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


RESULTS
In 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.


CONCLUSIONS
These 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.
Laboratorio de Genética, Departamento de Ciencias Ecológicas y Centro de Biotecnología, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Santiago Casilla 653, Chile.
Iris
I
Loto
María Soledad
MS
Gutiérrez
Salvador
S
Barahona
Dionisia
D
Sepúlveda
Pilar
P
Martínez-Moya
Marcelo
M
Baeza
Víctor
V
Cifuentes
Jennifer
J
Alcaíno
D001487:Basidiomycota / Q000235:genetics* / Q000378:metabolism*; D002338:Carotenoids / Q000096:biosynthesis*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004875:Ergosterol / Q000378:metabolism*; D055786:Gene Knockout Techniques; D060847:Metabolic Engineering; D053858:Metabolic Networks and Pathways / Q000235:genetics; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA
D004271:DNA, Fungal; D002338:Carotenoids; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D004875:Ergosterol