Cristian Gurgui

An environmental bacterial taxon with a large and distinct metabolic repertoire

Cultivated bacteria such as actinomycetes are a highly useful source of biomedically important natural products. However, such ‘talented’ producers represent only a minute fraction of the entire, mostly uncultivated, prokaryotic diversity. The uncultured majority is generally perceived as a large, untapped resource of new drug candidates, but so far it is unknown whether taxa containing talented bacteria indeed exist. Here we report the single-cell- and metagenomics-based discovery of such producers. Two phylotypes of the candidate genus ‘Entotheonella’ with genomes of greater than 9 megabases and multiple, distinct biosynthetic gene clusters co-inhabit the chemically and microbially rich marine sponge Theonella swinhoei. Almost all bioactive polyketides and peptides known from this animal were attributed to a single phylotype. ‘Entotheonella’ spp. are widely distributed in sponges and belong to an environmental taxon proposed here as candidate phylum ‘Tectomicrobia’. The pronounced bioactivities and chemical uniqueness of ‘Entotheonella’ compounds provide significant opportunities for ecological studies and drug discovery.

Exploiting the mosaic structure of trans-acyltransferase polyketide synthases for natural product discovery and pathway dissection

Modular polyketide synthases (PKSs) are giant bacterial enzymes that synthesize many polyketides of therapeutic value. In contrast to PKSs that provide acyltransferase (AT) activities in cis, trans-AT PKSs lack integrated AT domains and exhibit unusual enzymatic features with poorly understood functions in polyketide assembly. This has retarded insight into the assembly of products such as mupirocin, leinamycin and bryostatin 1. We show that trans-AT PKSs evolved in a fundamentally different fashion from cis-AT systems, through horizontal recruitment and assembly of substrate-specific ketosynthase (KS) domains. The insights obtained from analysis of these KS mosaics will facilitate both the discovery of novel polyketides by genome mining, as we demonstrate for the thailandamides of Burkholderia thailandensis, and the extraction of chemical information from short trans-AT PCR products, as we show using metagenomic DNA of marine sponges. Our data also suggest new strategies for dissecting polyketide biosynthetic pathways and engineering polyketide assembly.

Metagenome Mining Reveals Polytheonamides as Posttranslationally Modified Ribosomal Peptides

Made and Modified The polytheonamides are 48-residue toxins derived from marine sponges that include 18 D-amino acids, as well as many other unusual amino acid modifications. Given the complexity, one might guess that these peptides are the product of nonribosomal, peptide synthetase (NRPS). However, Freeman et al. (p. 387, published online 13 September now show that polytheonamides are produced by a bacterial symbiont using a ribosomal pathway. Six candidate enzymes for the 48 posttranslational modifications were identified and three were functionally validated. Such ribosomal systems could be useful in bioengineering. Large toxins that comprise many modified and d-amino acids are ribosomally synthesized and then derivatized. It is held as a paradigm that ribosomally synthesized peptides and proteins contain only l-amino acids. We demonstrate a ribosomal origin of the marine sponge–derived polytheonamides, exceptionally potent, giant natural-product toxins. Isolation of the biosynthetic genes from the sponge metagenome revealed a bacterial gene architecture. Only six candidate enzymes were identified for 48 posttranslational modifications, including 18 epimerizations and 17 methylations of nonactivated carbon centers. Three enzymes were functionally validated, which showed that a radical S-adenosylmethionine enzyme is responsible for the unidirectional epimerization of multiple and different amino acids. Collectively, these complex alterations create toxins that function as unimolecular minimalistic ion channels with near-femtomolar activity. This study broadens the biosynthetic scope of ribosomal systems and creates new opportunities for peptide and protein bioengineering.

Polyketide assembly lines of uncultivated sponge symbionts from structure-based gene targeting

There is increasing evidence that uncultivated bacterial symbionts are the true producers of numerous bioactive compounds isolated from marine sponges. The localization and heterologous expression of biosynthetic genes could clarify this issue and provide sustainable supplies for a wide range of pharmaceuticals. However, identification of genes in the usually highly complex symbiont communities remains a challenging task. For polyketides, one of the most important groups of sponge-derived drug candidates, we have developed a general strategy that allows one to rapidly access biosynthetic gene clusters based on chemical moieties. Using this method, we targeted polyketide synthase genes from two different sponge metagenomes. We have obtained from a sponge-bacterial association a complete pathway for the rare and potent antitumor agent psymberin from Psammocinia aff. bulbosa. The data support the symbiont hypothesis and provide insights into natural product evolution in previously inaccessible bacteria.

Biosynthesis of the Myxobacterial Antibiotic Corallopyronin A

Corallopyronin A is a myxobacterial compound with potent antibacterial activity. Feeding experiments with labelled precursors resulted in the deduction of all biosynthetic building blocks for corallopyronin A and revealed an unusual feature of this metabolite: its biosynthesis from two chains, one solely PKS‐derived and the other NRPS/PKS‐derived. The starter molecule is believed to be carbonic acid or its monomethyl ester. The putative corallopyronin A biosynthetic gene cluster is a trans‐AT‐type mixed PKS/NRPS gene cluster, containing a β‐branching cassette. Striking features of this gene cluster are a NRPS‐like adenylation domain that is part of a PKS‐type module and is believed to be responsible for glycine incorporation, as well as split modules with individual domains occurring on different genes. It is suggested that CorB is a trans‐acting ketosynthase and it is proposed that it catalyses the Claisen condensation responsible for the interconnection of the two chains. Additionally, the stereochemistry of corallopyronin A was deduced by a combination of a modified Moshers method and ozonolysis with subsequent chiral GC analyses.

Metagenomic Approaches to Identify and Isolate Bioactive Natural Products from Microbiota of Marine Sponges

Many marine sponges harbor massive consortia of symbiotic bacteria belonging to diverse phyla. Sponges are also an unusually rich source of biologically active natural products, and evidence is accumulating that these compounds might often be synthesized by the symbionts. Since the study of sponge-associated bacteria is generally hampered by very low cultivation rates, cultivation-independent, metagenomic methods have recently been applied to sponges. These methods allow for the isolation of biosynthetic gene clusters that can ultimately be exploited to develop sustainable natural product sources by heterologous expression. However, general challenges encountered in sponge metagenomic research are the poor quality of the isolated DNA with respect to size and yield, the difficulty to identify genes of interest among numerous homologs, insufficient clone numbers in metagenomic libraries, and time-consuming screening procedures to identify and isolate rare positive clones. Here, we give an overview of methods that address these problems and can be used to streamline isolation of biosynthetic and other genes of interest.

Corrigendum: An environmental bacterial taxon with a large and distinct metabolic repertoire

This corrects the article DOI: 10.1038/nature12959

Chemical surprises from an uncultivated sponge symbiont

Marine sponges are a rich source of bioactive natural products and are promising sources for drug discovery and development An impressive example is the sponge Theonella swinhoei, which has yielded more than 120 compounds belonging to diverse structural types. Many sponges also harbor highly complex consortia of symbiotic bacteria that are suspected to be the true source of at least some of the secondary metabolites. In previous work, our group demonstrated a bacterial origin of onnamide- and psymberin-type polyketides for two different sponges (1–3), but there were no insights into the producer of compounds from other natural product families. In addition, the exact taxonomic identity of sponge-associated producers remained unknown. This talk will present new insights into these two issues. Isolation of genes encoding a peptide biosynthetic pathway from the T. swinhoei metagenome demonstrated a bacterial origin. Several genes were heterologously expressed and functionally characterized, which revealed unprecedented biosynthetic transformations. The novelty of these modifications suggests the existence of a structurally distinct natural product family, for which we propose the name proteusins. Using a strategy consisting of single-cell analysis and metagenomic sequencing, we identified the bacterial producer of onnamide polyketides in T. swinhoei. Surprisingly, the data suggest the symbiont to be a chemically exceptionally prolific bacterium, producing not only onnamides but most other compounds from this sponge chemotype, including the known and two previously unknown proteusins. Further biosynthetic studies and a survey of other sponges indicate that close relatives of the producer are widespread in these animals and vary with respect to their biosynthetic capabilities. These bacteria might therefore represent the first uncultivated taxon with a metabolic richness resembling that of major cultivated bacterial natural product sources. These results reveal a key role of symbiotic bacteria in the chemistry of their sponge hosts and provide new strategies to study uncultivated symbionts in a more systematic fashion.