Identification and functional characterization of the CYP51 gene from the yeast Xanthophyllomyces dendrorhous that is involved in ergosterol biosynthesis

Abstract

BackgroundXanthophyllomyces dendrorhous is a basidiomycetous yeast that synthesizes astaxanthin, a carotenoid with great biotechnological impact. The ergosterol and carotenoid synthetic pathways derive from the mevalonate pathway and involve cytochrome P450 enzymes. Among these enzymes, the CYP51 family, which is involved in ergosterol biosynthesis, is one of the most remarkable that has C14-demethylase activity.ResultsIn this study, the CYP51 gene from X. dendrorhous was isolated and its function was analyzed. The gene is composed of ten exons and encodes a predicted 550 amino acid polypeptide that exhibits conserved cytochrome P450 structural characteristics and shares significant identity with the sterol C14-demethylase from other fungi. The functionality of this gene was confirmed by heterologous complementation in S. cerevisiae. Furthermore, a CYP51 gene mutation in X. dendrorhous reduced sterol production by approximately 40% and enhanced total carotenoid production by approximately 90% compared to the wild-type strain after 48 and 120 h of culture, respectively. Additionally, the CYP51 gene mutation in X. dendrorhous increased HMGR (hydroxy-methylglutaryl-CoA reductase, involved in the mevalonate pathway) and crtR (cytochrome P450 reductase) transcript levels, which could be associated with reduced ergosterol production.ConclusionsThese results suggest that the CYP51 gene identified in X. dendrorhous encodes a functional sterol C14-demethylase that is involved in ergosterol biosynthesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0428-2) contains supplementary material, which is available to authorized users.

Comparative Transcriptome Analysis of Gill Tissue in Response to Hypoxia in Silver Sillago (Sillago sihama)

“…The CYP superfamily plays a critical role in the oxidative metabolism and the main enzyme is responsible for biosynthetic processes in the organism, such as steroid hormones, sterols and and vitamins [37,38]. In the present study, the up-regulated cytochrome P450 family 51 (cyp51) and cytochrome P450 family 24A1 (cyp24a1) genes were significantly enriched in the steroid biosynthesis pathway under hypoxia.…”

Regulation of carotenogenesis in the red yeast Xanthophyllomyces dendrorhous: the role of the transcriptional co-repressor complex Cyc8–Tup1 involved in catabolic repression

“…LEU2 gene auxotrophy marker. It has a Xba I restriction site between the Act4 constitutive promoter and the TDH3t terminator[41] YEpNP-CYC8 S. cerevisiae e xpression vector derived from YEp-NP containing at the Xba I site between the Act4 promoter and the TDH3t terminator the X. dendrorhous CYC8 cDNAThis work YEpNP-TUP1 S. cerevisiae e xpression vector derived from YEp-NP containing at the Xba I site between the Act4 promoter and the TDH3t terminator the X. dendrorhous TUP1 cDNAThis work Amp S sensitive to ampicillin, Hyg S sensitive to hygromycin B, Hyg R resistant to hygromycin B, Zeo S sensitive to zeocin, Zeo R resistant to zeocin, ATCC American Type Culture Collection, AmpR ampicillin resistance, ColE1 ori replication origin of E. coli ColE1 plasmid …”

“…Regarding the latter, it should be noted that in most yeasts, there is a single gene encoding cytochrome P450 reductase, crtR in the case of X. dendrorhous , and that the cytochrome P450 reductase acts as an electron donor for various cytochrome P450 monooxygenases [15]. Three genes encoding cytochrome P450 monooxygenases have been functionally described in X. dendrorhous , including crtS , which is involved in carotenogenesis, and CYP51 [41] and CYP61 [7], which are involved in ergosterol biosynthesis. The increased crtR transcript level may also favor the synthesis of compounds other than carotenoids that compete for carotenogenesis precursors, similar to the way ergosterol synthesis negatively affects carotenoid synthesis.…”

Functional characterization of thiolase-encoding genes from Xanthophyllomyces dendrorhous and their effects on carotenoid synthesis

“…The crtS gene encodes a cytochrome P450 enzyme, and it has been shown that in X. dendrorhous, the crtR gene that encodes the cytochrome P450 reductase is essential for the synthesis of astaxanthin in this yeast [13]. Then, considering that two cytochrome P450 enzymes are involved in ergosterol biosynthesis [16, 28], the reduced crtS transcript levels could be related to reduced carotenogenic activity to maintain the flux towards ergosterol biosynthesis.…”

“…PCR was performed with Pfu DNA polymerase (Promega, Madison, WI, USA) using 1 μl of total X. dendrorhous RT reaction obtained from the RNA of strain UCD 67–385 to obtain the cDNAs corresponding to each gene. The amplified products were inserted into the plasmid YEp-NP [28]; this plasmid corresponds to a modified YEp-ACT4 plasmid [32] in which a sequence corresponding to 300 bp downstream of the TDH3 gene of S. cerevisiae was inserted into the Hin dIII recognition site of the plasmid (Additional file 5: Figure S4, A). Gene orientation in the plasmid was confirmed by colony PCR in a reaction mixture of 25 μl containing 2 U of Taq DNA polymerase, Taq buffer, 0.2 mM dNTPs, 2 mM MgCl 2 and 1 μM of each primer.…”