Diversity of sesquiterpene synthases in the basidiomycete Coprinus cinereus

Abstract

Fungi are a rich source of bioactive secondary metabolites, and mushroom-forming fungi (Agaricomycetes) are especially known for the synthesis of numerous bioactive and often cytotoxic sesquiterpenoid secondary metabolites. Compared with the large number of sesquiterpene synthases identified in plants, less than a handful of unique sesquiterpene synthases have been described from fungi. Here we describe the functional characterization of six sesquiterpene synthases (Cop1 to Cop6) and two terpene-oxidizing cytochrome P450 monooxygenases (Cox1 and Cox2) from Coprinus cinereus. The genes were cloned and, except for cop5, functionally expressed in Escherichia coli and/or Saccharomyces cerevisiae. Cop1 and Cop2 each synthesize germacrene A as the major product. Cop3 was identified as an alpha-muurolene synthase, an enzyme that has not been described previously, while Cop4 synthesizes delta-cadinene as its major product. Cop6 was originally annotated as a trichodiene synthase homologue but instead was found to catalyse the highly specific synthesis of alpha-cuprenene. Coexpression of cop6 and the two monooxygenase genes next to it yields oxygenated alpha-cuprenene derivatives, including cuparophenol, suggesting that these genes encode the enzymes for the biosynthesis of antimicrobial quinone sesquiterpenoids (known as lagopodins) that were previously isolated from C. cinereus and other Coprinus species.

Fig. 1. Unrooted neighbor-joining tree of fungal sesquiterpene synthase homologs

Protein sequences of known bacterial sesquiterpene sequences were used for homology searches in NCBI’s non-redundant protein sequence database and in fungal genome sequences obtained from the Broad Institute and JGI. Sequences that did not contain the first conserved metal binding domain (DDXXDD motif) of terpene synthases or appeared to be incorrectly annotated were not included in the alignment. Only a few representatives of fungal genera with several sequenced genomes (e.g. Aspergillus) were included in the search. Branches are labeled with their respective bootstrap values, gene accession numbers and strain names. Functionally characterized sesquiterpene synthases (including the Cop enzymes in this study) are also indicated. Question marks indicate that the function of Cop5 could not be determined in this study.

Fig. 2. GC/MS analysis of C. cinereus culture

The culture was grown for 1 month in the dark at 28 °C with shaking at 125 rpm. Several different sesquiterpenes were detected including pentalenene 1, α-muurolene 2, α-cuprenene 3 and δ-cadinene 4.

Fig. 3. Structures and names of sesquiterpenes described in this study

Fig. 4. GC/MS analysis of volatile organic compounds produced by E. coli transformants expressing sesquiterpene synthases from C. cinereus

E. coli cells expressing Cop1 (A) or Cop2 (B) produced as a major compound germacrene A 6a which undergoes Cope rearrangement to β-elemene 6 under the high temperatures of the injection port. E. coli cultures expressing Cop3 (C) accumulated significant quantities of α-muurolene 2, while δ-cadinene 4 was the major product of E. coli cells transformed with Cop4 (D). Peaks are labeled with numbers that correspond to their identified structures shown Fig. 3. Mass spectra for individual peaks are shown in Supplementary Fig. 2. Peak assignments were confirmed by comparing retention indices with that of authentic compounds.

Fig. 5. GC/MS analysis of recombinant S. cerevisiae cultures expressing Cop6 and P450s Cox1 and Cox2

Culture media of S. cerevisiae expressing sesquiterpene synthase Cop6 alone (A) or together with Cox1 (B), or Cox2 (C), or both (D), were analyzed for the accumulation of sesquiterpene compounds. Recombinant yeast cultures transformed with Cop6 made almost exclusively α-cuprenene 3. α-cuparene 14 is detected as a minor compound in all P450 co-expressing yeast cultures. Cultures co-expressing Cox2 make α-cuparophenol 18, while mass spectra of other new peaks (numbers correspond to mass spectra in Supplementary Fig. 2) detected in P450 and Cop6 co-expressing cultures yielded no matches in perused spectral libraries (indicated by questions marks). Parent ions for peaks are shown.

Fig. 6. Proposed reaction mechanisms for the formation of major products of C. cinereus sesquiterpene synthases Cop1-4 and Cop6

Shown cyclization pathways are based on previous investigations on cyclization reactions catalyzed by various sesquiterpene synthases reported in the literature (Steele et al., 1998, Tholl et al., 2005, Vedula et al., 2008).