Abstract
BackgroundXanthophyllomyces dendrorhous is a basidiomycetous yeast that synthesizes astaxanthin, which is a carotenoid with a great biotechnological impact. The ergosterol and carotenoid synthesis pathways are derived from the mevalonate pathway, and in both pathways, cytochrome P450 enzymes are involved.ResultsIn this study, we isolated and described the X. dendrorhous CYP61 gene, which encodes a cytochrome P450 involved in ergosterol biosynthesis. This gene is composed of nine exons and encodes a 526 amino acid polypeptide that shares significant percentages of identity and similitude with the C22-sterol desaturase, CYP61, from other fungi. Mutants derived from different parental strains were obtained by disrupting the CYP61 gene with an antibiotic selection marker. These mutants were not able to produce ergosterol and accumulated ergosta-5,8,22-trien-3-ol and ergosta-5,8-dien-3-ol. Interestingly, all of the mutants had a more intense red color phenotype than their respective parental strains. The carotenoid composition was qualitatively and quantitatively analyzed by RP-HPLC, revealing that the carotenoid content was higher in the mutant strains without major changes in their composition. The expression of the HMGR gene, which encodes an enzyme involved in the mevalonate pathway (3-hydroxy-3-methylglutaryl-CoA reductase), was analyzed by RT-qPCR showing that its transcript levels are higher in the CYP61 mutants.ConclusionsThese results suggest that in X. dendrorhous, ergosterol regulates HMGR gene expression by a negative feedback mechanism and in this way; it contributes in the regulation of the carotenoid biosynthesis.
Highlights
Xanthophyllomyces dendrorhous is a basidiomycetous yeast that synthesizes astaxanthin, which is a carotenoid with a great biotechnological impact
Cloning and sequence analysis of the CYP61 gene from X. dendrorhous Our X. dendrorhous genomic database was analyzed with the BLAST tool of the CLC Genomics Workbench 5 software using as query several CYP61 gene sequences available in the GenBank database
The CYP61 gene is involved in the ergosterol biosynthesis, so we evaluated the sterol production and composition in the cyp61- mutants by RPHPLC
Summary
Xanthophyllomyces dendrorhous is a basidiomycetous yeast that synthesizes astaxanthin, which is a carotenoid with a great biotechnological impact. Unlike other astaxanthin-producing organisms, X. dendrorhous has a single astaxanthin synthase (encoded by the crtS gene) that catalyzes the ketolation and hydroxylation of beta-carotene to produce astaxanthin [15,16] This enzyme is related to a 3A subfamily member of the cytochrome P450 protein family [15,16]. Cytochrome P450 proteins (P450s) are hemecontaining monooxygenases that are present in organisms from all domains of life [17]; P450s have significant roles in the oxidative metabolism of many exogenous and endogenous substrates [18] In their active state, these enzymes are reduced by electrons that are supplied by NAD(P)H through a P450 redox partner [19], which in eukaryotes is a cytochrome P450 reductase [20]. The X. dendrorhous crtR gene is different from the crtR gene originally described in cyanobacterium Synechocystis sp., which encodes a betacarotene hydroxylase [22]
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