Genome mining of oxidation modules in trans-acyltransferase polyketide synthases reveals a culturable source for lobatamides

Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) are multimodular megaenzymes that biosynthesize many bioactive natural products. They contain a remarkable range of domains and module types that introduce different substituents into growing polyketide chains. As one such modification, we recently reported Baeyer–Villiger-type oxygen insertion into nascent polyketide backbones, thereby generating malonyl thioester intermediates. In this work, genome mining focusing on architecturally diverse oxidation modules in trans-AT PKSs led us to the culturable plant symbiont Gynuella sunshinyii, which harbors two distinct modules in one orphan PKS. The PKS product was revealed to be lobatamide A, a potent cytotoxin previously only known from a marine tunicate. Biochemical studies show that one module generates glycolyl thioester intermediates, while the other is proposed to be involved in oxime formation. The data suggest varied roles of oxygenation modules in the biosynthesis of polyketide scaffolds and support the importance of trans-AT PKSs in the specialized metabolism of symbiotic bacteria.</p

Biosynthetic potential of the global ocean microbiome

8 pages, 4 figures, supplementary information https://doi.org/10.1038/s41586-022-04862-3.-- This Article is contribution number 130 of Tara OceansNatural microbial communities are phylogenetically and metabolically diverse. In addition to underexplored organismal groups1, this diversity encompasses a rich discovery potential for ecologically and biotechnologically relevant enzymes and biochemical compounds2,3. However, studying this diversity to identify genomic pathways for the synthesis of such compounds4 and assigning them to their respective hosts remains challenging. The biosynthetic potential of microorganisms in the open ocean remains largely uncharted owing to limitations in the analysis of genome-resolved data at the global scale. Here we investigated the diversity and novelty of biosynthetic gene clusters in the ocean by integrating around 10,000 microbial genomes from cultivated and single cells with more than 25,000 newly reconstructed draft genomes from more than 1,000 seawater samples. These efforts revealed approximately 40,000 putative mostly new biosynthetic gene clusters, several of which were found in previously unsuspected phylogenetic groups. Among these groups, we identified a lineage rich in biosynthetic gene clusters (‘Candidatus Eudoremicrobiaceae’) that belongs to an uncultivated bacterial phylum and includes some of the most biosynthetically diverse microorganisms in this environment. From these, we characterized the phospeptin and pythonamide pathways, revealing cases of unusual bioactive compound structure and enzymology, respectively. Together, this research demonstrates how microbiomics-driven strategies can enable the investigation of previously undescribed enzymes and natural products in underexplored microbial groups and environmentsThis work was supported by funding from the ETH and the Helmut Horten Foundation; the Swiss National Science Foundation (SNSF) through project grants 205321_184955 to S.S., 205320_185077 to J.P. and the NCCR Microbiomes (51NF40_180575) to S.S.; by the Gordon and Betty Moore Foundation (https://doi.org/10.37807/GBMF9204) and the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 101000392 (MARBLES) to J.P.; by an ETH research grant ETH-21 18-2 to J.P.; and by the Peter and Traudl Engelhorn Foundation and by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 897571 to C.C.F. S.L.R. was supported by an ETH Zurich postdoctoral fellowship 20-1 FEL-07. M.L., L.M.C. and G.Z. were supported by EMBL Core Funding and the German Research Foundation (DFG, Deutsche Forschungsgemeinschaft, project no. 395357507, SFB 1371 to G.Z.). M.B.S. was supported by the NSF grant OCE#1829831. C.B. was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement Diatomic, no. 835067). S.G.A. was supported by the Spanish Ministry of Economy and Competitiveness (PID2020-116489RB-I00). M.K. and H.M. were funded by the SNSF grant 407540_167331 as part of the Swiss National Research Programme 75 ‘Big Data’. M.K., H.M. and A.K. are also partially funded by ETH core funding (to G. Rätsch)With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)Peer reviewe

Genome mining- and synthetic biology-enabled production of hypermodified peptides

ISSN:1755-4349ISSN:1755-433

Structure and mechanism for iterative amide N-methylation in the biosynthesis of channel-forming peptide cytotoxins

The polytheonamides are highly modified and potent, cytotoxic peptides with a unique β-helical structure (helical diameter ∼4 Å) that affords selective membrane permeation of monovalent cations. Toxicity has been linked to promiscuous ion-channel behavior in studies of the prototypical polytheonamide B. Specific structural features of the β-helical toxins include, among other modifications, Cα-epimerizations and Nγ-methylations, which have been highlighted as the early-stage modifications most critical for β-helix formation. Here, we interrogate Cα-epimerization and Nγ-methylation to understand the importance of these modifications for secondary structure. We characterize the mechanism of Nγ-methylations on the amide side chains of D-Asn, an enzymatic modification with little biochemical precedent. Crystal structures of the AerE methyltransferase in complex with its epimerized peptide substrate and S-adenosyl-homocysteine reveal features of substrate recognition and an unexpected metal-ion that may mediate methyl transfer to the poorly nucleophilic amide. These studies provide a framework for the engineering of novel β-helical peptides with ion and membrane selectivity.ISSN:0027-8424ISSN:1091-649

Genome-Based Identification of a Plant-Associated Marine Bacterium as a Rich Natural Product Source

ISSN:1433-7851ISSN:1521-3773ISSN:0570-083

Automated structure prediction of trans-acyltransferase polyketide synthase products

ISSN:1552-4450ISSN:1552-446

Genome-based discovery and total synthesis of janustatins, potent cytotoxins from a plant-associated bacterium

Host-associated bacteria are increasingly being recognized as underexplored sources of bioactive natural products with unprecedented chemical scaffolds. A recently identified example is the plant-root-associated marine bacterium Gynuella sunshinyii of the chemically underexplored order Oceanospirillales. Its genome contains at least 22 biosynthetic gene clusters, suggesting a rich and mostly uncharacterized specialized metabolism. Here, in silico chemical prediction of a non-canonical polyketide synthase cluster has led to the discovery of janustatins, structurally unprecedented polyketide alkaloids with potent cytotoxicity that are produced in minute quantities. A combination of MS and two-dimensional NMR experiments, density functional theory calculations of C-13 chemical shifts and semiquantitative interpretation of transverse rotating-frame Overhauser effect spectroscopy data were conducted to determine the relative configuration, which enabled the total synthesis of both enantiomers and assignment of the absolute configuration. Janustatins feature a previously unknown pyridodihydropyranone heterocycle and an unusual biological activity consisting of delayed, synchronized cell death at subnanomolar concentrations.ISSN:1755-4349ISSN:1755-433

BluePharmTrain:biology and Biotechnology of Marine Sponges

BluePharmTrain is a Marie Curie Initial Training Network of 17 European academic and industrial partners collaborating to train young scientists in multidisciplinary aspects of blue biotechnology. Harvesting marine sponges for the extraction of bioactive compounds is often highly unsustainable, and the chemical synthesis of promising compounds is often either too complex or very expensive. To find sustainable and economically feasible production methods of sponge-derived compounds, individual BluePharmTrain research projects explore innovative techniques, focusing on selected sponge species shown to harbour interesting active metabolites. The different techniques include sponge cell cultivation, cultivation of microbial symbionts, next-generation sequencing approaches (i.e. metagenomics and metatranscriptomics), in situ and ex situ cultivation of sponges, life cycle characterisation, chemical structure elucidation of compounds and compound metabolic pathway description. Altogether, these consorted efforts and collaborations lead to new insights on sponge metabolism, sponge-microbe interactions and bioactive compound production.</p

BluePharmTrain: biology and biotechnology of marine sponges

BluePharmTrain is a Marie Curie Initial Training Network of 17 European academic and industrial partners collaborating to train young scientists in multidisciplinary aspects of blue biotechnology. Harvesting marine sponges for the extraction of bioactive compounds is often highly unsustainable, and the chemical synthesis of promising compounds is often either too complex or very expensive. To find sustainable and economically feasible production methods of sponge-derived compounds, individual BluePharmTrain research projects explore innovative techniques, focusing on selected sponge species shown to harbour interesting active metabolites. The different techniques include sponge cell cultivation, cultivation of microbial symbionts, next-generation sequencing approaches (i.e. metagenomics and metatranscriptomics), in situ and ex situ cultivation of sponges, life cycle characterisation, chemical structure elucidation of compounds and compound metabolic pathway description. Altogether, these consorted efforts and collaborations lead to new insights on sponge metabolism, sponge-microbe interactions and bioactive compound production.</p