Discovery of Novel Terpenoids from the Basidiomycete Pleurotus ostreatus through Genome Mining and Coculture Optimization

Abstract

In our previous work, postredienes A−C, three unusual linear sesterterpenes with high antifungal activities, were isolated from Pleurotus ostreatus SY10 when cocultured with Trametes robiniophila SY636. However, their titers were low, and exploration of newly biosynthesized trace analogues is required. Herein, genome mining analysis predicted that 17 gene clusters are involved in terpenoid biosynthesis in P. ostreatus. Thus, coculture conditions for strains SY10 and SY636 were optimized using a single-factor test and Box−Behnken design. As a result, the titers of postredienes A−C were increased by over 2.5-fold, reaching 1.28 to 8.40 mg/L. Moreover, five new terpenoids, named postredienes D−H (1−5), were successfully isolated. Compound 1 exhibited activities against the human pathogenic fungi Candida albicans and Cryptococcus neoformans comparable to those of amphotericin B. Compound 2 represents a novel sesterterpene with a five-membered ring at C-7. The absolute configurations of 1−5 were elucidated by making the methoxyphenylacetic acid esters and acetonide derivatives, combined with ECD and NMR calculation. Two potential gene clusters and relevant biosynthetic pathways for 1−5 were subsequently proposed based on real-time reverse transcriptionquantitative PCR (RT-qPCR) analysis. The current study provides new insights into the research of terpenoid biosynthesis genes in P. ostreatus and other basidiomycetes.

Discovery of New Antimicrobial Metabolites in the Coculture of Medicinal Mushrooms

“…UV data were acquired using a UV-6000T spectrophotometer (Metash, Shanghai, China). HRESIMS data were obtained using an Orbitrap Fusion Lumos mass spectrometer (Thermo, Waltham, MA, USA), and LC–MS data were obtained using a 1290/6460 mass spectrometer (Agilent, Santa Clara, CA, USA) . ECD data were obtained by using a Chirascan CD spectrometer (Applied Photophysics, Surrey, U.K.).…”

“…ECD data were obtained by using a Chirascan CD spectrometer (Applied Photophysics, Surrey, U.K.). Optical rotations were measured using a Hanon P850 automatic polarimeter (Haineng, Jinan, China) …”

“…Compared to collecting or artificially cultivating the fruiting bodies of medicinal mushrooms, mycelium fermentation offers advantages such as a stable product spectrum, shorter cultivation time, and easy industrial scale-up, making it an ideal method for researching their bioactive substances . However, compared to their natural habitat, certain activating factors are often lacking under traditional axenic conditions, resulting in some gene clusters remaining silent. , Several strategies have been applied to awaken silent genes in fungi, including genetic engineering (such as overexpression or knockout of the genes for transcription factors and global regulators), , one strain many compounds (OSMAC, mainly by altering the culture conditions), , and coculture. , Among these methods, the coculture strategy can effectively activate silent metabolic pathways by simulating natural microbial relationships, such as competition and antagonism, thus avoiding the need to manipulate the complex genome of mushrooms and reducing the workload for screening . This strategy has been well applied to explore the metabolic potential of mushrooms, leading to the discovery of many novel antimicrobial compounds, including postredienes A–F, , pleurotusin A, copsin, and 23 R -hydroxy-(20 Z ,24 R )-ergosta-4,6,8­(14),20­(22)-tetraen-3-one .…”

“…However, compared to their natural habitat, certain activating factors are often lacking under traditional axenic conditions, resulting in some gene clusters remaining silent. , Several strategies have been applied to awaken silent genes in fungi, including genetic engineering (such as overexpression or knockout of the genes for transcription factors and global regulators), , one strain many compounds (OSMAC, mainly by altering the culture conditions), , and coculture. , Among these methods, the coculture strategy can effectively activate silent metabolic pathways by simulating natural microbial relationships, such as competition and antagonism, thus avoiding the need to manipulate the complex genome of mushrooms and reducing the workload for screening . This strategy has been well applied to explore the metabolic potential of mushrooms, leading to the discovery of many novel antimicrobial compounds, including postredienes A–F, , pleurotusin A, copsin, and 23 R -hydroxy-(20 Z ,24 R )-ergosta-4,6,8­(14),20­(22)-tetraen-3-one . Thus, conducting in-depth research on medicinal mushrooms through the coculture strategy will facilitate the discovery of their antimicrobial substances, which may provide a research foundation for the development of antimicrobial agents and alternatives to antibiotics in medicine and husbandry realms to address the gradually intensifying antibiotic resistance …”

“…During our continued research on the antimicrobial substances from medicinal mushrooms by coculture strategy, , a remarkable enhancement was observed in the antifungal efficacy of secondary metabolites from the coculture broths of T. versicolor SY630 with either Vanderbylia robiniophila SY341 or Ganoderma gibbosum SY1001 compared to their monocultures.…”