Unusual and Highly Bioactive Sesterterpenes Synthesized by Pleurotus ostreatus during Coculture with Trametes robiniophila Murr

Abstract

Candida albicans and Cryptococcus neoformans, human-pathogenic fungi found worldwide, are receiving increasing attention due to high morbidity and mortality in immunocompromised patients. In the present work, 110 fungus pairs were constructed by coculturing 16 wood-decaying basidiomycetes, among which coculture of Trametes robiniophila Murr and Pleurotus ostreatus was found to strongly inhibit pathogenic fungi through bioactivity-guided assays. A combination of metabolomics and molecular network analysis revealed that 44 features were either newly synthesized or produced at high levels in this coculture system and that 6 of the features that belonged to a family of novel and unusual linear sesterterpenes contributed to high activity with MICs of 1 to 32 g/ml against pathogenic fungi. Furthermore, dynamic 13 C-labeling analysis revealed an association between induced features and the corresponding fungi. Unusual sesterterpenes were 13 C labeled only in P. ostreatus in a time course after stimulation by the coculture, suggesting that these sesterterpenes were synthesized by P. ostreatus instead of T. robiniophila Murr. Sesterterpene compounds 1 to 3 were renamed postrediene A to C. Real-time reverse transcription-quantitative PCR (RT-qPCR) analysis revealed that transcriptional levels of three genes encoding terpene synthase, farnesyl-diphosphate farnesyltransferase, and oxidase were found to be 8.2-fold, 88.7-fold, and 21.6-fold higher, respectively, in the coculture than in the monoculture, indicating that biosynthetic gene cluster 10 was most likely responsible for the synthesis of these sesterterpenes. A putative biosynthetic pathway of postrediene A to postrediene C was then proposed based on structures of sesterterpenes and molecular network analysis.

Coculture, An Efficient Biotechnology for Mining the Biosynthesis Potential of Macrofungi via Interspecies Interactions

“…Compared with the complicated living conditions in nature, many genes or clusters of microorganisms are silent in standard culture under the laboratory conditions. To activate these silent genes or clusters, the coculture strategy is considered as an efficient approach, because it can mimic the symbiotic relationships of microorganisms in their intrinsic habitats (Moody, 2014 ; Xu et al, 2018 ; Shen et al, 2019 ). Secondary metabolites induced by cocultures, such as small molecular metabolites, polysaccharides, and polypeptides, usually display diverse bioactivities, including antimicrobial activity, antitumor activity, and antioxidant activity, and represent a potential and powerful resource to discover the leading compounds for medicinal development.…”

“…To mimic the relationships of fungi in nature, different coculture systems, including macrofungi-macrofungi, macrofungi-microalgae, macrofungi-cyanobacteria, macrofungi-filamentous fungi, macrofungi-bacteria, and macrofungi-actinomycetes, have been established. These cocultures have shown various bioactivities, such as antimicrobial activity, antitumor activity, and antioxidant activity (Zheng et al, 2011 ; Shen et al, 2019 ).…”

“…The fermentation broth from the coculture of Trametes robiniophila Murr. and Pleurotus ostreatus showed the strongest inhibitory activity against the human-pathogenic fungi C. albicans and C. neoformans (Shen et al, 2019 ). The combination of metabolomics analysis and activity-guided isolation led to the discovery of three unusual linear sesterterpenes, postredienes A–C ( 10 – 12 ) ( Table 1 ), with 80% minimum inhibitory concentrations (MIC 80 ) of 1 to 32 μg/ml against C. albicans and C. neoformans .…”

“…The mycelium of macrofungi can also be cultivated under submerged fermentation in shaking flasks. Both the fruiting body and liquid-cultivated mycelium have been reported to produce a number of useful constituents, including bioactive lead compounds (such as alkaloids, polyketides, terpenoids, and steroids) and degradation-related oxidative enzymes (such as laccase, lignin peroxidase, and manganese peroxidase) (Qu et al, 2019 ; Shen et al, 2019 ; Zhang et al, 2019 ; Lira-Pérez et al, 2020 ), which make macrofungi an important resource for agriculture, industry, and medicine.…”

“…Only a few macrofungi, including G. lucidum, Ganoderma lingzhi, Cordyceps militaris, Agaricus bisporus, Schizophyllum commune , and Coprinopsis cinerea , have been reported to be edited by the CRISPR-Cas9 system or to achieve an overexpression system using traditional plasmids (Zhou et al, 2012 ; Qin et al, 2017 ; Sugano et al, 2017 ; Chen et al, 2018 ; Jan Vonk et al, 2019 ; Shen et al, 2019 , 2020 ). By comparison, the coculture strategy can simulate the symbiotic relationship of microorganisms in nature and easily activate silenced gene clusters through the interaction between strains (Moody, 2014 ; Xu et al, 2018 ; Shen et al, 2019 ). Recently, coculturing has been widely used in mining unknown biosynthetic potential, such as discovering novel secondary metabolites and enzymes that might be applied in the fields of medicine, agriculture, and industry in the future ( Figure 1 ).…”