Integrative Analysis of Transcriptome and Metabolome Sheds Light on Flavonoid Biosynthesis in the Fruiting Body of Stropharia rugosoannulata.

Flavonoids are a diverse family of natural compounds that are widely distributed in plants and play a critical role in plant growth, development, and stress adaptation. In recent years, the biosynthesis of flavonoids in plants has been well-researched, with the successive discovery of key genes driving this process. However, the regulation of flavonoid biosynthesis in fungi remains unclear. Stropharia rugosoannulata is an edible mushroom known for its high nutritional and pharmacological value, with flavonoids being one of its main active components. To investigate the flavonoid content of S. rugosoannulata, a study was conducted to extract and determine the total flavonoids at four stages: young mushroom (Ym), gill (Gi), maturation (Ma), and parachute-opening (Po). The findings revealed a gradual increase in total flavonoid concentration as the fruiting body developed, with significant variations observed between the Ym, Gi, and Ma stages. Subsequently, we used UPLC-MS/MS and transcriptome sequencing (RNA-seq) to quantify the flavonoids and identify regulatory genes of Ym, Gi, and Ma. In total, 53 flavonoid-related metabolites and 6726 differentially expressed genes (DEGs) were identified. Through KEGG pathway enrichment analysis, we identified 59 structural genes encoding flavonoid biosynthesis-related enzymes, most of which were up-regulated during the development of the fruiting body, consistent with the accumulation of flavonoids. This research led to the establishment of a comprehensive transcriptional metabolic regulatory network encompassing flavonoids, flavonoid synthases, and transcription factors (TFs). This represents the first systematic exploration of the molecular mechanism of flavonoids in the fruiting of fungi, offering a foundation for further research on flavonoid mechanisms and the breeding of high-quality S. rugosoannulata.

The Liquid Fermentation Process for Mycelia of Poria cocos (Agaricomycetes) by Single-Factor Experimentation and Response Surface Methodology.

Liquid fermentation could yield substantial mycelia mass and valuable secondary metabolites in large-scale production within a short, fermented duration. The liquid fermented process of mycelia of Poria cocos was optimized using a combination of single-factor experimentation and response surface methodology (RSM) to obtain more extract of P. cocos. The optimal conditions were determined as follows: The carbon source concentration at 1%, the nitrogen source concentration at 1%, the inoculum volume at 7% and a culture time of 9 d. Under these conditions, the ethyl acetate extract mass of P. cocos mycelia reached 0.0577 ± 0.0041 mg. There were significant interactions between nitrogen source concentration and cultivation time. The predicted values by the mathematical model based on the response surface analysis showed a close agreement with experimental data.

Chemical Constituents from Mycelia of Lepista sordida (Agaricomycetes) and Their ABTS Radical Scavenging Activity.

Lepista sordida is an edible mushroom possessing high nutritional value and high medicinal value. The artificial cultivation technology of L. sordida made a breakthrough and has been popularized in Yunnan, Guizhou, Sichuan province with good economic benefits. The secondary metabolites were investigated from ethyl EtOAc (acetate extract) of solid cultures of L. sordida. Silica gel column chromatography, semi-preparation HPLC, recrystallization, and medium pressure column chromatography were applied to obtain 15 compounds. Nine compounds were first isolated from genus Lepista and 11 compounds were first isolated from species L. sordida. Moreover, compounds 13 and 14 exhibited strong scavenging activity of ABTS.

Advances in the Anti-Tumor Activity of Biflavonoids in Selaginella.

Despite the many strategies employed to slow the spread of cancer, the development of new anti-tumor drugs and the minimization of side effects have been major research hotspots in the anti-tumor field. Natural drugs are a huge treasure trove of drug development, and they have been widely used in the clinic as anti-tumor drugs. Selaginella species in the family Selaginellaceae are widely distributed worldwide, and they have been well-documented in clinical practice for the prevention and treatment of cancer. Biflavonoids are the main active ingredients in Selaginella, and they have good biological and anti-tumor activities, which warrant extensive research. The promise of biflavonoids from Selaginella (SFB) in the field of cancer therapy is being realized thanks to new research that offers insights into the multi-targeting therapeutic mechanisms and key signaling pathways. The pharmacological effects of SFB against various cancers in vitro and in vivo are reviewed in this review. In addition, the types and characteristics of biflavonoid structures are described in detail; we also provide a brief summary of the efforts to develop drug delivery systems or combinations to enhance the bioavailability of SFB monomers. In conclusion, SFB species have great potential to be developed as adjuvant or even primary therapeutic agents for cancer, with promising applications.

Six Sets of Aromatic Polyketides Differing in Size and Shape Derive from a Single Biosynthetic Gene Cluster.

One new aromatic polyketide, prealnumycin B (1), and four known aromatic polyketides, K1115A (2), 1,6-dihydroxy-8-propylanthraquinone (DHPA, 3), phaeochromycin B (4), and (R)-7-acetyl-3,6-dihydroxy-8-propyl-3,4dihydronaphthalen-1(2H)-one (5), were isolated from the marine-derived Streptomyces sundarbansensis SCSIO NS01; these compounds represent four sets of aromatic polyketides differing in size and shape. A type II polyketide synthase (PKS) cluster, als, was identified by complete genome sequencing and was shown, by in vivo gene inactivation experiments in the wild-type (WT) NS01 strain and heterologous expression experiments, to encode the biosynthesis of compounds 1-5. Moreover, heterologous expression of the als cluster afforded three additional aromatic polyketides representing two different carbon skeletons, the new phaeochromycin L (6) and two known aromatic polyketides, phaeochromycins D (7) and E (8). These findings expand our knowledge of type II PKS machineries and their versatility in generating structurally diverse aromatic polyketides and highlight the power of type II PKSs in accessing new polyketides via ectopic expression in heterologous hosts.

Chemical synthesis and mechanism of a natural product from endolichenic fungus with a broad-spectrum anti microorganism activity.

Background: The antibiotic resistance in various bacteria is consistently increasing and is posing a serious threat to human health, prompting the need for the discovery of novel structurally featured natural products with promising biological activities in drug research and development. Endolichenic microbes have been proven to be a fertile source to produce various chemical components, and therefore these microbes have been on a prime focus for exploring natural products. In this study, to explore potential biological resources and antibacterial natural products, the secondary metabolites of an endolichenic fungus have been investigated. Methods: The antimicrobial products were isolated from the endolichenic fungus using various chromatographic methods, and the antibacterial and antifungal activities of the compounds were evaluated by the broth microdilution method under in vitro conditions. The antimicrobial mechanism has been discussed with measuring the dissolution of nucleic acid and protein, as well as the activity of alkaline phosphatase (AKP) in preliminary manner. Chemical synthesis of the active product compound 5 was also performed, starting from commercially available 2,6-dihydroxybenzaldehyde through a sequence of transformations that included methylation, the addition of propylmagnesium bromide on formyl group, the oxidation of secondary alcohol, and the deprotection of methyl ether motif. Results: Among the 19 secondary metabolites of the endolichenic fungus, Daldinia childiae (compound 5) showed attractive antimicrobial activities on 10 of the 15 tested pathogenic strains, including Gram-positive bacteria, Gram-negative bacteria, and fungus. The Minimum Inhibitory Concentration (MIC) of compound 5 for Candida albicans 10213, Micrococcus luteus 261, Proteus vulgaris Z12, Shigella sonnet, and Staphylococcus aureus 6538 was identified as 16 µg/ml, whereas the Minimum Bactericidal Concentration (MBC) of other strains was identified as 64 µg/ml. Compound 5 could dramatically inhibit the growth of S. aureus 6538, P. vulgaris Z12, and C. albicans 10213 at the MBC, likely affecting the permeability of the cell wall and cell membrane. These results enriched the library of active strains and metabolites resources of endolichenic microorganisms. The chemical synthesis of the active compound was also performed in four steps, providing an alternative pathway to explore antimicrobial agents.

Characterization of Secondary Metabolites from Mycelial Cultures of Black Morel Mushroom Morchella importuna (Ascomycota).

Species of the genus Morchella are highly prized worldwide for their excellent flavor and high medicinal value. In recent years, artificial cultivations of medicinal fungi with many advantages have elicited great interest as a promising alternative to produce certain valuable metabolites. Therefore, the secondary metabolites of fermented M. importuna belonging to the black morel clade isolated from China were investigated. The strain was cultured in a fermentation tank in PDB liquid medium by two-step method. The mycelia and fermentation broth were extracted by ethyl acetate. The secondary metabolites were separated and purified by repeated silica gel column chromatography. Structures of compounds were determined by NMR data and references. One new natural compound (1) and six known compounds (2-7) were obtained. Compounds 1, 2, 4, and 5 were first isolated from genus Morchella and compounds 3, 6, and 7 are first isolated from species M. importuna.

Extraction of flavonoids from Citri Reticulatae Pericarpium Viride using a deep eutectic solvent.

Flavonoids are the main active ingredient in Citri Reticulatae Pericarpium Viride (CRPV). In this study, a deep eutectic solvent (DES) was explored to extract the main flavonoids from CRPV, including narirutin (NAR) and hesperidin (HES). A total of 30 DESs were prepared, and DES-20 (proline : urea) was selected as the optimal solvent. According to the single factor and response surface methodology experiments, the optimal extraction conditions for the flavonoids included a molar ratio of 1 : 2, water content of 30%, extraction time of 28 min, extraction power of 240 W, and a liquid-to-solid ratio of 70 mL g-1 and the optimal extraction yields of NAR and HES were 21 ± 2 mg g-1 and 60 ± 2 mg g-1, respectively. The recovery rate of NAR and HES with macroporous resin SP825 were 88 ± 3% and 86 ± 4%, respectively. Compared with traditional extraction methods, this novel method greatly improved extraction efficiency and saved time. Scanning electron microscopy results showed that this novel method could destroy the plant cell wall to enhance the extraction efficiency. Then, 21 flavonoids were identified through an ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) technique and nine of them were discovered for the first time in CRPV extracts. Furthermore, a high-performance liquid chromatography (HPLC) method was simultaneously used to determine NAR and HES in CRPV extracts upon methodological validation. Finally, antioxidant and cytotoxic experiments in vitro showed that the flavonoids extracted from the CRPV exhibited certain antioxidant and cytotoxic activities. The above results indicated that the DES was a green solvent, which can effectively extract NAR and HES from CRPV.

Investigation of Chemical Compounds and DPPH Radical Scavenging Activity of Oudemansiella raphanipes (Agaricomycetes) Based on Fermentation.

Oudemansiella raphanipes, an edible medicinal mushroom, is very popular in the market. With the development of technology, liquid fermentation is becoming more and more popular in edible fungi cultivation. Strain O. raphanipes was cultured by submerged fermentation and secondary metabolites were investigated simultaneously. Six chemical constituents including two unsaturated fatty acids (1-2), one ergosterol (3), one phenol (4), and two benzamides (5-6) were acquired from ethyl acetate extract. Their structures were mainly elucidated by NMR spectral data and known products. All compounds were isolated from O. raphanipes for the first time. DPPH radical scavenging activity was evaluated. Orcinol (4) exhibited remarkable antioxidant activity; its free radical scavenging rate can reach up to 80% of 20 mg/mL. 2-pyruvoybenzamide (5) and 2-(2-hydroxypropanamido) benzamide (6) showed less active antioxidant capacity.

Preparation of Uracil by bacteria isolated from Morchella.

Morchella is one of the most famous rare edible and medicinal fungi over the world. Highly nutritious and immature cultivation techniques led to the high price and the markets have remained tight. The pathogenic bacteria were serious in artificial cultivation of Morchella that affected the growth and yield of Morchella. Isolation of pathogenic bacteria and metabolites were investigated in order to improve the artificial cultivation technology. The isolated strain (YDJZ-01-01C) was identified by Gram staining and sequence of 16S rDNA. Structures of metabolites were confirmed based on NMR spectra and literatures. However, the main products were uracil and thymine that considered as important intermediate of anti-tumor 5-fluorouracil. Interestingly, a new synthetic pathway for preparation of uracil by microorganism was found except for chemical synthesis. The new preparation pathway provided mild, green, sustainable and environment friendly method to produce uracil that meets the needs of modern chemistry.

Dihydrophenanthrofurans and bisbibenzyl derivatives from the stems of Dendrobium nobile.

Two new dihydrophenanthrofurans (1 and 2) and two new bisbibenzyl derivatives (3 and 4) were isolated from the traditional Chinese medicinal plant Dendrobium nobile, along with four known compounds (5-8). The absolute configurations of compounds 1 and 4 were elucidated through extensive NMR and ECD spectroscopic analyses. New compounds showed no antimicrobial activity against four gram-positive bacterial strains and four gram-negative bacteria at the concentration of 1 mg/mL, but displayed significant cytotoxic activity against HepG2 human hepatic cell line with the IC50 values ranging from 1.25 µM to 19.47 µM.

Chemical constituents of fungus F03 belonging to Basidiomycota.

Filamentous fungus F03 belonging to Basidiomycota was obtained and identified as Phlebiopsis crassa based on ITS sequence when Morchella. sp was isolated from the wild fruiting body by spores releasing method. Chemical constituents were separated by gel chromatography, HPLC and recrystallization. Structures of compounds were confirmed by NMR data. Four products orsellinic acid (1), α-nigerose (2), uridine (3), N-(4-hydroxyphenyl)acetamide (4) were identified and all compounds were isolated from the genus Phlebiopsis for the first time.

Secondary Metabolites and Antiradical Activity of Liquid Fermentation of Morchella sp. Isolated from Southwest China.

Morels famous for their taste and nutrition are in short supply all over the world although they were considered as one of the most highly prized edible and medicinal mushrooms. Because of the limitation of resource and cultivation technology, fermentation of edible mushroom was gradually applied to nutrient, bioactivity and breeder seed preparation. At present, there are more reports on sugar and amino acid but less on other components. Morchella sp. YDJ-ZY-1 was isolated from the wild fruiting body by the spores releasing method in Zunyi Guizhou province in Southwest China and identified based on phenotype and genotype characteristics. Chemical compositions of YDJ-ZY-1 were investigated from liquid fermentation that will lay the foundation for further development and utilization. Four pyranoids (1-4) and 2-(1-oxo-2-hydroxyethyl) furan (5), linoleic acid (6), Morelin (2-hydroxy-cinnamic acid methyl ester, (7) and 1-O-ß-d-ribofuranose-Morelin (8) were obtained from EtOAc extraction and elucidated by spectral data. Product 4 and 8 were new compounds and 7 was isolated from nature for the first time. Antiradical activity was evaluated by free radical scavenging effect on DPPH (1,1-Diphenyl-2-picrylhydrazyl radical 2,2-Diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl). Compound 5 exhibited strong antiradical activity while compounds 1 and 2 exhibited moderate activity. Thus, incubation of Morchella sp YDJ-ZY-1 separated from the wild fruit body afforded eight compounds. Secondary metabolites with new structures were mined from fermentation of Morchella sp. and antiradical activity was evaluated.

Two new echinocystic acid derivatives catalyzed by filamentous fungus Gliocladium roseum CGMCC 3.3657.

Biotransformation of Echinocystic acid (EA,1) using G. roseum CGMCC 3.3657 has been investigated, which leads to the isolation and identification of two novel Echinocystic acid derivatives, 4, 16α-dihydroxy-3,4-seco-olean-12-en-3,28-dioic acid (2) and 16α-hydroxy, A-homo-3α-oxa-olean-12-en-3-one-28-oic acid (3). Their structures have been elucidated by analysis of spectroscopic data. This biocatalysis could serve as an efficient tool complementary to classical chemical methods for the transformation of EA.

Microbe-mediate transformation of echinocystic acid by whole cells of filamentous fungus Cunninghamella blakesleana CGMCC 3.910.

Structural modification of echinocystic acid (EA), a pentacyclic triterpenoid with wide spread biological activities was investigated by microbial transformation. Microbe-mediate transformation of EA was carried out by filamentous fungus Cunninghamella blakesleana CGMCC 3.910. Four metabolites 3ß, 7ß, 16α-trihydroxy-olean-12-en-28-oic acid (EA-2); 3ß, 7ß, 16ß,19ß-tetrahydroxy-olean-12-en-28-oic acid (EA-3); 3ß, 7ß, 16α, 21ß-tetrahydroxy-olean-12-en-28-oic acid (EA-4); 3ß, 7ß, 16α-trihydroxy-olean-11, 13(18)-dien-28-oic acid (EA-5) were produced. Structures of transformed products were elucidated by 1D and 2D NMR and HR-MS data. EA-3 and EA-4 were new compounds.

Mahimbrine A, a Novel Isoquinoline Alkaloid Bearing a Benzotropolone Moiety from Mahonia imbricata.

A novel isoquinoline alkaloid, mahimbrine A, possessing a rare benzotropolone framing scaffold, was isolated from the endemic plant of Mahonia imbricata. Its structure was established on the basis of extensive spectroscopic analysis. A plausible biosynthetic route of mahimbrine A was proposed. Mahimbrine A showed no antimicrobial activity at the concentration of 1 mg/mL.

Characterization and heterologous expression of the neoabyssomicin/abyssomicin biosynthetic gene cluster from Streptomyces koyangensis SCSIO 5802.

BACKGROUND: The deep-sea-derived microbe Streptomyces koyangensis SCSIO 5802 produces neoabyssomicins A-B (1-2) and abyssomicins 2 (3) and 4 (4). Neoabyssomicin A (1) augments human immunodeficiency virus-1 (HIV-1) replication whereas abyssomicin 2 (3) selectively reactivates latent HIV and is also active against Gram-positive pathogens including methicillin-resistant Staphylococcus aureus (MRSA). Structurally, neoabyssomicins A-B constitute a new subtype within the abyssomicin family and feature unique structural traits characteristic of extremely interesting biosynthetic transformations. RESULTS: In this work, the biosynthetic gene cluster (BGC) for the neoabyssomicins and abyssomicins, composed of 28 opening reading frames, was identified in S. koyangensis SCSIO 5802, and its role in neoabyssomicin/abyssomicin biosynthesis was confirmed via gene inactivation and heterologous expression experiments. Bioinformatics and genomics analyses enabled us to propose a biosynthetic pathway for neoabyssomicin/abyssomicin biosynthesis. Similarly, a protective export system by which both types of compounds are secreted from the S. koyangensis producer was identified, as was a four-component ABC transporter-based import system central to neoabyssomicin/abyssomicin biosynthesis. Furthermore, two regulatory genes, abmI and abmH, were unambiguously shown to be positive regulators of neoabyssomicin/abyssomicin biosynthesis. Consistent with their roles as positive regulatory genes, the overexpression of abmI and abmH (independent of each other) was shown to improve neoabyssomicin/abyssomicin titers. CONCLUSIONS: These studies provide new insight into the biosynthesis of the abyssomicin class of natural products, and highlight important exploitable features of its BGC for future efforts. Elucidation of the neoabyssomicin/abyssomicin BGC now enables combinatorial biosynthetic initiatives aimed at improving both the titers and pharmaceutical properties of these important natural products-based drug leads.

Biocatalysis of ursolic acid by the fungus Gliocladium roseum CGMCC 3.3657 and resulting anti-HCV activity.

Biocatalysis of ursolic acid (UA 1) by Gliocladium roseum CGMCC 3.3657 was investigated. Baeyer-Villiger oxidation was found to occur during the reaction. Four metabolites were isolated from the cultures and their structures were identified as 21-oxo,A-homo-3a-oxa-urs-12-en-3-one-28-oic acid (2), 21-oxo-3,4-seco-ursan-4(23),12-dien-3,28-dioic acid (3), 21ß-hydroxyl-A-homo-3a-oxa-urs-12-en-3-one-28-oic acid (4) and 21ß-hydroxyl-3,4-seco-ursan-4(23),12-dien-3,28-dioic acid (5), based on their NMR and MS spectral data. All of the four metabolites were new and their anti-HCV activity was tested. Their biotransformation pathway was also proposed.

Biotransformation of ursolic acid by Syncephalastrum racemosum CGMCC 3.2500 and anti-HCV activity.

Microbial transformation of ursolic acid (UA, 3ß-hydroxy-urs-12-en-28-oic acid, 1) by filamentous fungus Syncephalastrum racemosum CGMCC 3.2500 was conducted. Five metabolites 3ß, 7ß, 21ß-trihydroxy-urs-12-en-28-oic acid (2); 3ß, 21ß-dihydroxy-urs-11-en-28-oic acid-13-lactone (3); 1ß, 3ß, 21ß-trihydroxy-urs-12-en-28-oic acid (4); 3ß, 7ß, 21ß-trihydroxy-urs-1-en-28-oic acid-13-lactone (5); and 21-oxo-1ß, 3ß-dihydroxy-urs-12-en-28-oic acid (6) were afforded. Elucidation of the structures of these metabolites was primarily based on 1D and 2D NMR and HR-MS data. Metabolite 2 was a new compound. In addition, the anti-HCV activity of compounds 1-6 was evaluated.

Biotransformation of ursolic acid by an endophytic fungus from medicinal plant Huperzia serrata.

Endophytic fungi were used not only for their producing bioactive products but also for their ability to transform natural compounds. An endophytic fungus, isolated from medicinal plant Huperzia serrata, was identified as Umbelopsis isabellina based on the internal transcribed spacer of ribosomal DNA (rDNA-ITS) region. It was used to transform ursolic acid (1), a pentacyclic triterpene. Incubation of ursolic acid with U. isabellina afforded three products, 3ß-hydroxy-urs-11-en-28,13-lactone (2), 3ß,7ß-dihydroxy-urs-11-en-28,13-lactone (3), 1ß,3ß-dihydroxy-urs-11-en-28,13-lactone (4). Although product 2 was a known compound, it was first obtained by microbial transformation. Products 3 and 4 were new compounds. The structural elucidation of the three compounds was achieved mainly by the 1D- and 2D-NMR, MS, IR data. The endophytic fungus U. isabellina can hydroxyate the C12-C13 double bond at position 13 of ursolic acid 1 and form a five-member lactone effectively. In the meantime, this fungus can also introduce the hydroxyl group at C-1 or C-7 of ursolic acid 1.