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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.

Growth from spores activated a biosynthetic gene cluster in Actinomadura sp. RB29, resulting in the identification of two novel groups of halogenated polyketide natural products, named maduralactomycins and actinospirols. The unique tetracyclic and spirocyclic structures were assigned based on a combination of NMR analysis, chemoinformatic calculations, X-ray crystallography, and 13C labeling studies. On the basis of HRMS2 data, genome mining, and gene expression studies, we propose an underlying noncanonical angucycline biosynthesis and extensive post-polyketide synthase (PKS) oxidative modifications.

Filamentous cyanobacteria belong to the most prolific producers of structurally unique and biologically active natural products, yet the majority of biosynthetic gene clusters predicted for these multicellular collectives are currently orphan. Here, we present a systems analysis of secondary metabolite gene expression in the model strain Nostoc punctiforme PCC73102 using RNA-seq and fluorescence reporter analysis. Our data demonstrate that the majority of the cryptic gene clusters are not silent but are expressed with regular or sporadic pattern. Cultivation of N. punctiforme using high-density fermentation overrules the spatial control and leads to a pronounced upregulation of more than 50% of biosynthetic gene clusters. Our data suggest that a combination of autocrine factors, a high CO2 level, and high light account for the upregulation of individual pathways. Our overarching study not only sheds light on the strategies of filamentous cyanobacteria to share the enormous metabolic burden connected with the production of specialized molecules but provides an avenue for the genome-based discovery of natural products in multicellular cyanobacteria as exemplified by the discovery of highly unusual variants of the tricyclic peptide microviridin.

International audience; Genomics and metabolomics are widely used to explore specialized metabolite diversity. The Paired Omics Data Platform is a community initiative to systematically document links between metabolome and (meta)genome data, aiding identification of natural product biosynthetic origins and metabolite structures.

Sespenine is a structurally unusual indole sesquiterpenoid isolated from endophytic Streptomyces sp. HKI0595. Herein, we report a ten-step (the longest linear sequence) synthesis of this molecule from commercially available materials, on the basis of our first generation synthesis. Sharpless asymmetric epoxidation and Stille–Miyata coupling were used to construct a functionalized epoxy ester, which underwent Ti(III) mediated reductive radical cyclization to give a trans-decalin intermediate with a 2-methoxycarbonylindole side chain. Oxidation of this compound afforded a pair of epimeric 3-hydroxyindolenines, and the major isomer entered a bioinspired cascade of Prins cyclization/Friedel–Crafts/retro Friedel–Crafts under acidic conditions, to furnish the polycyclic core of sespenine. Sespenine analogues bearing different C2 substituents were prepared with similar chemistry. Xiamycin A, a carbazole congener of sespenine, was synthesized from the minor hydroxyindolenine epimer as well.

Sulfa drugs, such as sulfonilamide and dapsone, are classical antibiotics that have been in clinical use worldwide. Despite the relatively simple architectures, practically no natural products are known to feature such aromatic sulfonamide or diarylsulfone substructures. We report the unexpected discovery of three fully unprecedented, sulfonyl-bridged alkaloid dimers (sulfadixiamycins A-C) from recombinant Streptomyces species harboring the entire xiamycin biosynthesis gene cluster. Sulfadixiamycins exhibit moderate antimycobacterial activities and potent antibiotic activities even against multidrug-resistant bacteria. Gene inactivation, complementation, and biotransformation experiments revealed that a flavin-dependent enzyme (XiaH) plays a key role in sulfadixiamycin biosynthesis. XiaH mediates a radical-based, three-component reaction involving two equivalents of xiamycin and sulfur dioxide, which is reminiscent of radical styrene/SO2 copolymerization.

Divergolides are structurally diverse ansamycins produced by a bacterial endophyte (Streptomyces sp.) of the mangrove tree Bruguiera gymnorrhiza. By genomic analyses a gene locus coding for the divergolide pathway was detected. The div gene cluster encodes genes for the biosynthesis of 3-amino-5-hydroxybenzoate and the rare extender units ethylmalonyl-CoA and isobutylmalonyl-CoA, polyketide assembly by a modular type I polyketide synthase (PKS), and enzymes involved in tailoring reactions, such as a Baeyer-Villiger oxygenase. A detailed PKS domain analysis confirmed the stereochemical integrity of the divergolides and provided valuable new insights into the formation of the diverse aromatic chromophores. The bioinformatic analyses and the isolation and full structural elucidation of four new divergolide congeners led to a revised biosynthetic model that illustrates the formation of four different types of ansamycin chromophores from a single polyketide precursor.

Indoloterpenoid natural products play an eminent role as drugs, and their significance for medicine has propelled a plethora of synthetic and biosynthetic studies. Interestingly, these alkaloids are a hallmark of plant and fungal metabolism, but virtually nothing was known about the corresponding bacterial pathways. Only recently, in the context of profiling the bacterial endophytes of widespread mangrove trees, we discovered the unprecedented bacterial indolosesquiterpenes xiamycin (1), indosespene (2), and sespenine (3 ; Figure 1). Together with oridamycin (4) and oxiamycin (5) these multicyclic alkaloids constitute a new family of bacterial indoloterpenes. The co-occurrence of these diverse hybrid metabolites in a single organism implies a biogenetic relationship. Thus, we and another research group have independently analyzed xiamycin (xia) biosynthesis gene clusters. Through mutational analyses and heterologous expression, we found that the pathway involves an unparalleled cyclization sequence to yield diverse ring systems. The heterologous expression of the entire gene cluster also led to the discovery of N C and N N coupled xiamycin dimers, which had been overlooked in the wild-type strain owing to their low production. These structurally intriguing bixiamycins represent the first examples of bacterial bisindolosesquiterpenes (BIST), and their biosynthesis has remained enigmatic. Herein, we report the discovery and full characterization of a series of highly regiodivergent, N C and N N aryl-coupled xiamycin dimers, and reveal their potent antibacterial activities. Furthermore, we show that a single flavoprotein not only mediates diverse aryl couplings, but also ether formation. Finally, we support a radical-based mechanism by a biomimetic synthesis of the xiamycin derivatives. To reveal the range of bixiamycins produced, we inspected the metabolic profile of Streptomyces albus carrying the entire xia biosynthesis gene cluster. HPLC-HRMS analyses indicated that the strain produces a number of compounds that likely result from the dimerization of xiamycin (1), as they have the same molecular formula (C46H48N2O6), but differ in retention times. To obtain sufficient amounts of these new compounds for a full structural characterization, the culture was scaled up. Both mycelia and culture filtrate of a scaled-up fermentation (50 L) were extracted with ethyl acetate, and the combined extracts were subjected to fractionation by flash chromatography, first through silica, then through a Sephadex LH-20 column. Final purification by preparative HPLC yielded various dimers, including the atroposiomeric pair of N N coupled bixiamycins (6a/6b) and other types of dimers (Figure 2) as pure compounds: 6a (10 mg), 6b (5 mg), 7a (3 mg), 7b (1.0 mg), and 8 (23.1 mg). NMR analysis of the new dimers revealed that they all possess the xiamycin backbone. Compounds 7a and 7b were obtained as a pair of atropodiastereomers. Their H NMR spectra show two sets of signals, thus indicating that the coupling sites of the two moieties of the molecule are not identical. As only a signal for H21 is visible, whereas the counterpart (H21’) is missing in the H NMR spectrum, a N C coupling between N1 and C21’ was proposed. COSY and HMBC correlations confirmed the proposed structure for the two halves of the dimer. Comparison of the experimental CD spectra with the TDCAMB3LYP/6-31G*//B97D/TZVP calculated ones revealed that 7a has a P configuration, whereas the minor atropodiastereomer 7b has an M configuration at the N C axis (Figure 3; see the Supporting Information for details). Figure 1. Structures of bacterial indolosesquiterpenes, xiamycin (1), indosespene (2), sespenine (3), oridamycin (4), and oxiamycin (5).

Indole terpenoids encompass a highly diverse group of natural products, including infamous psychotropic agents such as lysergic acid derivatives, the aphrodisiac yohimbine, and the potassium channel blockers paxilline and lolitrem. What is remarkable about this multifarious class is that practically all indole terpene alkaloids have been isolated from plants and fungi. Stimulated by the importance of these synthetically challenging compounds, groundbreaking studies have been undertaken in recent years to understand and engineer terpene alkaloid pathways in fungi and plants. In light of the impressive number of known eukaryotic indole terpene metabolites, it is peculiar that only recently the first bacterial representatives of this group were discovered. We and others independently reported the structures of pentacyclic indolocarbazoles from Streptomyces spp., namely the diastereomers oridamycin (1) and xiamycin A (2 ; Scheme 1). Considering that these indolosesquiterpenes (IST) are reminiscent of plant metabolites, it is astounding that two xiamycin-producing strains are endophytes of widespread mangrove trees, Bruguiera gymnorrhiza and Kandelia candel. A more detailed metabolic investigation of the K. candel endophyte revealed three congeners of 2, xiamycin B (3), the seco-derivative indosespene (4), and the novel bridged spiro compound sespenine (5). These rare endophyte metabolites likely play an ecological role in their habitats because their diverse antiviral, antibacterial, and antifungal activities may contribute to the antibiotic reservoir of the mangrove plants. From a chemical point of view, the co-occurrence of these structurally novel alkaloids is intriguing because it suggests a common biogenetic origin. However, to date nothing is known about the biosynthesis of indole terpenes in bacteria. Herein we unveil the molecular basis for unprecedented bacterial indolosesquiterpene biosynthesis in a mangrove endophyte and show by heterologous gene expression and mutational analysis that the unusual pentacyclic ring systems of xiamycin and sespenine are formed by a novel cyclization sequence. Furthermore, we report the discovery of three new xiamycin dimers from a heterologously reconstituted IST pathway. To elucidate the genes required for IST biosynthesis in the mangrove endophyte, we subjected whole genomic DNA to shotgun sequencing. Bioinformatic mining (basic local alignment search tool (BLAST) analysis) of the genomic draft sequence of Streptomyces sp. HKI0576 for terpenoid biosynthesis genes revealed a gene cluster (GenBank accession No. HE815466) with a three-gene cassette (xiaABC) coding for canonical enzymes involved in the non-mevalonate (or deoxyxylulose, DOX) pathway: DXS (1-deoxy-d-xylulose 5-phosphate synthase), HDS (4-hydroxy-3-methylbut-2-enyl diphosphate synthase) and HDR (4-hydroxy-3-methylbut-2enyl diphosphate reductase); remaining DOX pathway genes were identified elsewhere in the genome. The isoprene unit biosynthesis genes are flanked by genes coding for regulatory components, two putative polyprenyl synthetases, and various oxidoreductases. An important clue in the identification of the biosynthetic gene cluster was the finding of a putative indole oxygenase gene, which suggested that this gene cluster could play a role in indole terpenoid biosynthesis. We could verify this assumption through various lines of evidence, specifically by targeted gene deletions and heterologous expression of the entire gene locus. First, we selected a cosmid (04B02) harboring the predicted xia biosynthesis gene cluster (Figure 1) and subcloned the insert (ca. 38 kb) into a Streptomyces–E. coli shuttle vector (pKJ55). The resulting construct, pXU472, was introduced into the heterologous host Streptomyces albus. By HPLC-HRMS (Exactive) monitoring we could detect 2 (C23H25NO3, m/z 362.176 [M H] ), 3 Scheme 1. Structures of bacterial indole sesquiterpenes oridamycin (1), xiamycin A (2) and B (3), indosespene (4), and sespenine (5).

[3+2] in the wild: Biomimetic natural product syntheses and theoretical considerations have indicated that 1,3-dipolar cycloadditions take place in nature. Now, the structure, biosynthesis, and function of a heavily modified prenylated flavin cofactor have been elucidated. In the azomethine ylide form, it undergoes [3+2] cycloadditions with aromatic acids and promotes their decarboxylation.

[3+2] in the wild: Biomimetic natural product syntheses and theoretical considerations have indicated that 1,3-dipolar cycloadditions take place in nature. Now, the structure, biosynthesis, and function of a heavily modified prenylated flavin cofactor have been elucidated. In the azomethine ylide form, it undergoes [3+2] cycloadditions with aromatic acids and promotes their decarboxylation.

Terpene and terpenoid cyclizations are counted among the most complex chemical reactions occurring in nature and contribute crucially to the tremendous structural diversity of this largest family of natural products. Many studies were conducted at the chemical, genetic, and biochemical levels to gain mechanistic insights into these intriguing reactions that are catalyzed by terpene and terpenoid cyclases. A myriad of these enzymes have been characterized. Classical textbook knowledge divides terpene/terpenoid cyclases into two major classes according to their structure and reaction mechanism. However, recent discoveries of novel types of terpenoid cyclases illustrate that nature's enzymatic repertoire is far more diverse than initially thought. This Review outlines novel terpenoid cyclases that are out of the ordinary.