Nonribosomal peptides protect Pseudomonas nunensis 4A2e from amoebal and nematodal predation.

The rhizosphere is a highly competitive environment forcing bacteria to evolve strategies to oppose their enemies. The production of toxic secondary metabolites allows bacteria to counteract predators. In this study, we describe the anti-predator armamentarium of the soil-derived bacterium Pseudomonas nunensis 4A2e. Based on a genome mining approach, we identified several biosynthetic gene clusters coding for nonribosomal peptide synthetases. Generation of gene deletion mutants of the respective clusters shows a loss of defense capabilities. We isolated the novel lipopeptides keanumycin D and nunapeptins B and C, and fully elucidated their structures by a combination of in-depth mass spectrometry experiments, stable isotope labelling, and chemical synthesis. Additionally, investigation of the quorum sensing-dependent biosynthesis allowed us to elucidate parts of the underlying regulation of the biosynthetic machinery. Ecology-inspired bioassays highlight the role of these peptides as a defence strategy against protozoans and led us to find a previously unknown function against the bacterivorous nematode Oscheius myriophilus.

Bifurcate evolution of quinone synthetases in basidiomycetes.

BACKGROUND: The terphenylquinones represent an ecologically remarkable class of basidiomycete natural products as they serve as central precursors of pigments and compounds that impact on microbial consortia by modulating bacterial biofilms and motility. This study addressed the phylogenetic origin of the quinone synthetases that assemble the key terphenylquinones polyporic acid and atromentin. RESULTS: The activity of the Hapalopilus rutilans synthetases HapA1, HapA2 and of Psilocybe cubensis PpaA1 were reconstituted in Aspergilli. Liquid chromatography and mass spectrometry of the culture extracts identified all three enzymes as polyporic acid synthetases. PpaA1 is unique in that it features a C-terminal, yet catalytically inactive dioxygenase domain. Combined with bioinformatics to reconstruct the phylogeny, our results demonstrate that basidiomycete polyporic acid and atromentin synthetases evolved independently, although they share an identical catalytic mechanism and release structurally very closely related products. A targeted amino acid replacement in the substrate binding pocket of the adenylation domains resulted in bifunctional synthetases producing both polyporic acid and atromentin. CONCLUSIONS: Our results imply that quinone synthetases evolved twice independently in basidiomycetes, depending on the aromatic α-keto acid substrate. Furthermore, key amino acid residues for substrate specificity were identified and changed which led to a relaxed substrate profile. Therefore, our work lays the foundation for future targeted enzyme engineering.

Natural products from reconstructed bacterial genomes of the Middle and Upper Paleolithic.

Major advances over the past decade in the field of ancient DNA are providing access to past paleogenomic diversity, but the diverse functions and biosynthetic capabilities of this growing paleome remain largely elusive. We investigated the dental calculus of 12 Neanderthals and 52 anatomically modern humans ranging from 100,000 years ago to the present and reconstructed 459 bacterial metagenome-assembled genomes. We identified a biosynthetic gene cluster shared by seven Middle and Upper Paleolithic individuals that allows for the heterologous production of a class of previously unknown metabolites that we name "paleofurans." This paleobiotechnological approach demonstrates that viable biosynthetic machinery can be produced from the preserved genetic material of ancient organisms, allowing access to natural products from the Pleistocene and providing a promising area for natural product exploration.

Correction: Tyrosine bioconjugation with hypervalent iodine.

[This corrects the article DOI: 10.1039/D2SC04558C.].

Ecological Niche-Inspired Genome Mining Leads to the Discovery of Crop-Protecting Nonribosomal Lipopeptides Featuring a Transient Amino Acid Building Block.

Investigating the ecological context of microbial predator-prey interactions enables the identification of microorganisms, which produce multiple secondary metabolites to evade predation or to kill the predator. In addition, genome mining combined with molecular biology methods can be used to identify further biosynthetic gene clusters that yield new antimicrobials to fight the antimicrobial crisis. In contrast, classical screening-based approaches have limitations since they do not aim to unlock the entire biosynthetic potential of a given organism. Here, we describe the genomics-based identification of keanumycins A-C. These nonribosomal peptides enable bacteria of the genus Pseudomonas to evade amoebal predation. While being amoebicidal at a nanomolar level, these compounds also exhibit a strong antimycotic activity in particular against the devastating plant pathogen Botrytis cinerea and they drastically inhibit the infection of Hydrangea macrophylla leaves using only supernatants of Pseudomonas cultures. The structures of the keanumycins were fully elucidated through a combination of nuclear magnetic resonance, tandem mass spectrometry, and degradation experiments revealing an unprecedented terminal imine motif in keanumycin C extending the family of nonribosomal amino acids by a highly reactive building block. In addition, chemical synthesis unveiled the absolute configuration of the unusual dihydroxylated fatty acid of keanumycin A, which has not yet been reported for this lipodepsipeptide class. Finally, a detailed genome-wide microarray analysis of Candida albicans exposed to keanumycin A shed light on the mode-of-action of this potential natural product lead, which will aid the development of new pharmaceutical and agrochemical antifungals.

Functional modulation of chemical mediators in microbial communities.

Interactions between microorganisms are often mediated by specialized metabolites. Although the structures and biosynthesis of these compounds may have been elucidated, microbes exist within complex microbiomes and chemical signals can thus also be subject to community-dependent modifications. Increasingly powerful chemical and biological tools allow to shed light on this poorly understood aspect of chemical ecology. We provide an overview of loss-of-function and gain-of-function chemical mediator (CM) modifications within microbial multipartner relationships. Although loss-of-function modifications are abundant in the literature, few gain-of-function modifications have been described despite their important role in microbial interactions. Research in this field holds great potential for our understanding of microbial interactions and may also provide novel tools for targeted interference with microbial signaling.

Tyrosine bioconjugation with hypervalent iodine.

Hypervalent iodine reagents have recently emerged as powerful tools for late-stage peptide and protein functionalization. Herein we report a tyrosine bioconjugation methodology for the introduction of hypervalent iodine onto biomolecules under physiological conditions. Tyrosine residues were engaged in a selective addition onto the alkynyl bond of ethynylbenziodoxolones (EBX), resulting in stable vinylbenziodoxolones (VBX) bioconjugates. The methodology was successfully applied to peptides and proteins and tolerated all other nucleophilic residues, with the exception of cysteine. The generated VBX were further functionalized by palladium-catalyzed cross-coupling and azide-alkyne cycloaddition reactions. The method could be successfully used to modify bioactive natural products and native streptavidin to enable thiol-mediated cellular uptake.

Yellow polyketide pigment suppresses premature hatching in social amoeba.

Low-molecular-weight natural products from microbes are indispensable in the development of potent drugs. However, their biological roles within an ecological context often remain elusive. Here, we shed light on natural products from eukaryotic microorganisms that have the ability to transition from single cells to multicellular organisms: the social amoebae. These eukaryotes harbor a large number of polyketide biosynthetic genes in their genomes, yet virtually none of the corresponding products can be isolated or characterized. Using complementary molecular biology approaches, including CRISPR-Cas9, we generated polyketide synthase (pks5) inactivation and overproduction strains of the social amoeba Dictyostelium discoideum. Differential, untargeted metabolomics of wild-type versus mutant fruiting bodies allowed us to pinpoint candidate metabolites derived from the amoebal PKS5. Extrachromosomal expression of the respective gene led to the identification of a yellow polyunsaturated fatty acid. Analysis of the temporospatial production pattern of this compound in conjunction with detailed bioactivity studies revealed the polyketide to be a spore germination suppressor.

The potential of amoeba-based processes for natural product syntheses.

The identification of novel platform organisms for the production and discovery of small molecules is of high interest for the pharmaceutical industry. In particular, the structural complexity of most natural products with therapeutic potential restricts an industrial production since chemical syntheses often require complex multistep routes. The amoeba Dictyostelium discoideum can be easily cultivated in bioreactors due to its planktonic growth behavior and contains numerous polyketide and terpene synthase genes with only a few compounds being already elucidated. Hence, the amoeba both bears a wealth of hidden natural products and allows for the development of new bioprocesses for existing pharmaceuticals. In this mini review, we present D. discoideum as a novel platform for the production of complex secondary metabolites and discuss its suitability for industrial processes. We also provide initial insights into future bioprocesses, both involving bacterial coculture setups and for the production of plant-based pharmaceuticals.

Total Synthesis and Structure Correction of the Cyclic Lipodepsipeptide Orfamide A.

A total synthesis of the cyclic lipodepsipeptide natural product orfamide A was achieved. By developing a synthesis format using an aminoacid ester building block and SPPS protocol adaptation, a focused library of target compounds was obtained, in high yield and purity. Spectral and LC-HRMS data of all library members with the isolated natural product identified the 5 Leu residue to be d- and the 3'-OH group to be R-configured. The structural correction of orfamide A by chemical synthesis and analysis was confirmed by biological activity comparison in Chlamydomonas reinhardtii, which indicated compound configuration to be important for bioactivity. Acute toxicity was also found against Trypanosoma brucei, the parasite causing African sleeping sickness.

Total Synthesis and Bioactivity Mapping of Geodiamolide H.

The first total synthesis of the actin-stabilizing marine natural product geodiamolide H was achieved. Solid-phase based peptide assembly paired with scalable stereoselective syntheses of polyketide building blocks and an optimized esterification set the stage for investigating the key ring-closing metathesis. Geodiamolide H and synthetic analogues were characterized for their toxicity and for antiproliferative effects in cellulo, by characterising actin polymerization induction in vitro, and by docking on the F-actin target and property computation in silico, for a better understanding of structure-activity relationships (SAR). A non-natural analogue of geodiamolide H was discovered to be most potent in the series, suggesting significant potential for tool compound design.

Lipopeptide-mediated bacterial interaction enables cooperative predator defense.

Bacteria are inherently social organisms whose actions should ideally be studied within an interactive ecological context. We show that the exchange and modification of natural products enables two unrelated bacteria to defend themselves against a common predator. Amoebal predation is a major cause of death in soil bacteria and thus it exerts a strong selective pressure to evolve defensive strategies. A systematic analysis of binary combinations of coisolated bacteria revealed strains that were individually susceptible to predation but together killed their predator. This cooperative defense relies on a Pseudomonas species producing syringafactin, a lipopeptide, which induces the production of peptidases in a Paenibacillus strain. These peptidases then degrade the innocuous syringafactin into compounds, which kill the predator. A combination of bioprospecting, coculture experiments, genome modification, and transcriptomics unravel this novel natural product-based defense strategy.

The Landscape of Recombination Events That Create Nonribosomal Peptide Diversity.

Nonribosomal peptides (NRP) are crucial molecular mediators in microbial ecology and provide indispensable drugs. Nevertheless, the evolution of the flexible biosynthetic machineries that correlates with the stunning structural diversity of NRPs is poorly understood. Here, we show that recombination is a key driver in the evolution of bacterial NRP synthetase (NRPS) genes across distant bacterial phyla, which has guided structural diversification in a plethora of NRP families by extensive mixing and matching of biosynthesis genes. The systematic dissection of a large number of individual recombination events did not only unveil a striking plurality in the nature and origin of the exchange units but allowed the deduction of overarching principles that enable the efficient exchange of adenylation (A) domain substrates while keeping the functionality of the dynamic multienzyme complexes. In the majority of cases, recombination events have targeted variable portions of the Acore domains, yet domain interfaces and the flexible Asub domain remained untapped. Our results strongly contradict the widespread assumption that adenylation and condensation (C) domains coevolve and significantly challenge the attributed role of C domains as stringent selectivity filter during NRP synthesis. Moreover, they teach valuable lessons on the choice of natural exchange units in the evolution of NRPS diversity, which may guide future engineering approaches.

Biosynthesis of Bacterial Natural Products and Small Molecules in Microbial Interactions.

Natural products are important mediators and effectors in complex microbial communities. This special collection is devoted to the multifaceted roles of these natural products as well as on understanding how, when, and why they are produced. (Picture created with biorender.com.).

Structure elucidation of bacterial nonribosomal lipopeptides.

Nonribosomal lipopeptides (NRLPs) are complex natural products of bacterial origin that not only fulfill important ecological functions but also serve as lead structures for the development of new pharmaceuticals. In order to carry out detailed structure-activity relationship studies and to decipher the biological activities of NRLPs, the primary structure, including stereochemical assignment, of every new member of this natural product family has to be established first. In this review, we want to focus on analytical techniques and tools that can be employed to elucidate the structure of bacterial NRLPs.

Chimeric LuxR Transcription Factors Rewire Natural Product Regulation.

LuxR-type transcriptional activator proteins frequently regulate the expression of biosynthetic gene clusters (BGCs). With only a fraction of bacterial BGCs being expressed under standard culturing conditions, modulation of LuxRs would provide a powerful approach to activate silent clusters. We show that by exploiting the modular nature of LuxR proteins, it is possible to construct functional chimeric LuxRs, which enables both the rewiring of quorum sensing systems and the activation of silent BGCs. Importantly, our strategy allowed us to identify the novel natural product pseudomonol from a bacterium of the genus Pseudomonas.

Biosynthesis of Pseudomonas-Derived Butenolides.

Butenolides are well-known signaling molecules in Gram-positive bacteria. Here, we describe a novel class of butenolides isolated from a Gram-negative Pseudomonas strain, the styrolides. Structure elucidation was aided by the total synthesis of styrolide A. Transposon mutagenesis enabled us to identify the styrolide biosynthetic gene cluster, and by using a homology search, we discovered the related and previously unknown acaterin biosynthetic gene cluster in another Pseudomonas species. Mutagenesis, heterologous expression, and identification of key shunt and intermediate products were crucial to propose a biosynthetic pathway for both Pseudomonas-derived butenolides. Comparative transcriptomics suggests a link between styrolide formation and the regulatory networks of the bacterium.

Structure, properties, and biological functions of nonribosomal lipopeptides from pseudomonads.

Bacteria of the genus Pseudomonas are ubiquitous in nature. Pseudomonads display a fascinating metabolic diversity, which correlates with their ability to colonize an extremely wide range of ecological niches. As a result, these bacteria are a prolific source of natural products. Biosynthesis of the latter is often orchestrated by arrays of chemical signals arising from intraspecies communication or interspecies relationships with bacteria, fungi, amoebae, plants, and insects. Especially nonribosomal lipopeptides, which have diverse biological activities, play important roles in the lifestyle of pseudomonads. In this review, we will focus on the molecular structures, properties, biosynthetic pathways, and biological functions of pseudomonal lipopeptides. This review is not only addressed to bio/chemists rather it serves as a comprehensive guide for all researchers (micro/biologists, ecologists, and environmental scientists) working in this multidisciplinary field.