Systems Biology Discussion Group
Systems Biology Approaches to Secondary Metabolites and Metabonomics
Posted May 02, 2014
Secondary metabolites, also known as natural products or idiolites, are small molecules that organisms produce to communicate and interact with their environment. Mainly produced by plants and microbes, these molecules are formed from simple precursors of the biosynthetic and chemical pathways in primary metabolism. A major challenge in secondary metabolite research is to develop detailed screening tools that allow for the discovery of new natural products and avoid simply identifying the most abundant molecules produced by each organism again and again. The better our understanding of the genetic and biosynthetic pathways that govern secondary metabolism, the better equipped researchers will be to find novel compounds and identify those with potential medicinal uses. On March 20, 2014, the Academy's Systems Biology Discussion Group presented Systems Biology Approaches to Secondary Metabolites and Metabonomics, convening speakers to discuss the functions of specific secondary metabolic pathways as well as general techniques for finding new natural products.
All metabolites occur as a result of activity within biological systems. Primary metabolites are intermediate nodes in metabolic pathways leading to the synthesis of high molecular weight biomolecules essential for growth, development, and reproduction. Precursor molecules can be diverted into alternative metabolic pathways to produce secondary metabolites, which are not required for life but instead provide protection and other survival advantages, such as repelling predators, attracting pollinators, or killing pathogens. Certain species of bacteria produce many secondary metabolites, mainly as toxins against other microorganisms that compete for the same resources. Secondary metabolites found in nature have long been a source of drugs, including antibiotics, antifungal agents, and chemotherapeutics, and it is likely that many more valuable compounds remain to be discovered.
The symposium began with an introduction by Manuel X. Duval, a senior scientist at Boehringer Ingelheim Pharmaceuticals and a professor at the University of New Haven. Duval stressed the importance of natural products as a rich source of potential drugs. But to exploit natural products, we need both to discover useful compounds and to reliably produce these compounds in large quantities. Manufacturing could be achieved by using the natural producers of secondary metabolites or by developing methods to synthesize the compounds in the lab; in either case, Duval cautioned researchers to keep the product manufacturing end goal in mind when screening for novel molecules.
Justin Nodwell of the University of Toronto presented strategies for finding new natural products. His lab works with Streptomyces coelicolor, a species of bacteria that produce pigmented secondary metabolites. By focusing on a blue-pigmented metabolite called actinorhodin, the group could easily detect changes in secondary metabolism by monitoring the color of the bacteria. After culturing S. coelicolor under conditions that do not allow it to produce actinorhodin efficiently, the group visually screened a library of small molecules, eventually focusing on ten that consistently increased actinorhodin production and naming them antibiotic remodeling compounds, or ARCs. They have since looked specifically at one family member, ARC2. This molecule causes bacterial cells to accumulate unsaturated fatty acids by inhibiting FabI, an enzyme involved in fatty acid biosynthesis. Data suggest the unsaturated fatty acids are incorporated into lipids that are deposited in the cell membrane, leading to cell wall damage. In response to the damage, the cell upregulates the afsS gene, which is a regulator of the secondary metabolism transcriptional network. Nodwell demonstrated this proposed pathway for ARC2 by showing that vancomycin and bacitracin, antibiotics that work by damaging the cell wall, had similar effects on actinorhodin production.
The identification of ARC2 as an inducer of secondary metabolism presented an opportunity to find new secondary metabolites produced by Streptomyces bacteria. Using a mass spectrometry-based approach, Nodwell's lab purified ARC2-induced molecules from multiple Streptomyces strains. ARC2 broadly activates secondary metabolism by targeting an upstream pathway, so it is possible to identify lesser-known compounds rather than detecting only the most abundant Streptomyces products. All the compounds the researchers have identified with this screen are rare products, and at least one is a completely novel molecule.
Surprisingly, most of the antimicrobial molecules detected were antifungal agents, not antibacterial compounds. The identification of molecules that are active against eukaryotic cells led the group to test several metabolites for toxicity against different eukaryotic model systems, including yeast, Drosophila melanogaster, and the nematode Caenorhabditis elegans. So far, they have found two compounds with activity against all organisms as well as one that is specific to fungi and another that is specific to C. elegans. The team hopes to elucidate the activity of many more Streptomyces-derived secondary metabolites in the coming months. Future studies will also include the use of genetically mutated strains of yeast, and gene pathway analysis to identify the functional targets of these natural products.
The next speakers were Kang Zhou and Steven Edgar from the laboratory of Dr. Gregory Stephanopoulos at Massachusetts Institute of Technology. Their related talks focused on efforts to optimize the biosynthetic pathway of taxadiene, an intermediate in the synthesis of the anti-cancer drug paclitaxel (Taxol). Zhou first shared his work to increase the production of isoprenoids, upstream precursors in the paclitaxel pathway. Isoprenoids are the largest group of natural products and are widely used in medicine. Extracting these compounds from plants usually results in low yield of the bioactive molecules. Although there are several options for production, such as semi-synthesis in the lab and production by plant cell cultures, more efficient methods are needed.
Zhou described an approach that uses E. coli as a host cell for isoprenoid synthesis. Researchers introduce the expression of foreign genes necessary for taxadiene formation and harness the DXP metabolic pathway endogenous to E. coli to accomplish taxadiene synthesis. This method allows for the study of the interactions between the endogenous DXP pathway and the taxadiene pathway to optimize taxadiene production in the bacteria. Interestingly, upregulating the DXP pathway to very high levels decreases rather than increases taxadiene production. Zhou found that the dxs protein is insoluble and can cause cell stress at high concentrations, inhibiting taxadiene synthesis. The addition of sorbitol, a protecting osmolyte, to the culture improved dxs solubility and allowed the bacterial cells to produce higher levels of taxadiene precursors.
The downstream synthesis of paclitaxel is very complex because the pathway is non-linear and has several branches. The most important steps in the pathway are catalyzed by cytochrome p450 enzymes, which activate carbon–hydrogen bonds. These enzymes are critical in reconstructing the paclitaxel biosynthetic pathway; thus E. coli is an excellent host for studying taxadiene synthesis because it has no endogenous p450 enzymes that could interfere in the pathway. Edgar found that the production of taxadiene can be optimized; however, the taxadiene product often goes through off-target side reactions rather than through the downstream reaction resulting in the desired end product. In an effort to improve pathway productivity, the researchers performed an enzyme mutagenesis screen, randomly mutating genes encoding p450 enzymes, and then screened the mutants for increased taxadiene production. One mutation successfully generated a di-hydroxylated compound, a promising result suggesting that a p450 enzyme is acting more than once. The ability of p450 enzymes to act multiple times may allow more steps in the taxadiene pathway to be completed with fewer enzymes, which is advantageous because p450 enzymes can cause stress to bacterial cells when expressed in large numbers.
The symposium concluded with a presentation by Sean F. Brady from The Rockefeller University. Brady discussed his lab's efforts to isolate natural products from soil bacteria. The majority of these bacterial species are not culturable, so his lab has developed new techniques to study their secondary metabolism. One method is to clone DNA from a bacterial species into culturable bacteria. This technique is quite straightforward because all the genes in a given secondary metabolite biosynthetic pathway are usually found in a cluster on the bacterial chromosome. But this strategy requires that the gene clusters of interest are known, and therefore does not allow for the identification of novel products. It is also limited because some secondary metabolism pathways may not be activated in culture.
To aid in the search for new natural products, Brady's group is constructing genomic libraries from soil collected around the world. Each DNA library is created by lysing the bacteria from a soil sample, and then isolating and purifying environmental DNA. The lab is creating a map of bacterial diversity that will help researchers identify where to find different types of compounds. Brady's lab has shown that the types of molecules produced by soil bacteria differ depending on environment and soil composition, highlighting the great potential certain soil types may have as sources of bioactive secondary metabolites. Brady also discussed a systematic method for probing environmental DNA libraries to find genes that are involved in specific biosynthetic pathways. This sequence-tag-guided screening approach is helpful in finding clinically relevant compounds related to known families of antibiotics and other drugs. It can also been used to find novel secondary metabolites by probing for more general biosynthetic steps that are common to a wide range of pathways and products.
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Presentations available from:
Steven Edgar (Massachusetts Institute of Technology)
Justin Nodwell, PhD (University of Toronto, Canada)
Kang Zhou, PhD (Massachusetts Institute of Technology)
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Manuel X. Duval, PhD
Boehringer Ingelheim Pharmaceuticals; University of New Haven
Manuel X. Duval is a bioinformatics scientist doing computational biology research in the Drug Discovery and Development section at Boehringer Ingelheim. His interest is in characterizing the mode of action of candidate therapeutics agents to cellular systems and identifying the source of inter-individual variability in drug response. Duval holds a PhD from the Université Joseph Fourier, France. He has worked in drug research and development since 2001, following research in genomics and bioinformatics, specifically agro-genomics, at Texas A&M University.
Thomas B. Freeman, MS
Boehringer Ingelheim Pharmaceuticals
Thomas B. Freeman is a scientist in the Computational Biology section of the Scientific Knowledge Discovery Department at Boehringer Ingelheim. He provides computational analysis in support of exploratory drug discovery programs, particularly in the biology of human disease for the immunology and inflammation and cardio-metabolic therapeutic areas, recently focusing on chronic kidney disease. He holds an MS from Virginia Polytechnic Institute, where he studied peripheral thyroid hormone metabolism in development, as well as molecular virology (bovine parvovirus replication) and plant molecular biology (ubiquitin-dependent proteolysis and apoptosis). He has worked in industry engineering insect resistance traits in corn, and later in pharmaceutical research on CNS and cardiovascular disease. Freeman participates in the Sage Bionetwork Congresses and the Genetic Alliance. He is working to exploit technical and conceptual advances to begin to understand the complexity of human physiology and pathophysiology and apply this work to drug discovery and development.
Jennifer Henry, PhD
The New York Academy of Sciences
Jennifer Henry is the director of Life Sciences at the New York Academy of Sciences. Henry joined the Academy in 2009, before which she was a publishing manager in the Academic Journals division at Nature Publishing Group. She also has eight years of direct editorial experience as editor of Functional Plant Biology for CSIRO Publishing in Australia. She received her PhD in plant molecular biology from the University of Melbourne, specializing in the genetic engineering of transgenic crops. As director of Life Sciences, she is responsible for developing scientific symposia across a range of life sciences, including biochemical pharmacology, neuroscience, systems biology, genome integrity, infectious diseases and microbiology. She also generates alliances with organizations interested in developing programmatic content.
Sean F. Brady, PhD
Sean F. Brady holds a PhD in organic chemistry from Cornell University. He completed a fellowship at the Institute of Chemistry and Cell Biology at Harvard Medical School, where he became an instructor in the Department of Biological Chemistry and Molecular Pharmacology in 2004. He moved in 2006 to The Rockefeller University as an assistant professor, and is now a Howard Hughes Medical Institute Early Career Scientist. His research interests focus on the discovery and functional characterization of new genetically encoded small molecules. One area of particular interest is the development of methods to access new biologically active small molecules from uncultured bacteria.
Massachusetts Institute of Technology
Steven Edgar is a doctoral candidate in the lab of Dr. Gregory Stephanopoulos at the Massachusetts Institute of Technology Department of Chemical Engineering. His doctoral work focuses on the production of isoprenoids in heterologous hosts, with an emphasis on the anti-cancer pharmaceutical paclitaxel and its early-pathway precursors. Edgar completed undergraduate research in the labs of Dr. Victor Breedveld and Dr. Mark Prausnitz at Georgia Institute of Technology and completed extended internships at Johnson & Johnson, Eli Lilly, and ExxonMobil.
Justin Nodwell, PhD
Justin R. Nodwell is professor and chair in the Department of Biochemistry at the University of Toronto, where he also received his PhD investigating gene regulation in bacteriophage lambda and E. coli, with Dr. Jack Greenblatt. He worked with Dr. Richard Losick as a postdoctoral fellow at Harvard University, investigating a mechanism of cell–cell signaling involved in sporulation in streptomycetes bacteria. As a Medical Research Council Scholar at McMaster University he continued to work on spore formation, culminating with the elucidation of the mechanism of biosynthesis of the SapB morphogenetic peptide. His lab then moved on to investigate secondary metabolism to identify novel drug leads. The lab was the first to use genetic perturbation of the general regulatory network for secondary metabolism to drive the expression of cryptic secondary metabolites, and later, the first to use chemical perturbation to the same end. Nodwell was a founding member of the Antimicrobial Research Centre and the Michael DeGroote Institute for Infectious Diseases Research at McMaster University. At the University of Toronto, his research focuses on the streptomycetes and the use of genetic and chemical tools to drive the identification and characterization of new metabolites with potential as therapeutic agents.
Kang Zhou, PhD
Kang Zhou obtained a PhD from the Singapore–MIT Alliance in 2012 and is now a postdoctoral associate in the lab of Dr. Gregory Stephanopoulos at the Massachusetts Institute of Technology Department of Chemical Engineering. His research has focused since 2007 on the overproduction of valuable isoprenoid pharmaceuticals in genetically engineered microbes.
Laura K. Fogli
Laura K. Fogli is a PhD candidate at New York University School of Medicine, where she is studying pathology and immunology. Her doctoral research is on mechanisms of T cell-driven lung inflammation. She plans to pursue a career in medical writing.