Functional analysis of environmental DNA-derived type II PKS reveals structurally diverse secondary metabolites

ArticleinProceedings of the National Academy of Sciences 108(31):12629-34 · August 2011with48 Reads
DOI: 10.1073/pnas.1103921108 · Source: PubMed
A single gram of soil is predicted to contain thousands of unique bacterial species. The majority of these species remain recalcitrant to standard culture methods, prohibiting their use as sources of unique bioactive small molecules. The cloning and analysis of DNA extracted directly from environmental samples (environmental DNA, eDNA) provides a means of exploring the biosynthetic capacity of natural bacterial populations. Environmental DNA libraries contain large reservoirs of bacterial genetic diversity from which new secondary metabolite gene clusters can be systematically recovered and studied. The identification and heterologous expression of type II polyketide synthase-containing eDNA clones is reported here. Functional analysis of three soil DNA-derived polyketide synthase systems in Streptomyces albus revealed diverse metabolites belonging to well-known, rare, and previously uncharacterized structural families. The first of these systems is predicted to encode the production of the known antibiotic landomycin E. The second was found to encode the production of a metabolite with a previously uncharacterized pentacyclic ring system. The third was found to encode the production of unique KB-3346-5 derivatives, which show activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. These results, together with those of other small-molecule-directed metagenomic studies, suggest that culture-independent approaches are capable of accessing biosynthetic diversity that has not yet been extensively explored using culture-based methods. The large-scale functional screening of eDNA clones should be a productive strategy for generating structurally previously uncharacterized chemical entities for use in future drug development efforts.
    • "The targeting of phylogenetically unique sequence tags as means of identifying gene clusters that encode structurally novel classes of natural products has also been explored using KS β genes from type II PKS biosynthesis. This work has led to the identification of a number of natural products with either new or rarely seen ring systems (Fig. 2, 19–24) [31, 53, 54, 57], further illustrating the utility of targeted screens in conjunction with a metagenomic natural product-mining pipeline to discover diverse new secondary metabolites. "
    [Show abstract] [Hide abstract] ABSTRACT: Bacterial natural products have proven to be invaluable starting points in the development of many currently used therapeutic agents. Unfortunately, traditional culture-based methods for natural product discovery have been deemphasized by pharmaceutical companies due in large part to high rediscovery rates. Culture-independent, or "metagenomic," methods, which rely on the heterologous expression of DNA extracted directly from environmental samples (eDNA), have the potential to provide access to metabolites encoded by a large fraction of the earth's microbial biosynthetic diversity. As soil is both ubiquitous and rich in bacterial diversity, it is an appealing starting point for culture-independent natural product discovery efforts. This review provides an overview of the history of soil metagenome-driven natural product discovery studies and elaborates on the recent development of new tools for sequence-based, high-throughput profiling of environmental samples used in discovering novel natural product biosynthetic gene clusters. We conclude with several examples of these new tools being employed to facilitate the recovery of novel secondary metabolite encoding gene clusters from soil metagenomes and the subsequent heterologous expression of these clusters to produce bioactive small molecules.
    Full-text · Article · Nov 2015
    • "symbionts) rather than clonal isolates, enrichment of specific populations followed by construction of long-insert libraries that can be functionally pre-screened for clones of interest prior to sequencing has been employed with success (Hildebrand et al., 2004; Sudek et al., 2007). While this approach is time consuming, it can also be employed with environmental DNA as an alternative to shotgun metagenomic sequencing (Feng, Kallifidas, & Brady, 2009; Gillespie et al., 2002). Often these approaches require the use of specialised cloning vectors to overcome issues with codon bias, but have the added benefit that they can be used for heterologous expression and functional screening of the TMS gene products (Brady, Simmons, Kim, & Schmidt, 2009; Piel, 2011). "
    [Show abstract] [Hide abstract] ABSTRACT: Microbes produce a huge array of secondary metabolites endowed with important ecological functions. These molecules, which can be catalogued as natural products, have long been exploited in medical fields as antibiotics, anticancer and anti-infective agents. Recent years have seen considerable advances in elucidating natural-product biosynthesis and many drugs used today are natural products or natural-product derivatives. The major contribution to recent knowledge came from application of genomics to secondary metabolism and was facilitated by all relevant genes being organised in a contiguous DNA segment known as gene cluster. Clustering of genes regulating biosynthesis in bacteria is virtually universal. Modular gene clusters can be mixed and matched during evolution to generate structural diversity in natural products. Biosynthesis of many natural products requires the participation of complex molecular machines known as polyketide synthases and non-ribosomal peptide synthetases. Discovery of new evolutionary links between the polyketide synthase and fatty acid synthase pathways may help to understand the selective advantages that led to evolution of secondary-metabolite biosynthesis within bacteria. Secondary metabolites confer selective advantages, either as antibiotics or by providing a chemical language that allows communication among species, with other organisms and their environment. Herewith, we discuss these aspects focusing on the most clinically relevant bioactive molecules, the thiotemplated modular systems that include polyketide synthases, non-ribosomal peptide synthetases and fatty acid synthases. We begin by describing the evolutionary and physiological role of marine natural products, their structural/functional features, mechanisms of action and biosynthesis, then turn to genomic and metagenomic approaches, highlighting how the growing body of information on microbial natural products can be used to address fundamental problems in environmental evolution and biotechnology.
    Full-text · Article · Jul 2015
    • "While these clades add uncertainty to our trait-based biogeographic analysis , they are one of our most exciting results: we identified what could be a natural source of novel antibiotics and should be an attractive target for bioprospecting. Heterologous expression of gene clusters with KSα sequences from the least characterized parts of the phylogeny could reveal new antibiotics [34, 35]. The phylogenetic approach for estimating unmeasured traits presented here should support future microbial trait-based biogeography studies. "
    [Show abstract] [Hide abstract] ABSTRACT: Understanding patterns in the distribution and abundance of functional traits across a landscape is of fundamental importance to ecology. Mapping these distributions is particularly challenging for species-rich groups with sparse trait measurement coverage, such as flowering plants, insects, and microorganisms. Here, we use likelihood-based character reconstruction to infer and analyze the spatial distribution of unmeasured traits. We apply this framework to a microbial dataset comprised of 11,732 ketosynthase alpha gene sequences extracted from 144 soil samples from three continents to document the spatial distribution of putative microbial polyketide antibiotic production. Antibiotic production is a key competitive strategy for soil microbial survival and performance. Additionally, novel antibiotic discovery is highly relevant to human health, making natural antibiotic production by soil microorganisms a major target for bioprospecting. Our comparison of trait-based biogeographical patterns to patterns based on taxonomy and phylogeny is relevant to our basic understanding of microbial biogeography as well as the pressing need for new antibiotics.
    Full-text · Article · Jun 2015
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