Genomic organization of the structural genes controlling the astaxanthin biosynthesis pathway of Xanthophyllomyces dendrorhous

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.

Study of the expression of carotenoid biosynthesis genes in wild-type and deregulated strains of Xanthophyllomyces dendrorhous (Ex: Phaffia rhodozyma)

The expression, at the mRNA level, of carotenoid biosynthetic genes from Xanthophyllomyces dendrorhous was studied by RT-PCR. The experimental conditions for the RT-PCR assay were standardized to quantify the relative transcript levels of idi, crtE, crtYB and crtI genes. This work attempted to correlate astaxanthin production with the transcript levels of carotenogenic genes in a wild-type strain (UCD 67-385) and two overproducer deregulated strains (atxS1 and atxS2). At 3 day cultures, the wild-type strain contained higher transcript levels from the crtE and crtYB genes on minimal medium than on rich medium. Similarly, carotenoid production was higher on minimal medium than on rich medium. However, carotenoid production in the atxS1 and atxS2 strains was not correlated with the transcript level of carotenogenic genes under the same experimental conditions. This result suggests that there is not a linear relationship between carotenogenic transcript levels and carotenoid biosynthesis.

Estudio genético molecular de levaduras de diversos ambientes / Molecular genetic study of yeasts from different environments

Se describe la presencia de polimorfísmo genético en levaduras nativas aisladas de diferentes ambientes, tales como: agua de mar, suelos forestales con poca intervención antrópica, suelos de viñedos, otros ambientes naturales y desde pacientes con fungemia severa. Se detectó la presencia de elementos genéticos extracromosómicos en cuatro cepas diferentes de levaduras. El análisis de amplificación de una región ITS utilizando partidores específicos para el ITS2 (partidores ITS3 e ITS4), permite determinar una banda de amplificación de rDNA que varia de tamaño entre 450 y 560 pb. dependiendo del género de levadura analizada. En una cepa de Cryptococcus terreus se observó la presencia de tres bandas de amplificación ITS que sugieren una organización compleja de los genes de rDNA en esa cepa. Finalmente el análisis del cariotipo electroforético de cepas ambientales y clínicas de Pichia anomala mostró un marcado polimorfísmo cromosómico en esta levadura emergente.

Aislamiento de genes carotenogénesis de xanthophyllomyces dendrorhous (ex. phaffia rhodozyma) mediante PCR / Isolation of carotenogenesis genes of xanthophyllomyces dendrohous (ex. phaffia rhodozyma) through PCR

Xanthophyllomyces dendrorhous (ex.phaffia rhodozyma) es una levadura basidiomicetica carotenogénica, en la cual aspectos importantes de su biología como la organización general de su genoma, número de cromosomas y nivel de ploidia aún no son completamente entendidos. En atención a esto, se han orientado esfuerzos en estudios moleculares con el objetivo de aumentar el conocimiento de su genética. En el presente trabajo, se describe un eficiente procedimiento para seleccionar y clonar genes a partir de una genoteca de DNA genómico de x. dendrorhous mediante la amplificación de DNA por PCR, utilizándo parejas de partidores específicos para el gen crtl. Adicionalmente, se describe la síntesis de cDNA a partir de RNA total de la levadura mediante transcripción reversa acoplada a amplificación de DNA (rt-pcr)