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

Abstract

Background: Xanthophyllomyces 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.

Results: In 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.

Conclusions: These results suggest that the CYP51 gene identified in X. dendrorhous encodes a functional sterol C14-demethylase that is involved in ergosterol biosynthesis.

Figure 1

Sequence alignment between X. dendrorhous CYP51 and S. cerevisiae ERG11 proteins and prediction of structurally conserved motifs in CYP51. Amino acid alignment between the deduced CYP51 sequence from X. dendrorhous (Xd) strain UCD 67–385 and the S. cerevisiae (Sc) strain S288c ERG11 (lanosterol C14-demethylase) protein [Swiss Protein: P10614]. Amino acid differences with the same properties are denoted with a plus (+). Structural elements are highlighted with the name of the corresponding feature above them: possible transmembrane helix (underlined and italics), alpha helices (red and italics), beta-sheets (blue and italics), meander loop and Cys pocket (white highlighted in black). The asterisks (*) indicate the three totally conserved amino acids among cytochromes P450. Secondary structural elements were predicted with the CYP450 Engineering database [26], and the potential transmembrane region was predicted with TMpred [52].

Figure 2

Three-dimensional model and docking of the X. dendrorhous CYP51 deduced protein. (A) The model was generated by the SwissModel web server [54] using the S. cerevisiae ERG11 protein with lanosterol as the template ligand (model code. 4 lxj.1). (B) The AutoDock4 program [31] was used to perform automated docking to predict the binding sites of lanosterol (red) and itraconazole (blue) in the obtained model of the CYP51 protein from X. dendrorhous.

Figure 3

PCR-based analyses of S. cerevisiae erg11 ⁻ strains carrying the YEpNP-gERG11 and YEpNP-cCYP51 vectors. PCR analyses to confirm the presence of the kanMX gene at the ERG11 locus (panel A: primers CYP51ScExt.F and KanMX4.R2 and panel B: primers KanMX4.F2 and CYP51ScExt.R), the presence of the X. dendrorhous CYP51 gene (panel C: primers RTCYP51.F and CYP51.Rb), the presence of the ERG11 gene (panel D: gERG11.F and gERG11.R) and the absence of the ERG11 gene in the S. cerevisiae genome (panel E: gERG11.F and CYP51ScExt.R). Template DNA in each lane: S. cerevisiae parental diploid strain erg11 +/− (Lane 1), S. cerevisiae S288c strain (Lane 2), S. cerevisiae Sc-hCYP51 strain (Lane 3), S. cerevisiae Sc-hERG11 strain (Lane 4), X. dendrorhous UCD 67–385 strain (Lane 5), negative control without DNA (Lane 6). Molecular size marker Lambda/Hind III (Lane M: 23.1, 9.4, 6.6, 4.4, 2.3, 2.0 and 0.6 kb). A schematic diagram is included to represent the primer sets (shown in arrows) that were used. The UP and DOWN regions correspond to regions located 300 bp upstream and downstream of the S. cerevisiae ERG11 gene, respectively. Region KanMX4 corresponds to the geneticin (G418) resistance module and the pACT4 and tTDH3 regions correspond to the S. cerevisiae promoter and terminator region in YEpNP.

Figure 4

RT-qPCR analysis of the HMGR, crtR and CYP51 genes in the wild-type and CBS-CYP51 ^+/− strains. The HMGR (A), crtR (B) and CYP51 (C) transcript levels were determined by RT-qPCR after 24 and 48 h of cultivation of the wild-type and the CBS-CYP51 ^+/−strains. Each transcript level was normalized with respect to the transcript level of the actin gene and then with respect to the wild-type strain: CBS 6938 (=1, black bars) and CBS-CYP51^+/− (gray bars). Values are the mean ± standard error of three independent experiments (* p ≤ 0.05; Student’s t test).