Enhancement of carotenoid production by disrupting the C22-sterol desaturase gene (CYP61) in Xanthophyllomyces dendrorhous

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.

Astaxanthin and Related Xanthophylls

“…This mutant also showed lower ergosterol content, suggesting that ergosterol might regulate the HMGR gene expression in X. dendrorhous and, in turn, affect the carotenoid biosynthesis. Recently, the X. dendrorhous CYP61 gene involved in the ergosterol biosynthesis was identifi ed and characterized [ 135 ]. The disruption of this gene abolished ergosterol production and the carotenoid content, including astaxanthin, was almost doubled relative to the parental strain.…”

Study on the relationship between intracellular metabolites and astaxanthin accumulation during Phaffia rhodozyma fermentation

“…Astaxanthin is an isoprenoid belonging to the mevalonate pathway in P. rhodozyma [9,17]. Carotenoid biosynthesis is inevitably affected by the primary and other secondary metabolisms, such as protein, fatty acids, and ethanol.…”

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

“…Genotype: MATa his3delta1 leu2delta0 met15delta0 ura3delta0 deltaTUP1 [38]; ATCC 4007198   Sctup1 − +YEpNP-TUP1 Sctup1 − strain carrying YEpNP-TUP1 (vector to express the X. dendrorhous TUP1 gene)This work   Sctup1 −  + YEpNP Sctup1 − strain carrying YEpNP (empty vector)This workPlasmids pBluescript II SK (−) XRCloning vector. (AmpR, pUC ori, white/blue colony selection)Stratagene, La Jolla, CA, USA pMN- hph pBluescript II SK (−) XR containing at the Eco RV site a 1.8 kb module that confers resistance to hygromycin B[39] pIR- zeo pBluescript II SK (−) XR containing at the Eco RV site a 1.2 kb module that confers resistance to zeocin[7] pgCYC8pBluescript II SK (−) XR containing at the Eco RV site a 2.4 kb X. dendrorhous genomic fragment of the CYC8 geneThis work pgCYC8-HygpgCYC8 containing at the Bmg BI site the hygromycin B resistance module disrupting the CYC8 gene fragmentThis work pgCYC8-ZeopgCYC8 containing between Pml I and Bmg BI sites the zeocin resistance module replacing 527 bp of the CYC8 gene fragmentThis work pgTUP1pBluescript II SK(−)XR containing at Eco RV site a 2.7 kb X. dendrorhous genomic fragment of the TUP1 geneThis work pgTUP1-HygpgTUP1 containing the hygromycin B resistance module replacing a 2.0 kb Eco RV fragment inside the TUP1 gene fragmentThis work pgTUP1-ZeopgTUP1 containing the zeocin resistance module replacing a 2.0 kb Eco RV fragment inside the TUP1 gene fragmentThis work YEpNP S. cerevisiae expression vector modified from YEpAct4 [40]. LEU2 gene auxotrophy marker.…”

“…1). As in other non-photosynthetic eukaryotes, carotenoid synthesis in X. dendrorhous derives from the mevalonate pathway [2, 7, 8], which produces isopentenyl pyrophosphate (IPP). IPP is isomerized to dimethylallyl pyrophosphate (DMAPP) by the enzyme IPP isomerase, encoded by the idi gene [9].…”