Our research interests concern the structural identification of secondary metabolites (natural “small molecules”) and the elucidation of their biological functions. We currently pursue three major projects, which employ three different approaches for linking small-molecule molecular structures with biological function or activity.
The goal of this project is to complement the highly developed genomics and proteomics of the important model organism Caenorhabditis elegans with a comprehensive structural and biological characterization of its secondary metabolome, which, surprisingly, has been explored only to a very limited extent. C. elegans, which is one of the first multicellular organisms with a completed genome sequence, is small enough for high-throughput operations, yet features a highly differentiated physiology, and it has become apparent that many signaling pathways in C. elegans show strong analogies to corresponding pathways in higher animals. Our recent studies demonstrated the importance of small molecule-based signalling cascades for virtually every aspect of C. elegans biology, and furtehr showed that these nematodes are amazingly skilled chemists: using simple building blocks from conserved primary metabolism and a strategy of modular assembly, C. elegans creates complex molecular architectures to regulate development and behaviour. These nematode-derived modular metabolites (NDMMs) are based on the dideoxysugars ascarylose or paratose, which serve as scaffolds for attachment of moieties from lipid, amino acid, carbohydrate, citrate, and nucleoside metabolism. Mutant screens and comparative metabolomics based on NMR spectroscopy and MS have so-far revealed several 100 different ascarylose ("ascarosides") and a few paratose ("paratosides") derivatives, many of which represent potent signalling molecules that can be active at femtomolar levels, regulating aging, development, behaviour, body shape, and many other life history traits. NDMM biosynthesis appears to be carefully regulated as assembly of different modules proceeds with very high specificity. Preliminary biosynthetic studies have confirmed the primary metabolism origin of some NDMM building blocks, whereas the mechanisms that underlie their highly specific assembly are not understood.
Considering their functions and biosynthetic origin, NDMMs represent a new class of natural products that cannot easily be classified as "primary" or "secondary". We believe that the identification of new variants of primary metabolism-derived structures that serve important signalling functions in C. elegans and other nematodes provides a strong incentive for a comprehensive re-analysis of metabolism in higher animals, including humans.
Our efforts towards characterizing the C. elegans metabolome rely on innovative comparative metabolomics based on 2D NMR spectroscopy and high-resolution MS/MS. This strategy greatly accelerates the process of identifying new chemical structures and elucidating their biological functions, and takes advantage of the extensive mutant and RNAi libraries that cover almost the entire C. elegans genome. Our C. elegans project integrates analytical chemistry, synthetic chemistry, molecular biology, and protein biochemistry, and relies on an extensive network of collaborators at Caltech, MIT, and several Max Planck Institutes.
Fungi are among the most prolific sources of pharmacologically relevant natural products, and bioinformatic analyses of fungal genomes have revealed 1000s of biosynthetic gene clusters (BGCs), of which ~40% contain NRPS (non-ribosomal peptide synthetase) or NRPS-like genes. However, only a fraction of the biosynthetic capabilities of fungi have been discovered, because expression of many, perhaps even most biosynthetic pathways depends strongly on environmental conditions, including the presence of specific elicitors derived from the presence of other organisms. As a result, most fungal BGCs have no known metabolites (orphan BGCs). To address this challenge, our research leverages major recent advances in heterologous gene expression and comparative metabolomics, aiming to develop an innovative discovery pipeline for the systematic annotation of the biosynthetic capabilities of fungi. In collaboration with the lab of Nancy Keller at the University of Wisconsin-Madison, we are creating bacterial artificial chromosome (BAC) libraries with insert sizes ?100 kb that are sufficiently large to accommodate gene clusters encoding entire biosynthetic pathways of complex natural products. The use of BACs and an induced yeast recombinatory system will enable the systematic survey of the biosynthetic capacity of diverse fungal species. For rapid chemical characterization of novel metabolites, we are combining concepts of comparative metabolomics, high-resolution MS/MS, and high dynamic range 2D NMR spectroscopy to develop a fast innovative structure elucidation process that is specifically tailored for surveying arrays of metabolite extracts generated from serial heterologous expression.
NIH Director's Transformative R01 Award to Frank Schroeder (BTI/Cornell) and Dennis Kim (MIT)
The dependence on antibiotics in medicine and agriculture has resulted in the emergence of bacteria resistant to all known antibiotics, posing a grave threat to human health. Whereas most currently used antibiotics are derived from the secondary metabolism of microorganisms, no systematic search for antimicrobial small molecules produced by animals in response to infection has been conducted. Based on our recent analyses of the Caenorhabditis elegans metabolome, we hypothesize that nematodes, simple animals that represent one of the most successful forms of life on Earth, synthesize antimicrobial small molecules that neutralize and/or kill pathogenic bacteria and fungi. To investigate this hypothesis, we focus on the nematode species C. elegans and Pristionchus pacificus, which feed on bacteria and are exposed to a wide variety of non-pathogenic and pathogenic bacteria and fungi in their natural environment. We will develop nematode metabolite libraries that are enriched for compounds produced by these nematode hosts in response to pathogenic bacteria and fungi, including the human pathogens Pseudomonas aeruginosa and Staphylococcus aureus. We will then screen these small molecule metabolite libraries for antimicrobial activity, taking advantage of established pathogenesis assays that follow microbial proliferation and survival of the nematode host, with subsequent definitive identification of active compounds via comparative metabolomics, and chemical synthesis. Our recent studies of the metabolomes of these nematode species have identified several novel classes of small molecules of yet undetermined function, some of which have chemical structures strongly suggestive of mediating interactions with bacteria. We anticipate to find compounds that directly affect bacterial and fungal growth as well as metabolites that function through the inhibition of microbial colonization and virulence mechanisms. Compounds that modulate aspects of virulence but that do not directly affect viability of bacteria represent potential therapeutic agents that may be less likely to give rise to microbial resistance.