Natural products profoundly impact many research areas, including medicine, organic chemistry, and cell biology. However, discovery of new natural products suffers from a lack of high throughput analytical techniques capable of identifying structural novelty in the face of a high degree of chemical redundancy. Methods to select bacterial strains for drug discovery have historically been based on phenotypic qualities or genetic differences and have not been based on laboratory production of secondary metabolites. Therefore, untargeted LC/MS-based secondary metabolomics was evaluated to rapidly and efficiently analyze marine-derived bacterial natural products using LC/MS-principal component analysis (PCA). A major goal of this work was to demonstrate that LC/MS-PCA was effective for strain prioritization in a drug discovery program. As proof of concept, we evaluated LC/MS-PCA for strain selection to support drug discovery, for the discovery of unique natural products, and for rapid assessment of regulation of natural product production.

Bioassay-guided metabolomic analyses led to the characterization of four new 20-membered glycosylated polyketide macrolactams – macrotermycins A-D – from a termite-associated actinomycete, Amycolatopsis sp. M39. M39’s sequenced genome revealed the macrotermycin’s putative biosynthetic gene cluster. Macrotermycins A and C had antibacterial activity against human-pathogenic S. aureus and of greater ecological relevance, they also had selective antifungal activity against a fungal parasite of the termite fungal garden.

GilOII has been unambiguously identified as the key enzyme performing the crucial C-C bond cleavage reaction responsible for the unique rearrangement of a benz[a]anthracene skeleton to the benzo[d]naphthopyranone backbone typical for the gilvocarcin type natural anticancer antibiotics. Further investigations of this enzyme led to the isolation of a hydroxy-oxepinone intermediate which allowed important conclusions regarding the cleavage mechanism.

Microtermolides A (1) and B (2) were isolated from a Streptomyces sp. strain associated with fungus-growing termites. The structures of 1 and 2 were determined by 1D- and 2D-NMR spectroscopy and high-resolution mass spectrometry. Structural elucidation of 1 led to the re-examination of the structure originally proposed for vinylamycin (3). Based on a comparison of predicted and experimental 1H and 13C NMR chemical shifts, we propose that vinylamycin’s structure be revised from 3 to 4.

Drug resistant infectious diseases are quickly becoming a global health crisis. While Streptomyces spp. have been a major source of antibiotics over the past 50 years, efficient methods are needed to identify new antibiotics and greatly improve the rate of discovery. LCMS-based metabolomics were applied to analyze extracts of 50 Streptomyes spp. Using this methodology, we discovered bottromycin D and used whole genome sequencing to determine its biosynthesis by a ribosomal pathway.

ABSTRACTEndocrocin is a simple anthraquinone frequently identified in extracts of numerous fungi. Several biosynthetic schemes for endocrocin synthesis have been hypothesized, but to date, no dedicated secondary metabolite gene cluster that produces this polyketide as its major metabolite has been identified. Here we describe our biosynthetic and regulatory characterization of the endocrocin gene cluster inAspergillus fumigatus. This is the first report of this anthraquinone in this species. The biosynthetic genes required for endocrocin production are regulated by the global regulator of secondary metabolism, LaeA, and encode an iterative nonreducing polyketide synthase (encA), a physically discrete metallo-β-lactamase type thioesterase (encB), and a monooxygenase (encC). Interestingly, the deletion of a gene immediately adjacent toencC, termedencDand encoding a putative 2-oxoglutarate-Fe(II) type oxidoreductase, resulted in higher levels of endocrocin production than in the wild-type strain, whereas overexpression ofencDeliminated endocrocin accumulation. We found that overexpression of theencAtranscript resulted in higher transcript levels ofencA-Dand higher production of endocrocin. We discuss a model of theenccluster as one evolutionary origin of fungal anthraquinones derived from a nonreducing polyketide synthase and a discrete metallo-β-lactamase-type thioesterase.

A marine Verrucosispora sp. isolated from the sponge Chondrilla caribensis f. caribensis was found to produce thiocoraline, a potent cytotoxic compound. Five new analogs of thiocoraline were isolated and represent the first analogs of thiocoraline. 22'-Deoxythiocoraline (2), thiochondrilline C (5), and 12'-sulfoxythiocoraline (6) demonstrated significant cytotoxicity against the A549 human cancer cell line with EC50 values of 0.13, 2.86, and 1.26 µM, respectively. The analogs provide insight into the SAR and biosynthesis of thiocoraline. The DP4 probability method was used to analyze ab initio NMR calculations to confirm stereochemical assignments.

Forazoline A, a novel antifungal polyketide with in vivo efficacy against Candida albicans, was discovered using LCMS-based metabolomics to investigate marine invertebrate-associated bacteria. Forazoline A had a highly unusual and unprecedented skeleton. Acquisition of 13C-13C gCOSY and 13C-15N HMQCNMR experiments provided the direct carbon-carbon and carbon-nitrogen connectivity, respectively; this represents the first example of determining direct 13C-15N connectivity for a natural product. Using yeast chemical genomics, we propose that forazoline A works via a new mechanism of action with a phenotypic outcome of disrupting membrane integrity.