Systems Biology Approaches to Secondary Metabolites and Metabonomics

Overview

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.

ARC2-mediated induction of secondary metabolism could identify novel Streptomyces-derived natural products. (Image courtesy of Justin Nodwell)

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.

Isoprenoid synthesis through the DXP pathway in E. coli is improved by increasing the solubility of pathway proteins. Sorbitol increased the solubility of the enzyme dxs, leading to decreased cell toxicity and improved DXP pathway productivity. (Image courtesy of Kang Zhou)

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.

Use the tab above to find multimedia from this event.

Presentations available from:
Steven Edgar (Massachusetts Institute of Technology)
Justin Nodwell, PhD (University of Toronto, Canada)
Kang Zhou, PhD (Massachusetts Institute of Technology)

The Systems Biology Discussion Group is proudly supported by

Mission Partner support for the Frontiers of Science program provided by