Characterisation of ascocorynin biosynthesis in the purple jellydisc fungus Ascocoryne sarcoides

Wieder, Carsten; Peres da Silva, Roberta; Witts, Jessica; Jaeger, Christof Martin; Geib, Elena; Brock, Matthias

Abstract

Background: Non-ribosomal peptide synthetase-like (NRPS-like) enzymes are highly enriched in fungal genomes and can be discriminated into reducing and non-reducing enzymes. Non-reducing NRPS-like enzymes possess a C-terminal thioesterase domain that catalyses the condensation of two identical aromatic α-keto acids under the formation of enzyme-specific substrate-interconnecting core structures such as terphenylquinones, furanones, butyrolactones or dioxolanones. Ascocoryne sarcoides produces large quantities of ascocorynin, which structurally resembles a terphenylquinone produced from the condensation of p-hydroxyphenylpyruvate and phenylpyruvate. Since the parallel use of two different substrates by a non-reducing NRPS-like enzyme appeared as highly unusual, we investigated the biosynthesis of ascocorynin in A. sarcoides. Results: Here, we searched the genome of A. sarcoides for genes coding for non-reducing NRPS-like enzymes. A single candidate gene was identified that was termed acyN. Heterologous gene expression confirmed that AcyN is involved in ascocorynin production but only produces the non-hydroxylated precursor polyporic acid. Although acyN is embedded in an ascocorynin biosynthesis gene cluster, a gene encoding a monooxygenase required for the hydroxylation of polyporic acid was not present. Expression analyses of all monooxygenase-encoding genes from A. sarcoides identified a single candidate that showed the same expression pattern as acyN. Accordingly, heterologous co-expression of acyN and the monooxygenase gene resulted in the production of ascocorynin. Structural modelling of the monooxygenase suggests that the hydrophobic substrate polyporic acid enters the monooxygenase from a membrane facing entry site and is converted into the more hydrophilic product ascocorynin, which prevents its re-entry for a second round of hydroxylation. Conclusion: This study characterises the first naturally occurring polyporic acid synthetase from an ascomycete. It confirms the high substrate and product specificity of this non-reducing NRPS-like enzyme and highlights the requirement of a monooxygenase to produce the terphenylquinone ascocorynin.

Citation

Wieder, C., Peres da Silva, R., Witts, J., Jaeger, C. M., Geib, E., & Brock, M. (2022). Characterisation of ascocorynin biosynthesis in the purple jellydisc fungus Ascocoryne sarcoides. Fungal Biology and Biotechnology, 9, Article 8. https://doi.org/10.1186/s40694-022-00138-7

• Journal Article Type: Article
• Acceptance Date: Feb 19, 2022
• Online Publication Date: Apr 27, 2022
• Publication Date: Apr 27, 2022
• Deposit Date: Mar 4, 2022
• Publicly Available Date: Apr 27, 2022
• Journal: Fungal Biology and Biotechnology
• Electronic ISSN: 2054-3085
• Publisher: Springer Science and Business Media LLC
• Peer Reviewed: Peer Reviewed
• Volume: 9
• Article Number: 8
• DOI: https://doi.org/10.1186/s40694-022-00138-7
• Keywords: Cell Biology; Molecular Biology; Applied Microbiology and Biotechnology; Ecology, Evolution, Behavior and Systematics; Biotechnology
• Public URL: https://nottingham-repository.worktribe.com/output/7536435
• Publisher URL: https://fungalbiolbiotech.biomedcentral.com/articles/10.1186/s40694-022-00138-7