Polycyclic Tetramate Macrolactams Research Articles

Critical analysis of polycyclic tetramate macrolactam biosynthetic gene cluster phylogeny and functional diversity.

Polycyclic tetramate macrolactams (PTMs) are bioactive natural products commonly associated with certain actinobacterial and proteobacterial lineages. These molecules have been the subject of numerous structure-activity investigations since the 1970s. New members continue to be pursued in wild and engineered bacterial strains, and advances in PTM biosynthesis suggest their outwardly simplistic biosynthetic gene clusters (BGCs) belie unexpected product complexity. To address the origins of this complexity and understand its influence on PTM discovery, we engaged in a combination of bioinformatics to systematically classify PTM BGCs and PTM-targeted metabolomics to compare the products of select BGC types. By comparing groups of producers and BGC mutants, we exposed knowledge gaps that complicate bioinformatics-driven product predictions. In sum, we provide new insights into the evolution of PTM BGCs while systematically accounting for the PTMs discovered thus far. The combined computational and metabologenomic findings presented here should prove useful for guiding future discovery.IMPORTANCEPolycyclic tetramate macrolactam (PTM) pathways are frequently found within the genomes of biotechnologically important bacteria, including Streptomyces and Lysobacter spp. Their molecular products are typically bioactive, having substantial agricultural and therapeutic interest. Leveraging bacterial genomics for the discovery of new related molecules is thus desirable, but drawing accurate structural predictions from bioinformatics alone remains challenging. This difficulty stems from a combination of previously underappreciated biosynthetic complexity and remaining knowledge gaps, compounded by a stream of yet-uncharacterized PTM biosynthetic loci gleaned from recently sequenced bacterial genomes. We engaged in the following study to create a useful framework for cataloging historic PTM clusters, identifying new cluster variations, and tracing evolutionary paths for these molecules. Our data suggest new PTM chemistry remains discoverable in nature. However, our metabolomic and mutational analyses emphasize the practical limitations of genomics-based discovery by exposing hidden complexity.

Rational construction of genome-minimized Streptomyces host for the expression of secondary metabolite gene clusters

Streptomyces offer a wealth of naturally occurring compounds with diverse structures, many of which possess significant pharmaceutical values. However, new product exploration and increased yield of specific compounds in Streptomyces have been technically challenging due to their slow growth rate, complex culture conditions and intricate genetic backgrounds. In this study, we screened dozens of Streptomyces strains inhabiting in a plant rhizosphere for fast-growing candidates, and further employed CRISPR/Cas-based engineering techniques for stepwise refinement of a particular strain, Streptomyces sp. A-14 that harbors a 7.47 Mb genome. After strategic removal of nonessential genomic regions and most gene clusters, we reduced its genome size to 6.13 Mb, while preserving its growth rate to the greatest extent. We further demonstrated that cleaner metabolic background of this engineered strain was well suited for the expression and characterization of heterologous gene clusters, including the biosynthetic pathways of actinorhodin and polycyclic tetramate macrolactams. Moreover, this streamlined genome is anticipated to facilitate directing the metabolic flux towards the production of desired compounds and increasing their yields.

A newpolycyclic tetramate macrolactam from Allostreptomyces RD068384: stereochemistry and antifungal potential.

A new polycyclic tetramate macrolactam designated allostreptamide (1), together with four known congeners, were isolated from the culture extract of Allostreptomyces RD068384. The planar structure of the new compound was elucidated through interpretation of NMR and MS data. The absolute configuration was determined through ROESY and ECD analyses. The isolated compounds revealed antifungal potential against fourteen Candida albicans isolates with minimum inhibitory concentrations (MICs) ranging from 64 to 2048 µg ml-1. Compound 3 showed antibiofilm action and considerably reduced the viability of five isolates (36%) in the formed biofilm. The qRT-PCR revealed that 3 downregulated the BCR1, PLB2, ALS1, and SAP5 biofilm related gene expression. Therefore, 3 could be a promising antifungal therapy for C. albicans infections.

New polycyclic tetramate macrolactams with antimycobacterial activity produced by marine-derived Streptomyces sp. KKMA-0239.

During our screening for anti-mycobacterial agents against Mycobacterium avium complex (MAC), two new polycyclic tetramate macrolactams (PTMs), named hydroxycapsimycin (1) and brokamycin (2), were isolated along with the known PTM, ikarugamycin (3), from the culture broth of marine-derived Streptomyces sp. KKMA-0239. The relative structures of 1 and 2 were elucidated by spectroscopic data analyses, including 1D and 2D NMR. Furthermore, the absolute configuration of 1 was confirmed by a single-crystal X-ray diffraction analysis. Compounds 2 and 3 exhibited moderate antimycobacterial activities against MAC, including clinically isolated drug-resistant M. avium.

Discovery of Aburatubolactams Reveals Biosynthetic Logic for Distinct 5/5-Type Polycyclic Tetramate Macrolactams.

A known polycyclic tetramate macrolactam (aburatubolactam C, 3) and three new ones (aburatubolactams D-F, 4-6, respectively) were isolated from the marine-derived Streptomyces sp. SCSIO 40070. The absolute configuration of 3 was established by X-ray analysis. A combinatorial biosynthetic approach unveiled biosynthetic enzymes dictating the formation of distinct 5/5-type ring systems (such as C7-C14 cyclization by AtlB1 in 5 and C6-C13 cyclization by AtlB2 in 6) in aburatubolactams.

The natural polycyclic tetramate macrolactam HSAF inhibit Fusarium graminearum through altering cell membrane integrity by targeting FgORP1

Fusarium graminearum is a dominant phytopathogenic fungus causing Fusarium head blight (FHB) in cereal crops. Heat-stable antifungal factor (HSAF) is a polycyclic tetramate macrolactam (PoTeM) isolated from Lysobacter enzymogenes that exhibits strong antifungal activity against F. graminearum. HSAF significantly reduces the DON production and virulence of F. graminearum. Importantly, HSAF exhibited no cross-resistance to carbendazim, phenamacril, tebuconazole and pydiflumetofen. However, the target protein of HSAF in F. graminearum is unclear. In this study, the oxysterol-binding protein FgORP1 was identified as the potential target of HSAF using surface plasmon resonance (SPR) combined with RNA-sequence (RNA-seq). The RNA-seq results showed cell membrane and ergosterol biosynthesis were significantly impacted by HSAF in F. graminearum. Molecular docking showed that HSAF binds with arginine 1205 and glutamic acid 1212, which are located in the oxysterol-binding domain of FgORP1. The two amino acids in FgORP1 are responsible for HSAF resistance in F. graminearum though site-directed mutagenesis. Furthermore, deletion of FgORP1 led to significantly decreased sensitivity to HSAF. Additionally, FgORP1 regulates the mycelial growth, conidiation, DON production, ergosterol biosynthesis and virulence in F. graminearum. Overall, our findings revealed the mode of action of HSAF against F. graminearum, indicating that HSAF is a promising fungicide for controlling FHB.

A Mechanistic Understanding of the Distinct Regio‐ and Chemoselectivity of Multifunctional P450s by Structural Comparison of IkaD and CftA Complexed with Common Substrates

AbstractRegio‐ and chemoselective C−H activation at multi‐positions of a single molecule is fascinating but chemically challenging. The homologous cytochrome P450 enzymes IkaD and CftA catalyze multiple C−H oxidations on the same polycyclic tetramate macrolactam (PoTeM) ikarugamycin, with distinct regio‐ and chemoselectivity. Herein we provide mechanistic understanding of their functional differences by solving crystal structures of IkaD and CftA in complex with ikarugamycin and unnatural substrates. Distinct conformations of the F/G region in IkaD and CftA are found to differentiate the orientation of PoTeM substrates, by causing different binding patterns with polar moieties to determine site selection, oxidation order, and chemoselectivity. Fine‐tuning the polar subpocket altered the regioselectivity of IkaD, indicating that substrate re‐orientation by mutating residues distal to the oxidation site could serve as an important method in future engineering of P450 enzymes.

A Mechanistic Understanding of the Distinct Regio- and Chemoselectivity of Multifunctional P450s by Structural Comparison of IkaD and CftA Complexed with Common Substrates.

Regio- and chemoselective C-H activation at multi-positions of a single molecule is fascinating but chemically challenging. The homologous cytochrome P450 enzymes IkaD and CftA catalyze multiple C-H oxidations on the same polycyclic tetramate macrolactam (PoTeM) ikarugamycin, with distinct regio- and chemoselectivity. Herein we provide mechanistic understanding of their functional differences by solving crystal structures of IkaD and CftA in complex with ikarugamycin and unnatural substrates. Distinct conformations of the F/G region in IkaD and CftA are found to differentiate the orientation of PoTeM substrates, by causing different binding patterns with polar moieties to determine site selection, oxidation order and chemoselectivity. Fine-tuning the polar subpocket altered the regioselectivity of IkaD, indicating that substrate re-orientation by mutating residues distal to the oxidation site could serve as an important method in future engineering of P450 enzymes.

Identification and Characterization of the 28-N-Methyltransferase Involved in HSAF Analogue Biosynthesis.

Polycyclic tetramate macrolactams (PoTeMs) are a family of structurally intriguing bioactive natural products. Although the presence of the N-28 methyl group is known to affect bioactivities of some PoTeMs, the mechanism for this methylation remains unclear. We report here the identification and characterization of the 28-N-methyltransferase for HSAF analogues, which is encoded by a gene located outside the HSAF (heat-stable antifungal factor) cluster in Lysobacter enzymogenes C3. Our data suggested that 28-N-methyltransferase utilizes S-adenosylmethionine (SAM) to methylate HSAF analogues, and acts after the dicyclic and tricyclic ring formation and prior to C-3 hydroxylation. Kinetic analysis showed that the optimal substrate for the enzyme is 3-dehydroxy HSAF (3-deOH HSAF). Moreover, it could also accept PoTeMs bearing a 5-6 or 5-6-5 polycyclic system as substrates. This is the first N-methyltransferase identified in the family of PoTeMs, and the identification of this enzyme provides a new tool to generate new PoTeMs as antibiotic lead compounds.

Discovery of Oxidized Polycyclic Tetramate Macrolactams Bearing One or Two Rings through Combinatorial Pathway Reassembly.

The structural diversity of polycyclic tetramate macrolactams (PoTeMs) are mainly generated by the cyclases and cytochrome P450s (CYPs). The PoTeM cluster sah in Saccharopolyspora hirsuta harboring two CYP genes was combinatorially reassembled and heterologously expressed in Streptomyces. As a result, six new cytotoxic PoTeMs, sahamides A-F (1-6), were discovered, and 1-3 are the first examples of oxidized one-ring PoTeMs. Remarkably, SahE represents the first CYP performing oxidative modification on the ornithine moiety of PoTeMs.

Multifunctional P450 Monooxygenase CftA Diversifies the Clifednamide Pool through Tandem C-H Bond Activations.

Polycyclic tetramate macrolactams (PTMs) are a class of structurally complex hybrid polyketide-nonribosomal peptide (PK-NRP) natural products produced by diverse bacteria. Several PTMs display pharmaceutically interesting bioactivities, and the early stages of PTM biosynthesis involving polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) enzymology are well studied. However, the timing and mechanisms of post PKS-NRPS oxidations by P450 monooxygenases encoded in PTM biosynthetic gene clusters (BGCs) remain poorly characterized. Here we demonstrate that CftA, encoded in clifednamide-type PTM BGCs, is a multifunctional P450 monooxygenase capable of converting the C29-C30 ethyl side chain of ikarugamycin to either a C29-C30 methyl ketone or a C29-C30 hydroxymethyl ketone through C-H bond activation, resulting in the formation of clifednamide A or clifednamide C, respectively. We also report the complete structure of clifednamide C solved via multidimensional NMR (COSY, HSQC, HMBC, NOESY, and TOCSY) using material purified from an engineered Streptomyces strain optimized for production. Finally, the in vitro reconstitution of recombinant CftA catalytic activity revealed the oxidation cascade for sequential conversion of ikarugamycin to clifednamide A and clifednamide C. Our findings confirm prior genetics-based predictions on the origins of clifednamide complexity via P450s encoded in PTM BGCs and place CftA into a growing group of multifunctional P450s that tailor PTM natural products through late-stage regioselective C-H bond activation.

Polycyclic Tetramate Macrolactams-A Group of Natural Bioactive Metallophores.

New infectious diseases and increase in drug-resistant microbial pathogens emphasize the need for antibiotics with novel mode-of-action. Tetramates represented by fungi-derived tenuazonic acid and bacterial polycyclic tetramate macrolactams (PTMs) are an important family of natural products with a broad spectrum of antimicrobial activities. Despite their potential application as new antibiotics, it remains unknown how PTMs function. In this study, genomic mining revealed that PTM biosynthetic gene clusters (BGCs) are widespread in both Gram-positive and Gram-negative bacteria, and we investigated a sponge endosymbiont Actinoalloteichus hymeniacidonis harboring a potential PTM-BGC. Xanthobaccin A that previously has only been isolated from a Gram-negative bacterium was obtained after a scale-up fermentation, isolation, and structure elucidation through mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Xanthobaccin A as well as two previously reported tetramates, equisetin and ikarugamycin, exhibited antibacterial activities against Bacillus subtilis. In addition, these three tetramates were for the first time to be confirmed as metallophores and the stoichiometry of the complexes were shown to be Fe(III)(equisetin)3/Fe(III)(equisetin)2 and Fe(III)(ikarugamycin)2, respectively. Meanwhile, we found that all three tetramates could reduce ferric into ferrous iron, which triggers the Fenton chemistry reaction. Their antibacterial activity was reduced by adding the radical scavenger, vitamin C. Altogether, our work demonstrates that equisetin and PTMs can act as metallophores and their antimicrobial mechanism is possibly mediated through Fenton chemistry.

Genome Mining and Metabolic Profiling Uncover Polycyclic Tetramate Macrolactams from Streptomyces koyangensis SCSIO 5802.

We have previously shown deep-sea-derived Streptomyces koyangensis SCSIO 5802 to produce two types of active secondary metabolites, abyssomicins and candicidins. Here, we report the complete genome sequence of S. koyangensis SCSIO 5802 employing bioinformatics to highlight its potential to produce at least 21 categories of natural products. In order to mine novel natural products, the production of two polycyclic tetramate macrolactams (PTMs), the known 10-epi-HSAF (1) and a new compound, koyanamide A (2), was stimulated via inactivation of the abyssomicin and candicidin biosynthetic machineries. Detailed bioinformatics analyses revealed a PKS/NRPS gene cluster, containing 6 open reading frames (ORFs) and spanning ~16 kb of contiguous genomic DNA, as the putative PTM biosynthetic gene cluster (BGC) (termed herein sko). We furthermore demonstrate, via gene disruption experiments, that the sko cluster encodes the biosynthesis of 10-epi-HSAF and koyanamide A. Finally, we propose a plausible biosynthetic pathway to 10-epi-HSAF and koyanamide A. In total, this study demonstrates an effective approach to cryptic BGC activation enabling the discovery of new bioactive metabolites; genome mining and metabolic profiling methods play key roles in this strategy.

A comparative metabologenomic approach reveals mechanistic insights into Streptomyces antibiotic crypticity

Streptomyces genomes harbor numerous, biosynthetic gene clusters (BGCs) encoding for drug-like compounds. While some of these BGCs readily yield expected products, many do not. Biosynthetic crypticity represents a significant hurdle to drug discovery, and the biological mechanisms that underpin it remain poorly understood. Polycyclic tetramate macrolactam (PTM) antibiotic production is widespread within the Streptomyces genus, and examples of active and cryptic PTM BGCs are known. To reveal further insights into the causes of biosynthetic crypticity, we employed a PTM-targeted comparative metabologenomics approach to analyze a panel of S. griseus clade strains that included both poor and robust PTM producers. By comparing the genomes and PTM production profiles of these strains, we systematically mapped the PTM promoter architecture within the group, revealed that these promoters are directly activated via the global regulator AdpA, and discovered that small promoter insertion-deletion lesions (indels) differentiate weaker PTM producers from stronger ones. We also revealed an unexpected link between robust PTM expression and griseorhodin pigment coproduction, with weaker S. griseus-clade PTM producers being unable to produce the latter compound. This study highlights promoter indels and biosynthetic interactions as important, genetically encoded factors that impact BGC outputs, providing mechanistic insights that will undoubtedly extend to other Streptomyces BGCs. We highlight comparative metabologenomics as a powerful approach to expose genomic features that differentiate strong, antibiotic producers from weaker ones. This should prove useful for rational discovery efforts and is orthogonal to current engineering and molecular signaling approaches now standard in the field.

A synthetic approach to 5/5/6-polycyclic tetramate macrolactams of the discodermide type

A flexible synthetic route to the 16-membered tetramate-embedding macrocyclic scaffold present in various polycyclic tetramate macrolactams (PTMs) was developed which differs from the seminal synthesis of ikarugamycin by Boeckman Jr. in protecting groups and the order of connections. We also devised a short approach to various stereoisomers of the 5/5/6-tricarbocyclic motif found in discodermide and other PTMs, starting from the Weiss’ diketone.

Evaluation of Apple Root-Associated Endophytic Streptomyces pulveraceus Strain ES16 by an OSMAC-Assisted Metabolomics Approach

The One Strain Many Compounds approach (OSMAC) is a powerful and comprehensive method that enables the chemo-diversity evaluation of microorganisms. This is achieved by variations of physicochemical cultivation parameters and by providing biotic and abiotic triggers to mimic microorganisms' natural environment in the lab. This approach can reactivate the silent biosynthetic routes of specific metabolites typically not biosynthesized under standard laboratory conditions. In the present study, we combined the OSMAC approach with static headspace solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS), high-performance liquid chromatography-high-resolution tandem mass spectrometry (HPLC-HRMSn), and matrix-assisted laser desorption/ionization high-resolution mass spectrometry imaging (MALDI-HRMSI) to evaluate the chemoecological significance of an apple root-associated endophytic Streptomyces pulveraceus strain ES16. We employed the OSMAC approach by cultivating the endophyte in six different media conditions and performed temporal studies over 14 days. Analysis of the volatilome revealed that only under stressful conditions associated with sporulation, endophytic S. pulveraceus ES16 produces geosmin, a volatile semiochemical known to attract the soil arthropods Collembola (springtails) specifically. Subsequently, targeted metabolic profiling revealed polycyclic tetramate macrolactams (PTMs) production by the endophyte under stress, which are bioactive against various pathogens. Additionally, the endophyte produced the iron-chelating siderophore, mirubactin, under the same conditions. The structures of the compounds were evaluated using HRMSn and by comparison with literature data. Finally, MALDI-HRMSI revealed the produced compounds' spatial-temporal distribution over 14 days. The compounds were profusely secreted into the medium after production. Our results indicate that endophytic S. pulveraceus ES16 can release the signal molecule geosmin, chemical defense compounds such as the PTMs, as well as the siderophore mirubactin into the host plant apoplast or the soil for ecologically meaningful purposes, which are discussed.

Expression of the Clifednamide Biosynthetic Pathway in Streptomyces Generates 27,28-seco-Derivatives.

Polycyclic tetramate macrolactams (PoTeMs) are a group of hybrid PK-NRP natural products having a variable set of carbocyclic rings, a conserved assembly pathway, and diverse bioactivities. We report here the identification of seven new PoTeMs, clifednamides D-J (3-9), along with the known clifednamides A (1) and B (2) through rational pathway refactoring and heterologous expression. Remarkably, clifednamides D (3), G (6), and H (7) feature an unprecedented 27,28-seco skeleton. The cytotoxic activities of compounds 1-9 indicated that the hydroxy group of C-25, the methyl group of C-30, the inner five-membered ring, and the intact macrocycle are all critical for the activities. Meanwhile, the cytochrome P450 enzyme CftS023A and the hydroxylase CftS023E involved in oxidative tailoring of clifednamides were found to decorate the fused 5-6 bicyclic intermediates. Accordingly, the biosynthetic pathway for clifednamides was proposed.

Chemical Diversity of Metabolites and Antibacterial Potential of Actinomycetes Associated with Marine Invertebrates from Intertidal Regions of Daya Bay and Nansha Islands

Marine actinobacteria particularly from marine environments are believed to be inexhaustible sources of biologically active molecules for biomedical and industrial applications. We isolated 126 strains of marine actinomycetes from marine invertebrates such as corals, sponges and gastropod molluscs from Daya Bay and Nansha Islands. The chemical diversity of the metabolites from the isolated strains was evaluated using HPLC-UV fingerprinting and their antibacterial activity was estimated. Preliminary chemical screening and antibacterial activity clearly illustrate that the marine invertebrates-associated actinomycetes are a rich source of novel biologically active compounds. Among the isolates, strain SCSIO-PTE-L054 was further investigated for its unique metabolic profile and antibacterial activity. The bioassay-guided isolation rendered a polycyclic tetramate macrolactam (PTM) as a major chemical constituent with potent antibacterial activity. Our results revealed that the Streptomyces sp., strain SCSIO-L054 is a new source of PTMs which could pave a way to isolation of new series of PTMs with a broad spectrum of activity. In summary, the present investigation makes it possible to select marine actinomycetes from Daya Bay and Nansha Islands as new potential sources to produce potent compounds for a wide range of applications in future drug discovery.

Targeted isolation of new polycyclic tetramate macrolactams from the deepsea-derived Streptomyces somaliensis SCSIO ZH66.

With a combined strategy of bioinformatics analysis, gene manipulation coupled with variation of growth conditions, the targeted activation of polycyclic tetramate macrolactams (PTMs) in the deepsea-derived Streptomyces somaliensis SCSIO ZH66 was conducted, which afforded a new (1) PTM, named somamycin A, along with three enol-type tetramic acid tautomers (2-4, somamycins B-D) of 10-epi-hydroxymaltophilin, 10-epi-maltophilin and 10-epi-HSAF, respectively. The structures of compounds 1-4 were elucidated by extensive spectroscopic analyses together with ECD calculations. Compound 1 exhibited notable growth inhibition against plant pathogenic fungi Fusariumoxysporum MHKW and Alternariabrassicae BCHB with the MIC values of 1.6 and 3.1μg/mL, respectively, which were more potent than those of the positive control nystatin; and compounds 3 and 4 displayed moderate antifungal activities. Moreover, compounds 1-4 exhibited moderate cytotoxicity against the human cancer cell lines of HCT116 and K562.

Engineered Biosynthesis of 5/5/6 Type Polycyclic Tetramate Macrolactams in an Ikarugamycin (5/6/5 Type)-Producing Chassis.

Combinatorial biosynthesis of 5/5/6 type polycyclic tetramate macrolactams (PoTeMs) was achieved in an engineered ikarugamycin (5/6/5 type) producer by introducing a set of 5/5/6 type PoTeM biosynthetic genes from Streptomyces griseus. This study supports the idea that PoTeMs share a common polyene tetramate precursor generated by the hybrid polyketide synthase/nonribosomal peptide synthetase enzymes. The X-ray crystal structure of pactamide G (7) sets an example for resolving the absolute configuration of 5/5/6 type PoTeMs.