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

Kritsye Leiva,Víctor Cifuentes,Nicole Werner,Marcelo Baeza,Dionisia Sepúlveda,Salvador Barahona,Jennifer Alcaíno

doi:10.1186/s12866-015-0428-2

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.

Highlights

Xanthophyllomyces dendrorhous is a basidiomycetous yeast that synthesizes astaxanthin, a carotenoid with great biotechnological impact
Cloning and sequence analysis of the X. dendrorhous CYP51 gene Using bioinformatic analyses of the genomic and transcriptomic data of X. dendrorhous available in our laboratory with the S. cerevisiae ERG11 gene as a query [GenBank: NM_001179137], we identified a putative X. dendrorhous CYP51 gene
The CYP51 gene was predicted to encode a 550 amino acid protein with an expected molecular weight of 61.8 kDa and pI of 6.64. This protein shares 48% identity and 65% similarity at 91% coverage with the S. cerevisiae C14-demethylase [ERG11, Swiss-Prot: P10614] (Figure 1), which belongs to the cytochrome P450 protein family and is involved in the third step of ergosterol biosynthesis from squalene: the conversion of lanosterol to 4,4-dimethylcholesta8,14,24-trienol [22,23]

Summary

Introduction

Xanthophyllomyces dendrorhous is a basidiomycetous yeast that synthesizes astaxanthin, a carotenoid with great biotechnological impact. CYP enzymes are involved in the biosynthesis of many physiologically important compounds, such as sterols, steroid hormones, fatty acids and vitamins [16], and a vast array of secondary metabolites in plants, insects and fungi [17]. These enzymes are the main catalysts involved in the activation and detoxification of different xenobiotics, such as drugs, pesticides, carcinogens and environmentally contaminating chemicals [18]. CYP enzymes require the reducing equivalents from NADPH or NADH, which are generally transferred to CYPs through a redox partner to catalyze the general reaction RH + O2 + 2e− + 2H+ → ROH + H2O [19]

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