Yeast-based heterologous production of the Colletochlorin family of fungal secondary metabolites.

Transcriptomic studies have revealed that fungal pathogens of plants activate the expression of numerous biosynthetic gene clusters (BGC) exclusively when in presence of a living host plant. The identification and structural elucidation of the corresponding secondary metabolites remain challenging. The aim was to develop a polycistronic system for heterologous expression of fungal BGCs in Saccharomyces cerevisiae. Here we adapted a polycistronic vector for efficient, seamless and cost-effective cloning of biosynthetic genes using in vivo assembly (also called transformation-assisted recombination) directly in Escherichia coli followed by heterologous expression in S. cerevisiae. Two vectors were generated with different auto-inducible yeast promoters and selection markers. The effectiveness of these vectors was validated with fluorescent proteins. As a proof-of-principle, we applied our approach to the Colletochlorin family of molecules. These polyketide secondary metabolites were known from the phytopathogenic fungus Colletotrichum higginsianum but had never been linked to their biosynthetic genes. Considering the requirement for a halogenase, and by applying comparative genomics, we identified a BGC putatively involved in the biosynthesis of Colletochlorins in C. higginsianum. Following the expression of those genes in S. cerevisiae, we could identify the presence of the precursor Orsellinic acid, Colletochlorins and their non-chlorinated counterparts, the Colletorins. In conclusion, the polycistronic vectors described herein were adapted for the host S. cerevisiae and allowed to link the Colletochlorin compound family to their corresponding biosynthetic genes. This system will now enable the production and purification of infection-specific secondary metabolites of fungal phytopathogens. More widely, this system could be applied to any fungal BGC of interest.

An Aminopyrimidone and Aminoimidazoles Alkaloids from the Rodrigues Calcareous Marine Sponge Ernsta naturalis.

A chemical study of the CH2Cl2-MeOH (1:1) extract from the sponge Ernsta naturalis collected in Rodrigues (Mauritius) based on a molecular networking dereplication strategy highlighted one novel aminopyrimidone alkaloid compound, ernstine A (1), seven new aminoimidazole alkaloid compounds, phorbatopsins D-E (2, 3), calcaridine C (4), naamines H-I (5, 7), naamidines J-K (6, 8), along with the known thymidine (9). Their structures were established by spectroscopic analysis (1D and 2D NMR spectra and HRESIMS data). To improve the investigation of this unstudied calcareous marine sponge, a metabolomic study by molecular networking was conducted. The isolated molecules are distributed in two clusters of interest. Naamine and naamidine derivatives are grouped together with ernstine in the first cluster of twenty-three molecules. Phorbatopsin derivatives and calcaridine C are grouped together in a cluster of twenty-one molecules. Interpretation of the MS/MS spectra of other compounds of these clusters with structural features close to the isolated ones allowed us to propose a structural hypothesis for 16 compounds, 5 known and 11 potentially new.

Isolation of an Extract from the Soft Coral Symbiotic Microorganism Salinispora arenicola Exerting Cytoprotective and Anti-Aging Effects.

Cells have developed a highly integrated system responsible for proteome stability, namely the proteostasis network (PN). As loss of proteostasis is a hallmark of aging and age-related diseases, the activation of PN modules can likely extend healthspan. Here, we present data on the bioactivity of an extract (SA223-S2BM) purified from the strain Salinispora arenicola TM223-S2 that was isolated from the soft coral Scleronephthya lewinsohni; this coral was collected at a depth of 65 m from the mesophotic Red Sea ecosystem EAPC (south Eilat, Israel). Treatment of human cells with SA223-S2BM activated proteostatic modules, decreased oxidative load, and conferred protection against oxidative and genotoxic stress. Furthermore, SA223-S2BM enhanced proteasome and lysosomal-cathepsins activities in Drosophila flies and exhibited skin protective effects as evidenced by effective inhibition of the skin aging-related enzymes, elastase and tyrosinase. We suggest that the SA223-S2BM extract constitutes a likely promising source for prioritizing molecules with anti-aging properties.

Solid-Phase Extraction Embedded Dialysis (SPEED), an Innovative Procedure for the Investigation of Microbial Specialized Metabolites.

Solid-phase extraction embedded dialysis (SPEED technology) is an innovative procedure developed to physically separate in-situ, during the cultivation, the mycelium of filament forming microorganisms, such as actinomycetes and fungi, and the XAD-16 resin used to trap the secreted specialized metabolites. SPEED consists of an external nylon cloth and an internal dialysis tube containing the XAD resin. The dialysis barrier selects the molecular weight of the trapped compounds, and prevents the aggregation of biomass or macromolecules on the XAD beads. The external nylon promotes the formation of a microbial biofilm, making SPEED a biofilm supported cultivation process. SPEED technology was applied to the marine Streptomyces albidoflavus 19-S21, isolated from a core of a submerged Kopara sampled at 20 m from the border of a saltwater pond. The chemical space of this strain was investigated effectively using a dereplication strategy based on molecular networking and in-depth chemical analysis. The results highlight the impact of culture support on the molecular profile of Streptomyces albidoflavus 19-S21 secondary metabolites.

Inhibition of jasmonate-mediated plant defences by the fungal metabolite higginsianin B.

Infection of Arabidopsis thaliana by the ascomycete fungus Colletotrichum higginsianum is characterized by an early symptomless biotrophic phase followed by a destructive necrotrophic phase. The fungal genome contains 77 secondary metabolism-related biosynthetic gene clusters, whose expression during the infection process is tightly regulated. Deleting CclA, a chromatin regulator involved in the repression of some biosynthetic gene clusters through H3K4 trimethylation, allowed overproduction of three families of terpenoids and isolation of 12 different molecules. These natural products were tested in combination with methyl jasmonate, an elicitor of jasmonate responses, for their capacity to alter defence gene induction in Arabidopsis. Higginsianin B inhibited methyl jasmonate-triggered expression of the defence reporter VSP1p:GUS, suggesting it may block bioactive jasmonoyl isoleucine (JA-Ile) synthesis or signalling in planta. Using the JA-Ile sensor Jas9-VENUS, we found that higginsianin B, but not three other structurally related molecules, suppressed JA-Ile signalling by preventing the degradation of JAZ proteins, the repressors of jasmonate responses. Higginsianin B likely blocks the 26S proteasome-dependent degradation of JAZ proteins because it inhibited chymotrypsin- and caspase-like protease activities. The inhibition of target degradation by higginsianin B also extended to auxin signalling, as higginsianin B treatment reduced auxin-dependent expression of DR5p:GUS. Overall, our data indicate that specific fungal secondary metabolites can act similarly to protein effectors to subvert plant immune and developmental responses.

Osirisynes G-I, New Long-Chain Highly Oxygenated Polyacetylenes from the Mayotte Marine Sponge Haliclona sp.

Chemical study of the CH2Cl2-MeOH (1:1) extract from the sponge Haliclona sp. collected in Mayotte highlighted three new long-chain highly oxygenated polyacetylenes, osirisynes G-I (1-3) together with the known osirisynes A (4), B (5), and E (6). Their structures were elucidated by 1D and 2D NMR spectra and HRESIMS and MS/MS data. All compounds were evaluated on catalase and sirtuin 1 activation and on CDK7, proteasome, Fyn kinase, tyrosinase, and elastase inhibition. Five compounds (1; 3-6) inhibited proteasome kinase and two compounds (5-6) inhibited CDK7 and Fyn kinase. Osirisyne B (5) was the most active compound with IC50 on FYNB kinase, CDK7 kinase, and proteasome inhibition of 18.44 µM, 9.13 µM, and 0.26 µM, respectively.

Degradation Mechanism of 2,4-Dichlorophenol by Fungi Isolated from Marine Invertebrates.

2,4-Dichlorophenol (2,4-DCP) is a ubiquitous environmental pollutant categorized as a priority pollutant by the United States (US) Environmental Protection Agency, posing adverse health effects on humans and wildlife. Bioremediation is proposed as an eco-friendly, cost-effective alternative to traditional physicochemical remediation techniques. In the present study, fungal strains were isolated from marine invertebrates and tested for their ability to biotransform 2,4-DCP at a concentration of 1 mM. The most competent strains were studied further for the expression of catechol dioxygenase activities and the produced metabolites. One strain, identified as Tritirachium sp., expressed high levels of extracellular catechol 1,2-dioxygenase activity. The same strain also produced a dechlorinated cleavage product of the starting compound, indicating the assimilation of the xenobiotic by the fungus. This work also enriches the knowledge about the mechanisms employed by marine-derived fungi in order to defend themselves against chlorinated xenobiotics.

Deleting a Chromatin Remodeling Gene Increases the Diversity of Secondary Metabolites Produced by Colletotrichum higginsianum.

Colletotrichum higginsianum is the causal agent of crucifer anthracnose disease, responsible for important economic losses in Brassica crops. A mutant lacking the CclA subunit of the COMPASS complex was expected to undergo chromatin decondensation and the activation of cryptic secondary metabolite biosynthetic gene clusters. Liquid-state fermentation of the Δ cclA mutant coupled with in situ solid-phase extraction led to the production of three families of compounds, namely, colletorin and colletochlorin derivatives with two new representatives, colletorin D (1) and colletorin D acid (2), the diterpenoid α-pyrone higginsianin family with two new analogues, higginsianin C (3) and 13- epi-higginsianin C (4), and sclerosporide (5) coupling a sclerosporin moiety with dimethoxy inositol.

Impact of the Cultivation Technique on the Production of Secondary Metabolites by Chrysosporium lobatum TM-237-S5, Isolated from the Sponge Acanthella cavernosa.

The fungi Chrysosporium lobatum TM-237-S5 was isolated from the sponge Acanthella cavernosa, collected from the mesophotic coral ecosystem of the Red Sea. The strain was cultivated on a potato dextrose agar (PDA) medium, coupling solid-state fermentation and solid-state extraction (SSF/SSE) with a neutral macroreticular polymeric adsorbent XAD Amberlite resin (AMBERLITE XAD1600N). The SSF/SSE lead to high chemodiversity and productivity compared to classical submerged cultivation. Ten phenalenone related compounds were isolated and fully characterized by one-dimensional and two-dimensional NMR and HRMS. Among them, four were found to be new compounds corresponding to isoconiolactone, (-)-peniciphenalenin F, (+)-8-hydroxyscleroderodin, and (+)-8-hydroxysclerodin. It is concluded that SSF/SSE is a powerful strategy, opening a new era for the exploitation of microbial secondary metabolites.

Unraveling the Detoxification Mechanism of 2,4-Dichlorophenol by Marine-Derived Mesophotic Symbiotic Fungi Isolated from Marine Invertebrates.

Chlorophenols (CPs) are environmental pollutants that are produced through various anthropogenic activities and introduced in the environment. Living organisms, including humans, are exposed to these toxic xenobiotics and suffer from adverse health effects. More specifically, 2,4-dichlorophenol (2,4-DCP) is released in high amounts in the environment and has been listed as a priority pollutant by the US Environmental Protection Agency. Bioremediation has been proposed as a sustainable alternative to conventional remediation methods for the detoxification of phenolic compounds. In this work, we studied the potential of fungal strains isolated as symbionts of marine invertebrates from the underexplored mesophotic coral ecosystems. Hence, the unspecific metabolic pathways of these fungal strains are being explored in the present study, using the powerful analytical capabilities of a UHPLC-HRMS/MS. The newly identified 2,4-DCP metabolites add significantly to the knowledge of the transformation of such pollutants by fungi, since such reports are scarce.

Neuroprotective Effect of CR-777, a Glutathione Derivative of Withaferin A, Obtained through the Bioconversion of Withania somnifera (L.) Dunal Extract by the Fungus Beauveria bassiana.

The bioconversion of Withania somnifera extract by the fungus Beauveria bassiana leads to cysteine and glutathione derivatives of withaferin A at the C-6 position. The compounds were purified and fully characterized by 1D-NMR, 2D-NMR, and HRMS analysis. The glutathione derivative CR-777 was evaluated as a neuroprotective agent from damage caused by different neurotoxins mimicking molecular symptoms in Parkinson´s disease (PD), including 1-methyl-4-phenylpyridinium (MPP+), 6-hydroxydopamine (6-OHDA), and α-synuclein (α-Syn). CR-777, at nanomolar concentrations, protected dopaminergic and cortical neurons. In 6-OHDA-treated neurons, CR-777 increased cell survival and neurite network and decreased the expression of α-Syn. Using specific inhibitors of cell toxicity signaling pathways and specific staining experiments, the observed role of CR-777 seemed to involve the PI3K/mTOR pathway. CR-777 could be considered as a protective agent against a large panel of neuronal stressors and was engaged in further therapeutic development steps.

Osmanicin, a Polyketide Alkaloid Isolated from Streptomyces osmaniensis CA-244599 Inhibits Elastase in Human Fibroblasts.

The strain Streptomyces osmaniensis CA-244599 isolated from the Comoros islands was submitted to liquid-state fermentation coupled to in situ solid-phase extraction with amberlite XAD-16 resin. Elution of the trapped compounds on the resin beads by ethyl acetate afforded seven metabolites, osmanicin (1), streptazolin (2), streptazone C (3), streptazone B1 (4), streptenol C (5), nocardamine (6) and desmethylenylnocardamine (7). Osmanicin (1) is a newly reported unusual scaffold combining streptazolin (2) and streptazone C (3) through a Diels-Alder type reaction. Experimental evidence excluded the spontaneous formation of 1 from 2 and 3. The isolated compounds were evaluated for their ability to inhibit elastase using normal human diploid fibroblasts. Compound 1 exhibited the most potent activity with an IC50 of 3.7 µM.

Innovative Approach to Sustainable Marine Invertebrate Chemistry and a Scale-Up Technology for Open Marine Ecosystems.

Isolation of marine compounds from living invertebrates represents a major challenge for sustainable and environmentally friendly exploitation of marine bio-resources. To develop innovative technology to trap invertebrate compounds in the open sea, the proof of concept of a system combining external continuous circulation of water with XAD-amberlite solid-phase extraction was validated in an aquarium. In this work, we reported the elicitation of guanidine alkaloid production of Crambe crambe in the presence of Anemonia sulcata, both collected from the Mediterranean Sea. Besides the previously reported crambescidin 359 (1), and crambescidin acid (2), three new compounds were isolated; one carboxylated analog of 1 named crambescidin 401 (3), and two analogs of crambescin B, crambescin B 281 (4) and crambescin B 253 (5). Based on these results, a technology named Somartex® for “Self Operating MARine Trapping Extractor” was patented and built to transfer the concept from closed aquarium systems to open marine ecosystems.

Sporothriolide-Related Compounds from the Fungus Hypoxylon monticulosum CLL-205 Isolated from a Sphaerocladina Sponge from the Tahiti Coast.

Two sporothriolide-related compounds were obtained from an extract of the fungus Hypoxylon monticulosum CLL-205, isolated from a Sphaerocladina sponge collected from the Tahiti coast. Compound 2 is a deoxy analogue of sporothric acid (4). Compound 3 is a newly reported unusual scaffold combining sporothriolide (1) and trienylfuranol A (5) moieties, through a Diels-Alderase-type reaction. Various experimental and analytical arguments supported the biocatalytic origin of compound 3. The structures of the isolated compounds were elucidated using 1D and 2D NMR, HRMS, and IR data. The structure and the absolute configuration of 3 were unambiguously confirmed by a single-crystal X-ray diffraction analysis.

Natural hydrazine-containing compounds: Biosynthesis, isolation, biological activities and synthesis.

Hydrazine, hydrazone and hydrazide derivatives are nitrogen-nitrogen bond containing compounds. Such molecules are relatively scarce in nature and have been isolated from plants, marine organisms and microorganisms. These compounds exhibit remarkable structural diversity and relevant biological activities. The enzymes involved in the formation of the N-N bond are still unknown, but many lines of evidence support the involvement of N-nitrosation and N-hydroxylation activating steps. Beside the challenging N-N bond, N-acylases catalyzing the C-N bond formation contribute to the chemical diversity of N-N-containing natural products (N2NP). This review examines the state of knowledge regarding the biosynthesis of N2NP, for which only two biosynthetic gene clusters have been investigated. Biological properties and chemical synthesis of hydrazines, hydrazones and hydrazides are also reported.

Synteruptor: mining genomic islands for non-classical specialized metabolite gene clusters.

Microbial specialized metabolite biosynthetic gene clusters (SMBGCs) are a formidable source of natural products of pharmaceutical interest. With the multiplication of genomic data available, very efficient bioinformatic tools for automatic SMBGC detection have been developed. Nevertheless, most of these tools identify SMBGCs based on sequence similarity with enzymes typically involved in specialised metabolism and thus may miss SMBGCs coding for undercharacterised enzymes. Here we present Synteruptor (https://bioi2.i2bc.paris-saclay.fr/synteruptor), a program that identifies genomic islands, known to be enriched in SMBGCs, in the genomes of closely related species. With this tool, we identified a SMBGC in the genome of Streptomyces ambofaciens ATCC23877, undetected by antiSMASH versions prior to antiSMASH 5, and experimentally demonstrated that it directs the biosynthesis of two metabolites, one of which was identified as sphydrofuran. Synteruptor is also a valuable resource for the delineation of individual SMBGCs within antiSMASH regions that may encompass multiple clusters, and for refining the boundaries of these SMBGCs.

Isolation and characterization of unusual hydrazides from Streptomyces sp. impact of the cultivation support and extraction procedure.

Three novel hydrazides, geralcins C-E (1-3), were isolated from Streptomyces sp. LMA-545, together with MH-031 and geralcins A and B. This unusual family of compounds was isolated from liquid-state and agar-supported fermentation using Amberlite XAD-16 solid-phase extraction during the cultivation step. The use of such neutral resin during the cultivation step allowed the specific adsorption of microbial secondary metabolites, avoiding any contamination of the crude extracts by the constituents of the culture medium. The trapped compounds were eluted from the resin with methanol, and their structures elucidated using (1)H, (13)C, and (15)N NMR spectroscopic analysis and high-resolution mass spectrometry. Molecular modeling calculations were applied in order to support structural attributions. No antimicrobial, cytotoxic, or DnaG-inhibition activities were detected for geralcins D and E. Geralcin C has no antimicrobial activity but exhibited an IC(50) of 0.8 µM against KB and HCT116 cancer cell lines. Furthermore, geralcin C inhibited the E. coli DnaG primase, a Gram-negative antimicrobial target, with an IC(50) of 0.7 mM.

Application of solid-phase extraction to agar-supported fermentation.

Agar-supported fermentation (Ag-SF), a variant of solid-state fermentation, has recently been improved by the development of a dedicated 2 m(2) scale pilot facility, Platotex. We investigated the application of solid-phase extraction (SPE) to Ag-SF in order to increase yields and minimize the contamination of the extracts with agar constituents. The selection of the appropriate resin was conducted on liquid-state fermentation and Diaion HP-20 exhibited the highest recovery yield and selectivity for the metabolites of the model fungal strains Phomopsis sp. and Fusarium sp. SPE applied to Ag-SF resulted in a particular compartmentalization of the culture. The mycelium that requires oxygen to grow migrates to the top layer and formed a thick biofilm. The resin beads intercalate between the agar surface and the mycelium layer, and trap directly the compounds secreted by the mycelium through a "solid-solid extraction" (SSE) process. The resin/mycelium layer is easily recovered by scraping the surface and the target metabolites extracted by methanol. Ag-SF associated to SSE represents an ideal compromise for the production of bioactive secondary metabolites with limited economic and environmental impact.

Total biosynthesis of fungal tetraketide pyrones.

Fungal tetraketide pyrones possess important and potent bioactivities, but their detailed biosynthetic pathways are unknown and synthetic routes to their production are lengthy. Here we investigated the fungal pathways to the multiforisins and compounds related to islandic acid. Heterologous expression experiments yield high titres of these compounds and pathway intermediates. The results both elucidate the pathway and offer a platform for the total biosynthesis of this class of metabolites.

Isolation and characterization of α,ß-unsaturated γ-lactono-hydrazides from Streptomyces sp.

Two novel α,ß-unsaturated γ-lactono-hydrazides, geralcin A (2) and geralcin B (3), were isolated from Streptomyces sp. LMA-545. This unusual scaffold consists of the condensation of alkyl-hydrazide with an α,ß-unsaturated γ-lactone, 3-(5-oxo-2H-furan-4-yl)propanoic acid (1), which was isolated from the same broth culture. Amberlite XAD-16 solid-phase extraction was used during the cultivation step, and the trapped compounds (1-3) were eluted from the resin with methanol. The structures were elucidated using (1)H, (13)C, and (15)N NMR spectroscopic analysis and high-resolution mass spectrometry. Geralcin B (3) was cytotoxic against MDA231 breast cancer cells with an IC(50) of 5 µM.