The biosynthetic logic and enzymatic machinery of approved fungi-derived pharmaceuticals and agricultural biopesticides.

Covering: 2000 to 2023The kingdom Fungi has become a remarkably valuable source of structurally complex natural products (NPs) with diverse bioactivities. Since the revolutionary discovery and application of the antibiotic penicillin from Penicillium, a number of fungi-derived NPs have been developed and approved into pharmaceuticals and pesticide agents using traditional "activity-guided" approaches. Although emerging genome mining algorithms and surrogate expression hosts have brought revolutionary approaches to NP discovery, the time and costs involved in developing these into new drugs can still be prohibitively high. Therefore, it is essential to maximize the utility of existing drugs by rational design and systematic production of new chemical structures based on these drugs by synthetic biology. To this purpose, there have been great advances in characterizing the diversified biosynthetic gene clusters associated with the well-known drugs and in understanding the biosynthesis logic mechanisms and enzymatic transformation processes involved in their production. We describe advances made in the heterogeneous reconstruction of complex NP scaffolds using fungal polyketide synthases (PKSs), non-ribosomal peptide synthetases (NRPSs), PKS/NRPS hybrids, terpenoids, and indole alkaloids and also discuss mechanistic insights into metabolic engineering, pathway reprogramming, and cell factory development. Moreover, we suggest pathways for expanding access to the fungal chemical repertoire by biosynthesis of representative family members via common platform intermediates and through the rational manipulation of natural biosynthetic machineries for drug discovery.

Aspertides A-E: Antimicrobial Pentadepsipeptides with a Unique p-Methoxycinnamoyl Amide Group from the Marine Isolates Aspergillus tamarii MA-21 and Aspergillus insuetus SD-512.

Marine fungus-derived natural products are an important source of antimicrobial compounds against marine aquatic pathogens. Here, we describe the isolation and characterization of five new pentadepsipeptides, aspertides A-E (1-5), containing a unique p-methoxycinnamoyl amide group, from the marine fungi Aspergillus tamarii MA-21 and Aspergillus insuetus SD-512. Among them, aspertides B-E (2-5) also possessed uncommon amino acid residues, such as 3-hydroxyproline, 2,3-dihydroxyproline, or pipecolinic acid. The structures of these compounds were elucidated on the basis of NMR and mass spectroscopic analyses. The absolute configurations of them were established by chiral HPLC analyses of the acidic hydrolysates and NMR calculations with DP4+ probability analysis. In bio-activity assays, compounds 4 and 5 exhibited antibacterial activities against aquatic-pathogenic bacteria, including Edwardsiella tarda, Vibrio alginolyticus, Vibrio anguillarum, Vibrio vulnificus, and Staphylococcus aureus, with MIC values of 8-32 µg/mL.

Unnatural activities and mechanistic insights of cytochrome P450 PikC gained from site-specific mutagenesis by non-canonical amino acids.

Cytochrome P450 enzymes play important roles in the biosynthesis of macrolide antibiotics by mediating a vast variety of regio- and stereoselective oxidative modifications, thus improving their chemical diversity, biological activities, and pharmaceutical properties. Tremendous efforts have been made on engineering the reactivity and selectivity of these useful biocatalysts. However, the 20 proteinogenic amino acids cannot always satisfy the requirement of site-directed/random mutagenesis and rational protein design of P450 enzymes. To address this issue, herein, we practice the semi-rational non-canonical amino acid mutagenesis for the pikromycin biosynthetic P450 enzyme PikC, which recognizes its native macrolide substrates with a 12- or 14-membered ring macrolactone linked to a deoxyamino sugar through a unique sugar-anchoring mechanism. Based on a semi-rationally designed substrate binding strategy, non-canonical amino acid mutagenesis at the His238 position enables the unnatural activities of several PikC mutants towards the macrolactone precursors without any sugar appendix. With the aglycone hydroxylating activities, the pikromycin biosynthetic pathway is rewired by the representative mutant PikCH238pAcF carrying a p-acetylphenylalanine residue at the His238 position and a promiscuous glycosyltransferase. Moreover, structural analysis of substrate-free and three different enzyme-substrate complexes of PikCH238pAcF provides significant mechanistic insights into the substrate binding and catalytic selectivity of this paradigm biosynthetic P450 enzyme.

Response of bacterial community metabolites to bacterial wilt caused by Ralstonia solanacearum: a multi-omics analysis.

The soil microbial community plays a critical role in promoting robust plant growth and serves as an effective defence mechanism against root pathogens. Current research has focused on unravelling the compositions and functions of diverse microbial taxa in plant rhizospheres invaded by Ralstonia solanacearum, however, the specific mechanisms by which key microbial groups with distinct functions exert their effects remain unclear. In this study, we employed a combination of amplicon sequencing and metabolomics analysis to investigate the principal metabolic mechanisms of key microbial taxa in plant rhizosphere soil. Compared to the healthy tobacco rhizosphere samples, the bacterial diversity and co-occurrence network of the diseased tobacco rhizosphere soil were significantly reduced. Notably, certain genera, including Gaiella, Rhodoplanes, and MND1 (Nitrosomonadaceae), were found to be significantly more abundant in the rhizosphere of healthy plants than in that of diseased plants. Eight environmental factors, including exchangeable magnesium, available phosphorus, and pH, were found to be crucial factors influencing the composition of the microbial community. Ralstonia displayed negative correlations with pH, exchangeable magnesium, and cation exchange flux, but showed a positive correlation with available iron. Furthermore, metabolomic analysis revealed that the metabolic pathways related to the synthesis of various antibacterial compounds were significantly enriched in the healthy group. The correlation analysis results indicate that the bacterial genera Polycyclovorans, Lysobacter, Pseudomonas, and Nitrosospira may participate in the synthesis of antibacterial compounds. Collectively, our findings contribute to a more in-depth understanding of disease resistance mechanisms within healthy microbial communities and provide a theoretical foundation for the development of targeted strategies using beneficial microorganisms to suppress disease occurrence.

Two New Phenol Derivatives from the Cold Seep-Derived Fungus Aspergillus insuetus SD-512.

Two new phenol derivatives, namely insphenol A (1) and acetylpeniciphenol (2), along with seven known analogs (3-9), were isolated from the deep-sea cold seep-derived fungus, Aspergillus insuetus SD-512. The structures of 1 and 2 were established by extensive interpretation of NMR and mass spectroscopic data. The absolute configuration of 1 was determined by the combination of coupling constant analysis and acid hydrolysis. Among the isolated compounds, insphenol A (1) represents the first example of isopentenyl phenol derivative with a unique 1-glycosylation from the species Aspergillus insuetus. The isolated new compounds were evaluated for antibacterial activities against six human or aquatic pathogens, while compound 2 exhibited inhibitory effect against Edwardsiella tarda, Vibrio alginolyticus, and V. vulnificus, with MIC values of 4, 8, and 8 µg/mL, respectively.

Six New Antimicrobial Metabolites from the Deep-Sea Sediment-Derived Fungus Aspergillus fumigatus SD-406.

Six new metabolites, including a pair of inseparable mixtures of secofumitremorgins A (1a) and B (1b), which differed in the configuration of the nitrogen atom, 29-hydroxyfumiquinazoline C (6), 10R-15-methylpseurotin A (7), 1,4,23-trihydroxy-hopane-22,30-diol (10), and sphingofungin I (11), together with six known compounds (2-5 and 8-9), were isolated and identified from the deep-sea sediment-derived fungus Aspergillus fumigatus SD-406. Their structures were determined by detailed spectroscopic analysis of NMR and MS data, chiral HPLC analysis of the acidic hydrolysate, X-ray crystallographic analysis, J-based configuration analysis, and quantum chemical calculations of ECD, OR, and NMR (with DP4+ probability analysis). Among the compounds, 1a/1b represent a pair of novel scaffolds derived from indole diketopiperazine by cleavage of the amide bond following aromatization to give a pyridine ring. Compounds 1, 4, 6, 7, 10 and 11 showed inhibitory activities against pathogenic bacteria and plant pathogenic fungus, with MIC values ranging from 4 to 64 µg/mL.

A new steroid with 7ß,8ß-epoxidation from the deep sea-derived fungus Aspergillus penicillioides SD-311.

7ß,8ß-epoxy-(22E,24R)-24-methylcholesta-4,22-diene-3,6-dione (1), a new steroid, along with five known analogues (2-6), was isolated from the deep sea-derived fungus, Aspergillus penicillioides SD-311. Strikingly, 1 possessed a rare 7,8-epoxidation moiety. Meanwhile, this is the first time to report natural products from this fungus species. The structures were established by extensive spectroscopic analysis. The absolute configuration was determined by X-ray diffraction experiments. Compound 1 showed antibacterial activity against Vibrio anguillarum with MIC value of 32.0 µg/mL, while 2 displayed inhibitions against Edwardsiella tarda and Micrococcus luteus with MIC values both of 16 µg/mL.

Ophiobolin Sesterterpenoids and Farnesylated Phthalide Derivatives from the Deep Sea Cold-Seep-Derived Fungus Aspergillus insuetus SD-512.

Three new ophiobolin sesterterpenoids, (6R)-16,17,21,21-O-tetrahydroophiobolin G (1), (6R)-16,17-dihydroophiobolin H (2), and (5S,6S)-16,17-dihydroophiobolin H (3), and three new farnesylated phthalide derivatives farnesylemefuranones D-F (9-11), along with five known ophiobolin analogues (4-8), were isolated and identified from the culture extract of Aspergillus insuetus SD-512, a deep-sea-derived fungus obtained from cold seep sediments collected at a depth of 1331 m. Among them, compounds 9-11 are rare examples of phthalide derivatives linked with farnesyl moieties via ether bonds. Their structures were established on the basis of detailed interpretation of the NMR spectroscopic and mass spectrometric data. X-ray crystallographic analysis, ECD calculations, and DP4+ probability analysis were performed to confirm the structures and establish the relative and absolute configurations of compounds 1-4. Compounds 3 and 9-11 showed broad-spectrum antibacterial activities, and differences in potencies could be assigned to structural modifications. This is the first report of secondary metabolites obtained from a deep sea cold-seep-derived fungus.

New Antibacterial Thiodiketopiperazines from the Deep Sea Sediment-Derived Fungus Epicoccum nigrum SD-388.

Two new antibacterial thiodiketopiperazine derivatives (TDKPs), 7-dehydroxyepicoccin H and 7-hydroxyeutypellazine F, along with seven known TDKP analogs, were isolated and identified from Epicoccum nigrum SD-388, a deep-sea-sediment-derived fungus. The structures of these compounds were elucidated on the basis of detailed spectroscopic analysis. The absolute configuration of 7-dehydroxyepicoccin H was established by X-ray crystallographic analysis, while 7-hydroxyeutypellazine F was determined by ECD experiment and TDDFT-ECD calculation. The antibacterial activities against human and aquatic pathogens were evaluated. 7-Dehydroxyepicoccin H and 7-hydroxyeutypellazine F displayed inhibitory activities against aquatic pathogens Vibrio vulnificus, V. alginolyticus, and Edwardsiella tarda, with MIC values ranging from 4.0 to 8.0 µg/mL.

Cytotoxic Thiodiketopiperazine Derivatives from the Deep Sea-Derived Fungus Epicoccum nigrum SD-388.

Four new thiodiketopiperazine alkaloids, namely, 5'-hydroxy-6'-ene-epicoccin G (1), 7-methoxy-7'-hydroxyepicoccin G (2), 8'-acetoxyepicoccin D (3), and 7'-demethoxyrostratin C (4), as well as a pair of new enantiomeric diketopiperazines, (±)-5-hydroxydiphenylalazine A (5), along with five known analogues (6-10), were isolated and identified from the culture extract of Epicoccum nigrum SD-388, a fungus obtained from deep-sea sediments (-4500 m). Their structures were established on the basis of detailed interpretation of the NMR spectroscopic and mass spectrometric data. X-ray crystallographic analysis confirmed the structures and established the absolute configurations of compounds 1-3, while the absolute configurations for compounds 4 and 5 were determined by ECD calculations. Compounds 4 and 10 showed potent activity against Huh7.5 liver tumor cells, which were comparable to that of the positive control, sorafenib, and the disulfide bridge at C-2/C-2' is likely essential for the activity.

Stereochemical Elucidation of Natural Products from Residual Chemical Shift Anisotropies in a Liquid Crystalline Phase.

Determination of the stereochemistry of organic molecules still represents one of the major obstacles in the structure elucidation procedure in drug discovery. Although the application of residual dipolar couplings (RDCs) has revolutionized this field, residual chemical shift anisotropies (RCSAs) which contain valuable structural information for nonprotonated carbons have only been scarcely employed so far. In this study, we present a simple but highly effective solution to extract RCSAs of the analytes in a liquid crystalline phase formed by AAKLVFF oligopeptides. This method does not require any special instruments, devices, or correction during postacquisition data analysis and thus can be easily applied in any chemistry laboratory. To illustrate the potential of this method, the relative configurations of four known natural products (1-4) belonging to different structural classes were confirmed. Moreover, we unambiguously elucidated the stereochemistry of spiroepicoccin A (5), a rare thiodiketopiperazine marine natural product whose configuration could not be assigned based on conventional NMR methods.

Two New Diketomorpholine Derivatives and a New Highly Conjugated Ergostane-Type Steroid from the Marine Algal-Derived Endophytic Fungus Aspergillus alabamensis EN-547.

Chemical investigation of the marine algal-derived endophytic fungus Aspergillus alabamensis EN-547 resulted in the isolation of 4-epi-seco-shornephine A methyl ester (1) and 4-epi-seco-shornephine A carboxylic acid (2), two new secondary metabolites having a rare diketomorpholine motif, and 28-acetoxy-12ß,15α,25-trihydroxyergosta-4,6,8(14),22-tetraen-3-one (3), a new highly conjugated ergostane-type steroid, together with four known metabolites (4-7). Their chemical structures were elucidated by detailed analysis of their NMR spectra, ECDs, HRESIMS, optical rotation, and X-ray crystallographic data, and by comparison with literature data as well. The antimicrobial activities of compounds 1-7 were evaluated.

Generally detected genes in comparative transcriptomics in bivalves: toward the identification of molecular markers of cellular stress response.

The specificity and representativeness of protein-coding genes identified by transcriptomics as biomarkers for environmental toxicological stress is crucial. We extracted the differential gene expression profile data from 49 published comparative transcriptomic studies of bivalves from January 2004 till November 2014 performed in 15 different bivalve species. Among the studies, 77 protein-coding genes were frequently detected when we use threefold of the average detection frequency as cut-off. Cellular organization and communication, protein and energy metabolism, stress response are the main functional classes of these proteins. We consider if these protein-coding genes represent common cellular stress responses of bivalves.