, were mixed with the same volume of ethyl acetate and shacked vigorously at room temperature for 1 h. The organic phase was collected and evaporated to dryness with a Speed-Vac. The residue was re-dissolved in 60 µl of methanol:DMSO (1:1), clarified by centrifugation and then subjected to UPLC and LC/MS analysis as described elsewhere [49]. To assess the PM100117/18 production level, SMS medium was vacuum -filtrated with nitrocellulose filters of 0.45 µm pore size (Millipore).…”

“…Gene hyg allows recognizing clones in which a complete gene replacement by a double cross-over has taken place (Hyg S Apm R ) from those in which a single cross-over event has integrated the deletion plasmid into the chromosome (Hyg R Apm R ). To construct the pSETH plasmid backbone, used to achieve gene complementation and over-expression experiments, a 1.6-kb DNA fragment was amplified from plasmid pDR4 [53] with the primer pair NcoI-dHG R /NcoI-rvHG R and cloned in the NcoI site of plasmid pSETec [49], which harbors the constitutive ermE*p promoter. Plasmids intended to accomplish gene complementation and over-expression were constructed by inserting the respective target ORF in the BamHI/EcoRV sites of pSETH (Additional file 4: Table S6).…”

Direct Capture Technologies for Genomics-Guided Discovery of Natural Products

“…For example, the cluster for nataxazole, a cytotoxic benzoxazole, was captured by TAR but expression of the cluster caused significant growth inhibition in heterologous hosts. Moreover, some hosts could not be transformed due to the cytotoxicity of nataxazole pathway intermediates [53]. …”