Unraveling the Molecular Basis of Mycosporine Biosynthesis in Fungi.

The Phaffia rhodozyma UCD 67-385 genome harbors a 7873 bp cluster containing DDGS, OMT, and ATPG, encoding 2-desmethy-4-deoxygadusol synthase, O-methyl transferase, and ATP-grasp ligase, respectively, of the mycosporine glutaminol (MG) biosynthesis pathway. Homozygous deletion mutants of the entire cluster, single-gene mutants, and the Δddgs-/-;Δomt-/- and Δomt-/-;Δatpg-/- double-gene mutants did not produce mycosporines. However, Δatpg-/- accumulated the intermediate 4-deoxygadusol. Heterologous expression of the DDGS and OMT or DDGS, OMT, and ATPG cDNAs in Saccharomyces cerevisiae led to 4-deoxygadusol or MG production, respectively. Genetic integration of the complete cluster into the genome of the non-mycosporine-producing CBS 6938 wild-type strain resulted in a transgenic strain (CBS 6938_MYC) that produced MG and mycosporine glutaminol glucoside. These results indicate the function of DDGS, OMT, and ATPG in the mycosporine biosynthesis pathway. The transcription factor gene mutants Δmig1-/-, Δcyc8-/-, and Δopi1-/- showed upregulation, Δrox1-/- and Δskn7-/- showed downregulation, and Δtup6-/- and Δyap6-/- showed no effect on mycosporinogenesis in glucose-containing medium. Finally, comparative analysis of the cluster sequences in several P. rhodozyma strains and the four newly described species of the genus showed the phylogenetic relationship of the P. rhodozyma strains and their differentiation from the other species of the genus Phaffia.

Isolation and Selection of New Astaxanthin-Producing Strains of Phaffia rhodozyma.

Astaxanthin is a xanthophyll pigment of high economic value for its use as a feeding component in aquaculture. Phaffia rhodozyma (Xanthophyllomyces dendrorhous) is a basidiomycetous fungi able to synthesize astaxanthin as its major carotenoid, the only known yeast species bearing the capability to produce this type of carotenoid and the only tremellomycetes with biotechnological application. Recently, the habitat and intraspecific variability of this species have been found to be wider than previously expected, encouraging the search for new wild strains with potential biotechnological applications. Here we describe effective procedures for isolation of P. rhodozyma from environmental samples, accurate identification of the strains, analysis of their astaxanthin content, and proper conservation of the isolates.

Un método basado en la PCR para la identificación rápida de levaduras acumuladoras de astaxantina (Phaffia spp. ) / PCR-based method for the rapid identification of astaxanthin-accumulating yeasts (Phaffia spp.)

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.

PCR-based method for the rapid identification of astaxanthin-accumulating yeasts (Phaffia spp.).

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.

UV Sunscreens of Microbial Origin: Mycosporines and Mycosporine- like Aminoacids.

Exposure to ultraviolet radiation (UVR) is harmful to living organisms, causing damage to macromolecules such as DNA, RNA, proteins and lipids. Depending on the wavelength, the injury could be direct or indirect through reactive oxygen intermediates, so it is desirable to find compounds that can reduce both. Many organic chemicals used in commercial sunscreen possess estrogenic activity in vivo. In this report we analyzed recent patents related to UV sunscreens of microbial origin, in particular mycosporines (MYC) and mycosporine-like aminoacids (MAA). Both are promising natural alternatives for both direct (UV-absorption) and indirect (antioxidant) protection, given they show strong photostability and absence of cytotoxicity. It becomes clear that although the search for natural photoprotective molecules is relatively recent, efforts have been invested mainly in marine environments, remaining still many potential photoprotective molecules to find in other type of habitats. Furthermore, unicellular microorganisms have several advantages for the production of metabolites of interest, since they improve the production costs due to its simplicity of culture and easy genetic manipulation. The knowledge of the biosynthesis pathway of MYC and MAA is essential to improve rationally their expression levels. Currently, only the MAA pathway in bacteria has been reported, remaining the MYC pathway unclear. Future perspectives include the heterologous expression of MYC and/or MAA in industrially friendly microorganisms (bacteria and yeast) in order to co-produce different UV-protective molecules and thus cover a broader UV spectrum and simplify the production process.