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Natural products modulate interactions between gut microbiota and host.

Edible mushrooms are important nutraceutical sources of foods and drugs, which can produce various nutritional ingredients including all essential amino acids. The method of rapid screening for the strains producing specific functional components is very indispensable. Homocitrate synthase is one of the key enzymes in the α-aminoadipate pathway for lysine biosynthesis and has preferable sequence conservation in Agaricales. Based on the blast of homocitrate synthase homologous genes of strains of Agaricales, we achieved combinations of degenerate primers as molecular markers to rapidly screen the lysine-producing edible mushrooms. The experimental results revealed that the consistency between PCR amplification and HPLC analysis attained 82 and 75% in strains of Agaricales and Polyporales, respectively. The finding showed that the molecular marker has higher universality for screening edible mushroom resources of Agaricales. This PCR-based approach shows excellent potential in evaluating and discriminating edible wild-grown mushrooms with high lysine content in Agaricales.

Two new sesquiterpenoids, 1–2, together with three known compounds, were isolated from Trichoderma hypoxylon. Among the known compounds, compound 4 was isolated as naturally occurring compound for the first time. The structures of these new compounds were characterized by HR-ESI-MS and spectroscopic methods including 1D and 2D NMR. The absolute configurations of 1–2 were assigned by electronic circular dichroism (ECD) calculations.

Expression of a nonribosomal peptide synthetase-nonreducing polyketide synthase hybrid gene pcr10109 from Penicillium crustosum PRB-2 in Aspergillus nidulans led to the accumulation of 4-hydroxy-6-(4-hydroxyphenyl)-α-pyrone (1). Adding para-hydroxybenzoic acid into the medium in which the overexpressing mutant is growing increased the product yield up to 5-fold. This strategy could be helpful for heterologous gene expression experiments requiring special substrates for product formation.

Fungi have evolved a plethora of functionally diverse secondary metabolites (SMs) to enhance their adaptation to various environments. To understand how structurally diverse metabolites contribute to fungal adaptation, we elucidate fungus-mycohost specific interactions mediated by a family of polypeptides, i.e., peptaibols. We specified that peptaibol structural diversification was attributed to the nonspecific substrate recognition by the highly conserved peptaibol synthetases (PSs) in dead wood inhabiting mycoparasitic fungi from the genus Trichoderma. Exemplified by investigation of T. hypoxylon, we characterized a library of 19 amino acid residue peptaibols, named trichohypolins, containing 42 derivatives synthesized by a single PS enzyme (NPS1Th). Elimination of trichohypolin production by the deletion of nps1Th reduced the inhibitory activities of T. hypoxylon on at least 15 saprotrophic host fungi, indicating that peptaibols are essential for interactions of Trichoderma spp. with their mycohosts. Different antagonistic effects of five trichohypolin subfractions SF1–SF5 and two pure compounds trichohypolins A (1) and B (2) on saprotrophic host fungi revealed specific activities of peptaibol derivatives in mediating fungus-mycohost interaction. Our study provides insights into the role of metabolic diversity of biosynthetic pathways in interfungal interactions.

Siderophores are small molecular iron chelators and participate in the multiple cellular processes in fungi. In this study, we discovered and identified five amphiphilic coprogen siderophores including three new natural products according to LC-MS-guided separation strategy from Trichoderm hypoxylon. The structures of three new coprogens were elucidated by NMR spectroscopy, and high-resolution (HR)-ESI-MS analysis. Genetic deletions of dfcA and dfcB abolished the production of compounds 1-5 that implied their involvement in the biosynthesis of coprogens. Interestingly, cultivations of ΔdfcA and ΔdfcB mutants with pathogenic fungi Fusarium oxysporum and Mucor corcinelloides showed the weaker inhibitions in comparison to wild type that demonstrated coprogen's role in combating the pathogenic fungi. Our study not only enriched the diversities of siderophores but also provided an approach for finding the rare amphiphilic coprogen siderophores in fungi. Furthermore, this work provided a basis for investigation on the biosynthesis of fungal amphiphilic siderophores and their ecological roles in nature. KEY POINTS: • A series of amphiphilic coprogens were found. • The gene cluster of amphiphilic coprogens and ecological roles were elucidated.

Two potential non-ribosomal peptide synthetases (NRPSs) were identified in the genome of a guanophilic fungus Amphichorda guana by bioinformatics analysis and gene knockout experiments. Liquid chromatography coupled with mass spectrometry (LC-MS) guided isolation led to the discovery of a new cyclodepsipeptide isaridin H (1) and seven known analogs, desmethylisaridin E (2), isaridin E (3), isariin A (4), iso-isariin B (5), iso-isariin D (6), isariin E (7), and nodupetide (8). The absolute configuration of isaridin H (1) was achieved by Marfey's method. Isaridin H (1) showed significant antifungal activity against Botrytis cinerea and Alternaria solani.

2-Alkenyl-tetrahydropyrans belong to a rare class of natural products that exhibit broad antifungal activities. Their structural instability and rareness in nature have restrained their discovery and drug development. In this study, the heterologous expression of a single highly reducing polyketide synthase (HR-PKS, App1) from Trichoderma applanatum in Aspergillus nidulans leads to the formation of seven 2-alkenyl-tetrahydropyran derivatives including one known compound virensol C (1) and six new compounds (2–7). However, introducing App1 into Saccharomyces cerevisiae resulted in the identification of additional two 2-alkenyl-tetrahydropyrans lacking the hydroxyl or methoxyl group at the C-2 position (8 and 9). The structures of the isolated compounds were elucidated by extensive spectroscopic analysis using NMR and HR-ESI-MS.

Characterization of filamentous fungal regulatory elements remains challenging because of time-consuming transformation technologies and limited quantitative methods. Here we established a method for quantitative assessment of filamentous fungal promoters based on flow cytometry detection of the superfolder green fluorescent protein at single-cell resolution. Using this quantitative method, we acquired a library of 93 native promoter elements from Aspergillus nidulans in a high-throughput format. The strengths of identified promoters covered a 37-fold range by flow cytometry. P

Microbial communication has attracted notable attention as an indicator of microbial interactions that lead to marked alterations of secondary metabolites (SMs) in varied environments. However, the mechanisms responsible for SM regulation are not fully understood, especially in fungal-fungal interactions. Here, cocultivation of an endophytic fungus Epicoccum dendrobii with the model fungus Aspergillus nidulans and several other filamentous fungi triggered widespread alteration of SMs. Multiple silent biosynthetic gene clusters in A. nidulans were activated by transcriptome and metabolome analysis. Unprecedentedly, gene deletion and replacement proved that a partial loss-of-function VeA1 protein, but not VeA, was associated with the widespread SM changes in both A. nidulans and A. fumigatus during cocultivation. VeA1 regulation required the transcription factor SclB and the velvet complex members LaeA and VelB for producing aspernidines as representative formation of SMs in A. nidulans . This study provides new insights into the mechanism that trigger metabolic changes during fungal-fungal interactions.

Fungal secondary metabolites play an important role in the biological control of pests. The secondary metabolites produced by fungi, which are the main substance in biocontrol, have attracted more and more attention. With the development of sequencing technology in recent years, the use of genomic and bioinformatics technology to excavate fungal secondary metabolites has become a new hot spot, which also provide new strategies for the discovery of new fungal biocontrol agents in the post-genomic era. In this review, several typical insecticidal fungi and their invasion and pathogenic mechanisms were presented. Typically, insecticidal toxins produced by several common insecticidal fungi and the biosynthetic pathways were systematically summarized. Our review provides insights into the further development of fungal biocontrol agents. Finally, the application of fungal secondary metabolites in biocontrol agents was prospected.

A genetic dereplication approach in combination with differential gene expression led to the discovery of three new sesquiterpenes, tricinoloniol acids (TRAs) A-C (1-3) and the known fusidilactone A (4) from T. hypoxylon. Comparative transcriptomic analysis and targeted deletion identified the biosynthetic route for TRAs. Our results demonstrate an alternative application of the genetic dereplication method for exploring the biosynthesis of cryptic secondary metabolites (SMs), which utilizes the coordinated expression of trichothecene (tri) and tra cluster genes.

Crustosic acid (1) differs from terrestric acid (2) by a 5β-carboxylmethyl at the tetronate ring instead of a 5α-methyl group in Penicillium crustosum. The formation of 1 via carboxylcrustic and viridicatic acid was confirmed by gene deletion and heterologous expression. The conversion of 1 to 2 requires a decarboxylation-mediated olefination by TraH and subsequent reduction by TraD. The redox-assisted decarboxylation and stereoisomerization proved the biosynthetic relationships of fungal acyltetronates with different stereochemistry.

Basic leucine zipper (bZIP) transcription factors play a crucial role in the environmental stress response of eukaryotes. In this work, we studied the effect of gene manipulations, including both deletions and overexpressions, of two selected bZIP transcription factors, NapA and RsmA, in the oxidative stress response and sterigmatocystin production of Aspergillus nidulans. We found that NapA was important in the oxidative stress response by negatively regulating intracellular reactive species production and positively regulating catalase activities, whereas RsmA slightly negatively regulated catalase activities. Concerning sterigmatocystin production, the highest concentration was measured in the ΔrsmAΔnapA double deletion mutant, but elevated sterigmatocystin production was also found in the OErsmA OEnapA strain. Our results indicate that NapA influences sterigmatocystin production via regulating reactive species level whereas RsmA modulates toxin production independently of the redox regulation of the cells.

Endophytic fungi are emerging as attractive producers of natural products with diverse bioactivities and novel structures. However, difficulties in the genetic manipulation of endophytic fungi limit the search of novel secondary metabolites. In this study, we improved the polyethylene glycol (PEG)-mediated protoplast transformation method by introducing the CRISPR/Cas9 system into endophytic fungus Pestalotiopsis fici. Using this approach, we performed genome editing such as site-specific gene insertion, dual-locus mutations, and long DNA fragment deletions in P. fici efficiently. The average efficiency for site-specific gene insertion and two-site gene editing was up to 48.0% and 44.4%, respectively. In addition, the genetic manipulation time with long DNA fragment (5–10 kb) deletion was greatly shortened to one week in comparison with traditional methods such as Agrobacterium tumefaciens-mediated transformation (ATMT). Taken together, the development of the CRISPR/Cas9 system in the endophytic fungus will accelerate the discovery of novel natural products and further biological study.

Epidithiodiketopiperazines (ETPs) are a group of bioactive fungal natural products and structurally feature unique transannular disulfide bridges between α, α or α, β carbons. However, no enzyme has yet been demonstrated to catalyse α, β-disulfide bond formation in these molecules. Through genome mining and gene deletion approaches in Trichoderma hypoxylon, we identified a putative biosynthetic gene cluster of pretrichodermamide A (1), which requires a FAD-dependent oxidoreductase, TdaR, for the irregular α, β-disulfide formation in 1 biosynthesis. In vitro assays of TdaR, together with AclT involved in aspirochlorine and GliT involved in gliotoxin biosynthesis, proved that all three enzymes catalyse not only the conversion of red-pretrichodermamide A (4) to α, β-disulfide-containing 1 but also that of red-gliotoxin (5) to α, α-disulfide-containing gliotoxin (6). These results provide new insights into the thiol-disulfide oxidases responsible for the disulfide bond formation in natural products with significant substrate and catalytic promiscuities., A FAD-dependent oxidoreductase TdaR was responsible for α, β-disulfide formation in the biosynthesis of pretrichodermamide A. TdaR, together with its homologs AclT and GliT, catalysed not only α, α- but also α, β-disulfide formation in fungi.

Two new diterpenoids, hypoxyterpoids A (1) and B (2), and four new isocoumarin derivatives, hypoxymarins A–D (4–7), together, with seven known metabolites (3 and 8–13) were obtained from the crude extract of the mangrove-derived fungus Hypoxylon sp. The structures of the new compounds were elucidated on the basis of 1- and 2-dimensional (1D/2D) nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric analysis. The absolute configurations of compounds 1, 2, 4, 5, and 7 were determined by comparison of experimental and calculated electronic circular dichroism (ECD) spectra, and the absolute configurations of C-4′ in 6 and C-9 in 7 were determined by [Rh2(OCOCF3)4]-induced ECD spectra. Compound 1 showed moderate α-glucosidase inhibitory activities with IC50 values of 741.5 ± 2.83 μM. Compounds 6 and 11 exhibited DPPH scavenging activities with IC50 values of 15.36 ± 0.24 and 3.69 ± 0.07 μM, respectively.

Filamentous fungi are excellent sources for the production of a group of bioactive small molecules which are often called secondary metabolites (SMs). The advanced genome sequencing technology combined with bioinformatics analysis reveals a large number of unexplored biosynthetic gene clusters (BGCs) in the fungal genomes. To unlock this fungal SM treasure, many approaches including heterologous expression are being developed and efficient cloning of the BGCs is a crucial step to do this. Here, we present an efficient strategy for the direct cloning of fungal BGCs. This strategy consisted of Splicing by Overlapping Extension (SOE)-PCR and yeast assembly in vivo. By testing 14 BGCs DNA fragments ranging from 7 kb to 52 kb, the average positive rate was over 80%. The maximal insertion size for fungal BGC assembly was 52 kb. Those constructs could be used conveniently for the heterologous expression leading to the discovery of novel natural products. Thus, our results provide an efficient and quick method for the low cost direct cloning of fungal BGCs.

Penilactones A and B consist of a γ-butyrolactone and two clavatol moieties. We identified two separate gene clusters for the biosynthesis of these key building blocks in Penicillium crustosum. Gene deletion, feeding experiments, and biochemical investigations proved that a nonreducing PKS ClaF is responsible for the formation of clavatol and the PKS-NRPS hybrid TraA is involved in the formation of crustosic acid, which undergoes decarboxylation and isomerization to the predominant terrestric acid. Both acids are proposed to be converted to γ-butyrolactones with involvement of a cytochrome P450 ClaJ. Oxidation of clavatol to hydroxyclavatol by a nonheme FeII/2-oxoglutarate-dependent oxygenase ClaD and its spontaneous dehydration to an ortho-quinone methide initiate the two nonenzymatic 1,4-Michael addition steps. Spontaneous addition of the methide to the γ-butyrolactones led to peniphenone D and penilactone D, which undergo again stereospecific attacking by methide to give penilactones A/B.

“Diversity-enhanced extracts” is an effective method of producing chemical libraries for the purpose of drug discovery. Three rare new cytochalasan derivative chaetoglobosins B1-B3 (1–3) were obtained from chemically engineered crude broth extracts of Chaetomium madrasense 375 prepared by reacting with hydrazine monohydrate and four known metabolite chaetoglobosins (4–7) were also identified from the fungus. The structures were identified by NMR and MS analysis and electronic circular dichroism simulation. In addition, the antiproliferative activities of these compounds were also evaluated, and the drug-resistant activities of cytochalasans were evaluated for the first time. Compound 6 possessed potent activity against four human cancer cells (A549, HCC827, SW620, and MDA-MB-231), and two drug-resistant HCC827 cells (Gefitinib-resistant, Osimertinib-resistant) compared with the positive controls.

Mushroom-derived cyathane-type diterpenes possess unusual chemical skeleton and diverse bioactivities. To efficiently supply bioactive cyathanes for deep studies and explore their structural diversity, de novo synthesis of cyathane diterpenes in a geranylgeranyl pyrophosphate engineered Saccharomyces cerevisiae is investigated. Aided by homologous analyses, one new unclustered FAD-dependent oxidase EriM accounting for the formation of allyl aldehyde and three new NADP(H)-dependent reductases in the biosynthesis of cyathanes are identified and elucidated. By combinatorial biosynthetic strategy, S. cerevisiae strains generating twenty-two cyathane-type diterpenes, including seven “unnatural” cyathane xylosides (12, 13, 14a, 14b, 19, 20, and 22) are established. Compounds 12–14, 19, and 20 show significant neurotrophic effects on PC12 cells in the dose of 6.3–25.0 μmol/L. These studies provide new insights into the divergent biosynthesis of mushroom-originated cyathanes and a straightforward approach to produce bioactive cyathane-type diterpenes., Graphical abstract Cyathane diterpens are lead compounds for neurodegenerative diseases. This work convenes the de novo synthesis of twenty-two cyathane derivatives. A key and unclustered FAD-dependent oxidase was elucidated for the biosynthesis of erinacines.Image 1

Two potential non-ribosomal peptide synthetases (NRPSs) were identified in the genome of a guanophilic fungus Amphichorda guana by bioinformatics analysis and gene knockout experiments. Liquid chromatography coupled with mass spectrometry (LC-MS) guided isolation led to the discovery of a new cyclodepsipeptide isaridin H (1) and seven known analogs, desmethylisaridin E (2), isaridin E (3), isariin A (4), iso-isariin B (5), iso-isariin D (6), isariin E (7), and nodupetide (8). The absolute configuration of isaridin H (1) was achieved by Marfey's method. Isaridin H (1) showed significant antifungal activity against Botrytis cinerea and Alternaria solani.

Fungi from unique environments exhibit special physiological characters and plenty of bioactive natural products. However, the recalcitrant genetics or poor transformation efficiencies prevent scientists from systematically studying molecular biological mechanisms and exploiting their metabolites. In this study, we targeted a guanophilic fungus Amphichorda guana LC5815 and developed a genetic transformation system. We firstly established an efficient protoplast preparing method by conditional optimization of sporulation and protoplast regeneration. The regeneration rate of the protoplast is up to about 34.6% with 0.8 M sucrose as the osmotic pressure stabilizer. To develop the genetic transformation, we used the polyethylene glycol-mediated protoplast transformation, and the testing gene AG04914 encoding a major facilitator superfamily transporter was deleted in strain LC5815, which proves the feasibility of this genetic manipulation system. Furthermore, a uridine/uracil auxotrophic strain was created by using a positive screening protocol with 5-fluoroorotic acid as a selective reagent. Finally, the genetic transformation system was successfully established in the guanophilic fungus strain LC5815, which lays the foundation for the molecular genetics research and will facilitate the exploitation of bioactive secondary metabolites in fungi.

Tyrosine-decahydrofluorene derivatives feature a fused [6.5.6] tricarbocyclic core and a 13-membered para-cyclophane ether. Herein, we identified new xenoacremones A, B, and C (1−3) from the fungal strain Xenoacremonium sinensis ML-31 and elucidated their biosynthetic pathway using gene deletion in the native strain and heterologous expression in Aspergillus nidulans. The hybrid polyketide synthase–nonribosomal peptide synthetase (PKS−NRPS) XenE together with enoyl reductase XenG were confirmed to be responsible for the formation of the tyrosine-nonaketide skeleton. This skeleton was subsequently dehydrated by XenA to afford a pyrrolidinone moiety. XenF catalyzed a novel sigmatropic rearrangement to yield a key cyclohexane intermediate as a prerequisite for the formation of the multi-ring system. Subsequent oxidation catalyzed by XenD supplied the substrate for XenC to link the para-cyclophane ether, which underwent subsequent spontaneous Diels−Alder reaction to give the end products. Thus, the results indicated that three novel enzymes XenF, XenD, and XenC coordinate to assemble the [6.5.6] tricarbocyclic ring and para-cyclophane ether during biosynthesis of complex tyrosine-decahydrofluorene derivatives., Graphical abstract The biosynthetic pathway for xenoacremone A (1) was identified, in which a three-enzyme cascade with XenF-mediated sigmatropic rearrangement is required for tricarbocyclic core formation of 1 in fungi.Image 1

More and more available fungal genome sequence data reveal a large amount of secondary metabolite (SM) biosynthetic ‘dark matter’ to be discovered. Heterogeneous expression is one of the most effective approaches to exploit these novel natural products, but it is limited by having to clone entire biosynthetic gene clusters (BGCs) without errors. So far, few effective technologies have been developed to manipulate the specific large DNA fragments in filamentous fungi. Here, we developed a fungal BGC-capturing system based on CRISPR/Cas9 cleavage in vitro. In our system, Cas9 protein was purified and CRISPR guide sequences in combination with in vivo yeast assembly were rationally designed. Using targeted cleavages of plasmid DNAs with linear (8.5 kb) or circular (8.5 kb and 28 kb) states, we were able to cleave the plasmids precisely, demonstrating the high efficiency of this system. Furthermore, we successfully captured the entire Nrc gene cluster from the genomic DNA of Neosartorya fischeri. Our results provide an easy and efficient approach to manipulate fungal genomic DNA based on the in vitro application of Cas9 endonuclease. Our methodology will lay a foundation for capturing entire groups of BGCs in filamentous fungi and accelerate fungal SMs mining.

Heterologous expression has been proven to be a successful strategy for the identification of metabolites encoded by cryptic/silent genes. Expression of a nonreducing polyketide synthase (NR-PKS) gene from Penicillium crustosum in Aspergillus nidulans led to the accumulation of three isocoumarins 1-3. Feeding experiments revealed that the PKS product 1 can be converted by the host enzymes to its hydroxylated (2) and methylated (3) derivatives. These results provided one additional example that unexpected further modifications of an enzyme product can take place in a heterologous host.