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

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

SUBMITTER: Leiva K

PROVIDER: S-EPMC4415319 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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

Leiva Kritsye K Werner Nicole N Sepúlveda Dionisia D Barahona Salvador S Baeza Marcelo M Cifuentes Víctor V Alcaíno Jennifer J

BMC microbiology 20150425

<h4>Background</h4>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.<h4>Results</h4>In this study, the CYP51 gene from X. dendrorhous was isola ...[more]

PMID: 25906980

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Project description:BACKGROUND:Occurrence of extrachromosomal dsRNA elements has been described in the red-yeast Xanthophyllomyces dendrorhous, with numbers and sizes that are highly variable among strains with different geographical origin. The studies concerning to the encapsidation in viral-like particles and dsRNA-curing have suggested that some dsRNAs are helper viruses, while others are satellite viruses. However, the nucleotide sequences and functions of these dsRNAs are still unknown. In this work, the nucleotide sequences of four dsRNAs of the strain UCD 67-385 of X. dendrorhous were determined, and their identities and genome structures are proposed. Based on this molecular data, the dsRNAs of different strains of X. dendrorhous were analyzed. RESULTS:The complete sequences of L1, L2, S1 and S2 dsRNAs of X. dendrorhous UCD 67-385 were determined, finding two sequences for L1 dsRNA (L1A and L1B). Several ORFs were uncovered in both S1 and S2 dsRNAs, but no homologies were found for any of them when compared to the database. Instead, two ORFs were identified in each L1A, L1B and L2 dsRNAs, whose deduced amino acid sequences were homologous with a major capsid protein (5'-ORF) and a RNA-dependent RNA polymerase (3'-ORF) belonging to the Totiviridae family. The genome structures of these dsRNAs are characteristic of Totiviruses, with two overlapped ORFs (the 3'-ORF in the -1 frame with respect to the 5'-ORF), with a slippery site and a pseudoknot in the overlapped regions. These structures are essential for the synthesis of the viral polymerase as a fusion protein with the viral capsid protein through -1 ribosomal frameshifting. In the RNase protection analysis, all the dsRNAs in the four analyzed X. dendrorhous strains were protected from enzymatic digestion. The RT-PCR analysis revealed that, similar to strain UCD 67-385, the L1A and L1B dsRNAs coexist in the strains VKM Y-2059, UCD 67-202 and VKM Y-2786. Furthermore, determinations of the relative amounts of L1 dsRNAs using two-step RT-qPCR revealed a 40-fold increment of the ratio L1A/L1B in the S2 dsRNA-cured strain compared to its parental strain. CONCLUSIONS:Three totiviruses, named as XdV-L1A, XdV-L1B and XdV-L2, were identified in the strain UCD 67-385 of X. dendrorhous. The viruses XdV-L1A and XdV-L1B were also found in other three X. dendrorhous strains. Our results suggest that the smaller dsRNAs (named XdRm-S1 and XdRm-S2) of strain UCD 67-385 are satellite viruses, and particularly that XdRm-S2 is a satellite of XdV-L1A.

Project description:The cytochromes P450 (P450s) are a large superfamily of heme-containing monooxygenases involved in the oxidative metabolism of an enormous diversity of substrates. These enzymes require electrons for their activity, and the electrons are supplied by NAD(P)H through a P450 electron donor system, which is generally a cytochrome P450 reductase (CPR). The yeast Xanthophyllomyces dendrorhous has evolved an exclusive P450-CPR system that specializes in the synthesis of astaxanthin, a carotenoid with commercial potential. For this reason, the aim of this work was to identify and characterize other potential P450 genes in the genome of this yeast using a bioinformatic approach.Thirteen potential P450-encoding genes were identified, and the analysis of their deduced proteins allowed them to be classified in ten different families: CYP51, CYP61, CYP5139 (with three members), CYP549A, CYP5491, CYP5492 (with two members), CYP5493, CYP53, CYP5494 and CYP5495. Structural analyses of the X. dendrorhous P450 proteins showed that all of them have a predicted transmembrane region at their N-terminus and have the conserved domains characteristic of the P450s, including the heme-binding region (FxxGxRxCxG); the PER domain, with the characteristic signature for fungi (PxRW); the ExxR motif in the K-helix region and the oxygen-binding domain (OBD) (AGxDTT); also, the characteristic secondary structure elements of all the P450 proteins were identified. The possible functions of these P450s include primary, secondary and xenobiotic metabolism reactions such as sterol biosynthesis, carotenoid synthesis and aromatic compound degradation.The carotenogenic yeast X. dendrorhous has thirteen P450-encoding genes having potential functions in primary, secondary and xenobiotic metabolism reactions, including some genes of great interest for fatty acid hydroxylation and aromatic compound degradation. These findings established a basis for future studies about the role of P450s in the carotenogenic yeast X. dendrorhous and their potential biotechnological applications.

Project description:The yeast Xanthophyllomyces dendrorhous synthesizes the carotenoid astaxanthin, which has applications in biotechnology because of its antioxidant and pigmentation properties. However, wild-type strains produce too low amounts of carotenoids to be industrially competitive. Considering this background, it is indispensable to understand how the synthesis of astaxanthin is controlled and regulated in this yeast. In this work, the steps leading to the synthesis of the carotenoid precursor geranylgeranyl pyrophosphate (GGPP, C20) in X. dendrorhous from isopentenyl pyrophosphate (IPP, C5) and dimethylallyl pyrophosphate (DMAPP, C5) was characterized. Two prenyl transferase encoding genes, FPS and crtE, were expressed in E. coli. The enzymatic assays using recombinant E. coli protein extracts demonstrated that FPS and crtE encode a farnesyl pyrophosphate (FPP, C15) synthase and a GGPP-synthase, respectively. X. dendrorhous FPP-synthase produces geranyl pyrophosphate (GPP, C10) from IPP and DMAPP and FPP from IPP and GPP, while the X. dendrorhous GGPP-synthase utilizes only FPP and IPP as substrates to produce GGPP. Additionally, the FPS and crtE genes were over-expressed in X. dendrorhous, resulting in an increase of the total carotenoid production. Because the parental strain is diploid, the deletion of one of the alleles of these genes did not affect the total carotenoid production, but the composition was significantly altered. These results suggest that the over-expression of these genes might provoke a higher carbon flux towards carotenogenesis, most likely involving an earlier formation of a carotenogenic enzyme complex. Conversely, the lower carbon flux towards carotenogenesis in the deletion mutants might delay or lead to a partial formation of a carotenogenic enzyme complex, which could explain the accumulation of astaxanthin carotenoid precursors in these mutants. In conclusion, the FPS and the crtE genes represent good candidates to manipulate to favor carotenoid biosynthesis in X. dendrorhous.

Dataset Information

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

Publications

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

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