The complemented mutant ΔBcstc7, in the STC7 of Botrytis cinerea led to the characterization of 11,12,13-tri-nor-eremophilenols derivatives

Abstract

Botrytis cinerea has high potential for the production of specialized metabolites. The recent resequencing of the genome of the B05.10 strain using PacBio technology and the resulting update of the Ensembl Fungi (2017) database in the genome sequence have been instrumental in identifying new genes that could be involved in secondary metabolism. Thus, a new sesquiterpene cyclase (STC) coding gene (Bcstc7) has been included in the gene list from this phytopathogenic fungus. We recently constructed the null and complement transformants in STC7 which enabled us to functionally characterize this STC. Deletion of the Bcstc7 gene abolished (+)-4-epieremophilenol biosynthesis, and could then be re-established by complementing the null mutant with the Bcstc7 gene. Chemical analysis of the complemented transformant suggests that STC7 is the principal enzyme responsible for the key cyclization step of farnesyl diphosphate (FDP) to (+)-4-epi-eremophil-9-en-11-ols.A thorough analysis of the metabolites produced by two wild-type strains, B05.10 and UCA992, and the complemented mutant compl ΔBcstc7 niaD , revealed the isolation and structural characterization of six 11,12,13-trinor-eremophilene derivatives, in addition to a large number of known eremophilen-11-ol derivatives. The structural characterization was carried out by extensive spectroscopic techniques. The biosynthesis of these compounds is explained by a retroaldol reaction or by dehydration and oxidative cleavage of C11-C13 carbons. This is the first time that this interesting family of degraded eremophilenols has been isolated from the phytopathogenous fungus B. cinerea.

Unravelling the Function of the Sesquiterpene Cyclase STC3 in the Life Cycle of <em>Botrytis cinerea</em>

“…So, both transformants produced polyketide botcinins, as well as eremophilenol derivatives. Additionally, the ΔBcstc3 biosynthesized the keto derivative 16 and the 11,12,13-tri-nor-eremophilene 17, while the OvBcstc3 mutants produced the keto derivative 15 and the two 11,12,13-tri-nor-eremophilenes 17 and 18, all of them previously reported (Figure 11) [35][36][37]. Unfortunately, no new metabolites were detected/produced by the overexpressed transformant.…”

“…Bcstc1/Bcbot2 encodes for a pentalenene cyclase, which catalyzes the first step in the biosynthesis of the botrydial phytotoxin and other important derived sesquiterpenes, generating the precursor tricyclic alcohol presilphiperfolan-8β-ol [29,30]. The deletion of the Bcsct5 gene in the ABAoverproducer strain ATCC58025 let us conclude that BcStc5/BcAba5 is the key enzyme responsible for the sesquiterpenoid abscisic acid biosynthesis [31], and the sesquiterpene cyclase 7 (BcStc7) catalyzes the cyclization of farnesyl diphosphate to eremophil-9-en-11-ols [35][36][37]. Finally, the Bcstc2 gene is related to the cryptic metabolite botrycinereic acid synthesis [55].…”

“…The isolated metabolites (Figure 11) were characterized by extensive spectroscopic and spectrometric mass analyses. The metabolomic analysis of the null mutant ΔBcstc3 broth yielded the polyketide derivatives 1 (1.8 mg), 2 (2.2 mg) and 3 (1.5 mg) [53], the known eremophil-9-en-11-ols 5 (6.5 mg), 6 (0.5 mg), 7 (3.0 mg), 8 (3.0 mg), 9 (1.3 mg), 10 (2.0 mg), 11 (2.8 mg), 12 (0.8 mg), 13 (0.8 mg) and 14 (0.7 mg) [36,54], the 11-hydroxyeremophil-1(10)-en-2-one 16 (0.5 mg) [35] and the 11,12,13-tri-nor-eremophilene 17 (3.8 mg) [37].…”

“…The overexpressed mutant OvBcstc3 broth afforded the reported polyketide derivatives 2 (1.2 mg) and 4 (2.0 mg) and the known compounds 6 (3.4 mg), 7 (5.2 mg), 10 (1.5 mg), 11 (3.6 mg) [36,54], 15 (1.5 mg) [35], 17 (3.4 mg) and 18 (1.1 mg) [37]. The chemical structures were determined by a wide range of spectral analyses using mono-and bidimensional resonance experiments and they were confirmed by direct comparison of their spectroscopic constants with those of authentic samples.…”

“…Bcstc1 encodes for a pentalenene cyclase, which generates the tricyclic alcohol presilphiperfolan-8β-ol as a precursor of botrydial phytotoxins and other important derived sesquiterpenes [29,30]. On the other hand, BcSct5 is essential for the synthesis of the sesquiterpenoid abscisic acid [31][32][33][34], and BcStc7 is involved in (+)-4-epi-eremophilenol biosynthesis, a member of the family of eremophilene sesquiterpenes [35][36][37]. However, the biosynthesis of these two compounds is not fully understood yet [27].…”