Christopher M. Rath

Molecular Networking as a Dereplication Strategy

A major goal in natural product discovery programs is to rapidly dereplicate known entities from complex biological extracts. We demonstrate here that molecular networking, an approach that organizes MS/MS data based on chemical similarity, is a powerful complement to traditional dereplication strategies. Successful dereplication with molecular networks requires MS/MS spectra of the natural product mixture along with MS/MS spectra of known standards, synthetic compounds, or well-characterized organisms, preferably organized into robust databases. This approach can accommodate different ionization platforms, enabling cross correlations of MS/MS data from ambient ionization, direct infusion, and LC-based methods. Molecular networking not only dereplicates known molecules from complex mixtures, it also captures related analogues, a challenge for many other dereplication strategies. To illustrate its utility as a dereplication tool, we apply mass spectrometry-based molecular networking to a diverse array of marine and terrestrial microbial samples, illustrating the dereplication of 58 molecules including analogues.

Molecular cartography of the human skin surface in 3D

Significance The paper describes the implementation of an approach to study the chemical makeup of human skin surface and correlate it to the microbes that live in the skin. We provide the translation of molecular information in high-spatial resolution 3D to understand the body distribution of skin molecules and bacteria. In addition, we use integrative analysis to interpret, at a molecular level, the large scale of data obtained from human skin samples. Correlations between molecules and microbes can be obtained to further gain insights into the chemical milieu in which these different microbial communities live. The human skin is an organ with a surface area of 1.5–2 m2 that provides our interface with the environment. The molecular composition of this organ is derived from host cells, microbiota, and external molecules. The chemical makeup of the skin surface is largely undefined. Here we advance the technologies needed to explore the topographical distribution of skin molecules, using 3D mapping of mass spectrometry data and microbial 16S rRNA amplicon sequences. Our 3D maps reveal that the molecular composition of skin has diverse distributions and that the composition is defined not only by skin cells and microbes but also by our daily routines, including the application of hygiene products. The technological development of these maps lays a foundation for studying the spatial relationships of human skin with hygiene, the microbiota, and environment, with potential for developing predictive models of skin phenotypes tailored to individual health.

Meta-omic characterization of the marine invertebrate microbial consortium that produces the chemotherapeutic natural product ET-743.

In many macroorganisms, the ultimate source of potent biologically active natural products has remained elusive due to an inability to identify and culture the producing symbiotic microorganisms. As a model system for developing a meta-omic approach to identify and characterize natural product pathways from invertebrate-derived microbial consortia, we chose to investigate the ET-743 (Yondelis) biosynthetic pathway. This molecule is an approved anticancer agent obtained in low abundance (10(-4)-10(-5) % w/w) from the tunicate Ecteinascidia turbinata and is generated in suitable quantities for clinical use by a lengthy semisynthetic process. On the basis of structural similarities to three bacterial secondary metabolites, we hypothesized that ET-743 is the product of a marine bacterial symbiont. Using metagenomic sequencing of total DNA from the tunicate/microbial consortium, we targeted and assembled a 35 kb contig containing 25 genes that comprise the core of the NRPS biosynthetic pathway for this valuable anticancer agent. Rigorous sequence analysis based on codon usage of two large unlinked contigs suggests that Candidatus Endoecteinascidia frumentensis produces the ET-743 metabolite. Subsequent metaproteomic analysis confirmed expression of three key biosynthetic proteins. Moreover, the predicted activity of an enzyme for assembly of the tetrahydroisoquinoline core of ET-743 was verified in vitro. This work provides a foundation for direct production of the drug and new analogues through metabolic engineering. We expect that the interdisciplinary approach described is applicable to diverse host-symbiont systems that generate valuable natural products for drug discovery and development.

In Vivo and In Vitro Trans-Acylation by BryP, the Putative Bryostatin Pathway Acyltransferase Derived from an Uncultured Marine Symbiont

The putative modular polyketide synthase (PKS) that prescribes biosynthesis of the bryostatin natural products from the uncultured bacterial symbiont of the marine bryozoan Bugula neritina possesses a discrete open reading frame (ORF) (bryP) that encodes a protein containing tandem acyltransferase (AT) domains upstream of the PKS ORFs. BryP is hypothesized to catalyze in trans acylation of the PKS modules for polyketide chain elongation. To verify conservation of function, bryP was introduced into AT-deletion mutant strains of a heterologous host containing a PKS cluster with similar architecture, and polyketide production was partially rescued. Biochemical characterization demonstrated that BryP catalyzes selective malonyl-CoA acylation of native and heterologous acyl carrier proteins and complete PKS modules in vitro. The results support the hypothesis that BryP loads malonyl-CoA onto Bry PKS modules, and provide the first biochemical evidence of the functionality of the bry cluster.

Structural and functional analysis of AsbF: Origin of the stealth 3,4-dihydroxybenzoic acid subunit for petrobactin biosynthesis

Petrobactin, a virulence-associated siderophore produced by Bacillus anthracis, chelates ferric iron through the rare 3,4-isomer of dihydroxybenzoic acid (3,4-DHBA). Most catechol siderophores, including bacillibactin and enterobactin, use 2,3-DHBA as a biosynthetic subunit. Significantly, siderocalin, a factor involved in human innate immunity, sequesters ferric siderophores bearing the more typical 2,3-DHBA moiety, thereby impeding uptake of iron by the pathogenic bacterial cell. In contrast, the unusual 3,4-DHBA component of petrobactin renders the siderocalin system incapable of obstructing bacterial iron uptake. Although recent genetic and biochemical studies have revealed selected early steps in petrobactin biosynthesis, the origin of 3,4-DHBA as well as the function of the protein encoded by the final gene in the B. anthracis siderophore biosynthetic (asb) operon, asbF (BA1986), has remained unclear. In this study we demonstrate that 3,4-DHBA is produced through conversion of the common bacterial metabolite 3-dehydroshikimate (3-DHS) by AsbF—a 3-DHS dehydratase. Elucidation of the cocrystal structure of AsbF with 3,4-DHBA, in conjunction with a series of biochemical studies, supports a mechanism in which an enolate intermediate is formed through the action of this 3-DHS dehydratase metalloenzyme. Structural and functional parallels are evident between AsbF and other enzymes within the xylose isomerase TIM-barrel family. Overall, these data indicate that microbial species shown to possess homologs of AsbF may, like B. anthracis, also rely on production of the unique 3,4-DHBA metabolite to achieve full viability in the environment or virulence within the host.

Molecular analysis of model gut microbiotas by imaging mass spectrometry and nanodesorption electrospray ionization reveals dietary metabolite transformations.

The communities constituting our microbiotas are emerging as mediators of the health-disease continuum. However, deciphering the functional impact of microbial communities on host pathophysiology represents a formidable challenge, due to the heterogeneous distribution of chemical and microbial species within the gastrointestinal (GI) tract. Herein, we apply imaging mass spectrometry (IMS) to localize metabolites from the interaction between the host and colonizing microbiota. This approach complements other molecular imaging methodologies in that analytes need not be known a priori, offering the possibility of untargeted analysis. Localized molecules within the GI tract were then identified in situ by surface sampling with nanodesorption electrospray ionization Fourier transform ion cyclotron resonance-mass spectrometry (nanoDESI FTICR-MS). Products from diverse structural classes were identified including cholesterol-derived lipids, glycans, and polar metabolites. Specific chemical transformations performed by the microbiota were validated with bacteria in culture. This study illustrates how untargeted spatial characterization of metabolites can be applied to the molecular dissection of complex biology in situ.

Biocatalytic synthesis of pikromycin, methymycin, neomethymycin, novamethymycin, and ketomethymycin.

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.

Biochemical and Structural Characterization of Germicidin Synthase: Analysis of a Type III Polyketide Synthase that Employs Acyl-ACP as a Starter Unit Donor

Germicidin synthase (Gcs) from Streptomyces coelicolor is a type III polyketide synthase (PKS) with broad substrate flexibility for acyl groups linked through a thioester bond to either coenzyme A (CoA) or acyl carrier protein (ACP). Germicidin synthesis was reconstituted in vitro by coupling Gcs with fatty acid biosynthesis. Since Gcs has broad substrate flexibility, we directly compared the kinetic properties of Gcs with both acyl-ACP and acyl-CoA. The catalytic efficiency of Gcs for acyl-ACP was 10-fold higher than for acyl-CoA, suggesting a strong preference toward carrier protein starter unit transfer. The 2.9 Å germicidin synthase crystal structure revealed canonical type III PKS architecture along with an unusual helical bundle of unknown function that appears to extend the dimerization interface. A pair of arginine residues adjacent to the active site affect catalytic activity but not ACP binding. This investigation provides new and surprising information about the interactions between type III PKSs and ACPs that will facilitate the construction of engineered systems for production of novel polyketides.

Biochemical and structural characterization of the tautomycetin thioesterase: analysis of a stereoselective polyketide hydrolase.

Tautomycetin (TMC) is a polyketide metabolite produced by Streptomyces sp. CK4412 and Streptomyces griseochromogenes.[1] This intriguing molecule was previously shown to possess activated T-cell specific immunosuppressive activity with a novel mode of pharmacological action, both in vivo and in vitro.[2] More recent studies have also revealed promising anticancer activity in a variety of models.[3–5]

The bacterial chemical repertoire mediates metabolic exchange within gut microbiomes.

Microbial communities in the gut have been hypothesized to play key roles in the health of the host organism. Exploring the relationship between these populations and disease states has been a focus of the human microbiome project. However, the biological roles of the compounds produced by the gut bacteria are largely unknown. We hypothesize that these compounds act as metabolic exchange factors-mediating inter-species and intra-species interactions in the microbiome. This view is supported through this review of known bacterial metabolic exchange factors and evidence for uncharacterized metabolic exchange factors in the gut. The impact of model systems and technological developments in exploring this hypothesis are also discussed. Together, these investigations are revolutionizing our understanding of the gut microbiome-presenting the possibility of identifying new strategies for treating disease in the host.