ABSTRACT
It has been recently found that the natural distribution, habitat, and genetic diversity of astaxanthin-producing yeasts (i.e. Phaffia rhodozyma, synonym Xanthophyllomyces dendrorhous) is much greater than previously thought. P. rhodozyma is biotechnologically exploited due to its ability to produce the carotenoid pigment astaxanthin and thus, it is used as a natural source of this pigment for aquaculture. P. rhodozyma was also capable of synthesizing the potent UVB sunscreen mycosporine-glutaminol-glucoside (MGG). Therefore, further environmental studies are needed to elucidate its ecological aspects and detect new potential strains for the production of astaxanthin and MGG. However, obtaining new isolates of P. rhodozyma and related species is not always easy due to its low abundance and the presence of other sympatric and pigmented yeasts. In this work we report a successful development of a species-specific primer which has the ability to quickly and accurately detecting isolates representing all known lineages of the genus Phaffia (including novel species of the genus) and excluding closely related taxa. For this purpose, a primer of 20 nucleotides (called PhR) was designed to be used in combination with universal primers ITS3 and NL4 in a multiplex amplification. The proposed method has the sensitivity and specificity required for the precise detection of new isolates, and therefore represents an important tool for the environmental search for novel astaxanthin-producing yeasts.
Recientemente, se ha encontrado que la distribución natural, el hábitat y la diversidad genética de levaduras productoras de astaxantina (p. ej., Phaffia rhodozyma, sinónimo Xanthophyllomyces dendrorhous) son mucho mayores de lo que se pensaba. P. rhodozyma se explota biotecnológicamente debido a su capacidad para producir el pigmento carotenoide astaxantina y, por lo tanto, se utiliza como una fuente natural de este pigmento para la acuicultura. También se encontró que esta levadura es capaz de sintetizar el potente protector solar UVB micosporina-glutaminol-glucósido (MGG). Por lo tanto, más estudios ambientales para dilucidar sus aspectos ecológicos y detectar nuevas cepas potenciales productoras de astaxantina y MGG son necesarios. Sin embargo, la obtención de nuevos aislamientos de P. rhodozyma y especies relacionadas no siempre es fácil debido a su baja abundancia y a la presencia de otras levaduras simpátricas y pigmentadas. En este trabajo se describe el desarrollo exitoso de un cebador especie-específico que tiene la capacidad de detectar rápidamente y con precisión cepas representativas de todos los linajes del género Phaffia previamente reportados (incluyendo nuevas especies del género) y excluir especies estrechamente relacionadas. Para ello, se diseñó un cebador de 20 nucleótidos (denominado PhR) para ser utilizado en combinación con los cebadores universales ITS3 y NL4 en una amplificación multiplex. El método propuesto tiene la sensibilidad y la especificidad requerida para la detección precisa de nuevos aislamientos y, por lo tanto, representa una importante herramienta para la búsqueda ambiental de nuevas levaduras productoras de astaxantina.
Subject(s)
Yeasts/isolation & purification , Polymerase Chain Reaction/methods , Sensitivity and Specificity , Xanthophylls/isolation & purification , Methods , Nucleotides/analysisABSTRACT
The cloning and nucleotide sequence of the genes (idi, crtE, crtYB, crtl and crtS) controlling the astaxanthin biosynthesis pathway of the wild-type ATCC 24230 strain of Xanthophyllomyces dendrorhous in their genomic and cDNA version were obtained. The idi, crtE, crtYB, crtl and crtS genes were cloned, as fragments of 10.9, 11.5, 15.8, 5.9 and 4 kb respectively. The nucleotide sequence data analysis indicates that the idi, crtE, crtYB, crtl and crtS genes have 4, 8,4, 11, and 17 introns and 5, 9, 5, 12 and 18 exons respectively. In addition, a highly efficient site-directed mutagenesis system was developed by transformation by integration, followed by mitotic recombination (the double recombinant method). Heterozygote idi (idi+ / idi-::hph), crtE (crtE+ / crtE -::hph), crtYB (crtYB + / crtYB -::hph), crtI (crtI+ / crtI-::hph) and crtS (crtS +/crtS -::hph) and homozygote mutants crtYB (crtYB -::hph/crtYB -::hph), crtI (crtI -::hph/crtI -::hph) and crtS (crtS -::hph / crtS -::hph) were constructed. All the heterozygote mutants have a pale phenotype and produce less carotenoids than the wild-type strain. The genetic analysis of the crtYB, crtl and crtS loci in the wild-type, heterozygote, and homozygote give evidence of the diploid constitution of ATCC 24230 strains. In addition, the cloning of a truncated form of the crtYB that lacks 153 amino acids of the N-terminal region derived from alternatively spliced mRNA was obtained. Their heterologous expression in Escherichia coli carrying the carotenogenic cluster of Erwinia uredovora result in trans-complementation and give evidence of its functionality in this bacterium, maintaining its phytoene synthase activity but not the lycopene cyclase activity.
Subject(s)
Basidiomycota/genetics , Gene Expression Regulation, Fungal/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Complementary/genetics , Genes, Fungal/genetics , Polymerase Chain Reaction , RNA, Fungal/genetics , Xanthophylls/biosynthesis , Xanthophylls/geneticsABSTRACT
In the yeast Xanthophyllomyces dendrorhous the genes idi, crtE, crtYB, crtl and ast are involved in the biosynthesis of astaxanthin from isopentenyl pyrophosphate. The carotenoid production and the kinetics of mRNA expression of structural genes controlling the carotenogenesis in a wild-type ATCC 24230 and in carotenoid overproducer deregulated atxS2 strains were studied. The biosynthesis of carotenoid was induced at the late exponential growth phase in both strains. However, the cellular carotenoid concentration was four times higher in atxS2 than in the wild-type strain in the exponential growth phase, suggesting that carotenogenesis was deregulated in atxS2 at the beginning of growth. In addition, the maximum expression of the carotenogenesis genes at the mRNA level was observed during the induction period of carotenoid biosynthesis in the wild-type strain. The mRNA level of the crtYB, crtl, ast genes and to a lesser extent the idi gene, decayed at the end of the exponential growth phase. The mRNA levels of the crtE gene remained high along the whole growth curve of the yeast. In the atxS2 strain the mRNA levels of crtE gene were about two times higher than the wild-type strain in the early phase of the growth cycle.