Characterisation of ascocorynin biosynthesis in the purple jellydisc fungus Ascocoryne sarcoides

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.

New colours for old in the blue-cheese fungus Penicillium roqueforti

“…Transformants were analysed for metabolite production by inoculation of 25 mL of liquid GG20 medium with 1 × 10 6 conidia/mL followed by incubation for 40 h on a rotary shaker (150 rpm) at 28°C. Extracts from culture supernatants and mycelia were prepared as previously described 43 and subjected to HPLC analysis on a Dionex UltiMate3000 (Thermo Fisher Scienti c, UK) system using an analytical Eclipse XDB C18 (Agilent, 250 mm × 4.6 mm, particle size 5 µm) column 16 . High-resolution exact-mass determination was performed in negative mode on a Bruker impact II Ultra-High-Resolution LC-QTOF MS with an Eclipse XDB C18 column as speci ed above 43 .…”

“…Extracts from culture supernatants and mycelia were prepared as previously described 43 and subjected to HPLC analysis on a Dionex UltiMate3000 (Thermo Fisher Scienti c, UK) system using an analytical Eclipse XDB C18 (Agilent, 250 mm × 4.6 mm, particle size 5 µm) column 16 . High-resolution exact-mass determination was performed in negative mode on a Bruker impact II Ultra-High-Resolution LC-QTOF MS with an Eclipse XDB C18 column as speci ed above 43 . To visualise production of coloured metabolites from transformants, a minimal medium plate was point-inoculated with selected strains and incubated for 60 h at 28°C before photographs were taken.…”

Functional Application of the Single-Module NRPS-like d-Alanyltransferase in Maytansinol Biosynthesis

“…The analysis of the EIC peaks of the fermentation products of the HGFS01 strain unveiled the presence of distinct signals corresponding to D-alanylated products, e.g., N-desmethyl-3-O-D-alanyl maytansinol (6) and 3-O-D-alanyl maytansinol (7) (NMR spectroscopic data in Table S10), and their corresponding hydrolysis products N-desmethyl maytansinol (8) and maytansinol (5) (Figure 4A). The undetected signal of 4 could be notably observed in the fermentation of the HGFS03 strain, wherein the smastC gene was overexpressed in the asm11disrupted mutant HGFS02 (Figure S14).…”

“…To validate the usefulness of the engineered strain for scale-up fermentation, a 3-L fermentation with a lab-scale bioreactor was conducted to assess the performance of HGFS08. Utilizing LA media suitable for liquid fermentation of A. pretiosum, 46 maytansinol was obtained with the yield of 25 ± 2 mg/L after 10-day fermentation, and 32.8 mg of maytansinol was isolated together with 6.0 mg of 3-O-D-alanyl maytansinol (7). The bioreactor fermentation of HGFS08 resulted in a comparable maytansinol yield to solid-state fermentation and enabled the retention of previously inaccessible intermediate 7, attributed to the shortened fermentation period and maintenance of a neutral pH environment (Figures S24 and S25 desepoxymaytansinol owing to the substrate compatibility of the TE domains.…”

“…Essentially, adenylation (A) and thiolation (T) domains are commonplace in single-module NRPS-like enzymes, while alternative domains, such as condensation (C), reductase (R), thioesterase (TE), epimerase (E), and oxidase (Ox), further diversify their domain repertoires. Among these, A-T-TE tridomain NRPS-like enzymes have garnered recognition for their involvement in the formation of quinones , or lactones − through the dimerization of α-keto carboxylic acids, exemplified by AtrA, TdiA, and ApvA in fungi as well as EchA in Streptomyces . In particular, AstC, a unique A-T-TE tridomain NRPS-like enzyme discovered in Streptomyces sp.…”