Codon usage in Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma)

By sequence analysis of 96 randomly selected clones in a cDNA library of Xanthophyllomyces dendrorhous, ten novel, full-length clones encoding cytoplasmic ribosomal proteins (rp) were found. The deduced amino acid sequences showed significant homology to their counterparts from eukaryotic origin including mammals, fungi and plants. Some ribosmal protein encoding cDNAs appeared several times, but by Southern blot analysis it was shown they are encoded by a single copy gene. The nucleotide sequences of ten full length cDNAs were used to investigate the codon usage in X. dendrorhous.     

Fed-batch and continuous culture ofPhaffia rhodozyma (Xanthophyllomyces dendrorhous)

The basidiomycetous yeastPhaffia rhodozyma was grown in batch, fed-batch and continuous culture, and some parameters governing growth and total carotenoid production were determined.     

Co-production of carotenoids and xylitol by Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

Carotenoid production by Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) when growing on xylose depended on the initial substrate concentration. Astaxanthin was the main carotenoid, others were echinenone, 3-hydroxyechinenone and canthaxanthin. Xylitol (an intermediate of xylose metabolism, with application as sweetener) accumulates in the fermentation media. Xylitol concentrations up to 22 g/l were obtained in experiments carried out with an initial xylose concentration of 42 g/l. Xylitol was assimilated after xylose depletion.     

Molecular characterization of totiviruses in Xanthophyllomyces dendrorhous

ABSTRACT: BACKGROUND: Occurrence of extrachromosomal dsRNA elements has been described in the red-yeast Xanthophyllomyces dendrorhous, with numbers and sizes that are highly variable among strains with different geographical origin. The studies concerning to the encapsidation of viral-like particles and dsRNA-curing have suggested that some dsRNAs are helper viruses, while others are satellite viruses. However, the nucleotide sequences and functions of these dsRNA are still unknown. In this work, the nucleotide sequences of four dsRNAs of the strain UCD 67-385 of X. dendrorhous were determined, and their identities and genome structures are proposed. Based on this molecular data, the dsRNAs of different strains of X. dendrorhous were analyzed. RESULTS: The complete sequences of L1, L2, S1 and S2 dsRNAs of X. dendrorhous UCD 67-385 were determined, finding two sequences for L1 dsRNA (L1A and L1B). Several ORFs were uncovered in both S1 and S2 dsRNAs, but no homologies were found for any of them when compared to the database. Instead, two ORFs were identified in each L1A, L1B and L2 dsRNAs, whose deduced amino acid sequences were homologous with a major capsid protein (5'-ORF) and a RNA-dependent RNA polymerase (3'-ORF) belonging to the Totivirus family. The genome structures of these dsRNAs are characteristic of Totiviruses, with two overlapped ORFs (the 3'-ORF in the -1 frame with respect to the 5'-ORF), with a slippery site and a pseudoknot in the overlapped regions. These structures are essential for the synthesis of the viral polymerase as a fusion protein with the viral capsid protein through -1 ribosomal frameshifting. In the RNase protection analysis, all the dsRNAs in the four analyzed X. dendrorhous strains were protected from enzymatic digestion. The RT-PCR analysis revealed that, similar to strain UCD 67-385, the L1A and L1B dsRNAs coexist in the strains VKM Y-2059, UCD 67-202 and VKM Y-2786. Furthermore, determinations of the relative amounts of L1 dsRNAs using two-step RT-qPCR revealed a 40-fold increment of the ratio L1A/L1B in the S2 dsRNA-cured strain compared to its parental strain. CONCLUSIONS: Three totiviruses, named as XdV-L1A, XdV-L1B and XdV-L2, were identified in the strain UCD 67-385 of X. dendrorhous. The viruses XdV-L1A and XdV-L1B were also found in other three X. dendrorhous strains. Our results suggest that the smaller dsRNAs (named XdRm-S1 and XdRm-S2) of strain UCD 67-385 are satellite viruses, and particularly that XdRm-S2 is a satellite of XdV-L1A.     

Analysis of proteomic changes in colored mutants of Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The yeast Xanthophyllomyces dendrorhous synthesizes astaxanthin as its most prevalent xanthophyll derivative. Comparisons between the protein profiles of mutant lines of this yeast can provide insight into the carotenogenic pathway. Differently colored mutants (red, orange, pink, yellow, and white) were obtained from this yeast species, and their protein profiles were determined using two-dimensional polyacrylamide gel electrophoresis (2DE). Individual proteins differentially expressed were identified using mass spectrometry. The red mutants hyperproduced total carotenoids (mainly astaxanthin), while in white and orange mutants, mutagenesis affected the phytoene dehydrogenase activity as indicated by the accumulation of phytoene. Inactivation of astaxanthin synthase after the mutagenic treatment was evident in β-carotene accumulating mutants. Differences in the proteomic profiles of wild-type X. dendrorhous and its colored mutants were demonstrated using 2DE. Of the total number of spots detected in each gel (297–417), 128 proteins were present in all strains. The red mutant showed the greatest number of matches with respect to the wild type (305 spots), while the white and yellow mutants, which had reduced concentrations of total carotenoids, presented the highest correlation coefficient (0.6) between each other. A number of differentially expressed proteins were sequenced, indicating that tricarboxylic acid cycle and stress response proteins are closely related to the carotenogenic process.     

Genetic determination of ploidy level in Xanthophyllomyces dendrorhous

Xanthophyllomyces dendrorhous (formely Phaffia rhodozyma) is a basidiomycetous yeast-like fungus that produces carotenoids useful for the food industry. Recently, its sexual cycle was reported but little is known about its genetic constitution. To inquire into the ploidy state of X. dendrorhous, biased mutant spectrum, genetic complementation and mitotic recombination analysis were used. A wild-type strain was subjected to N-methyl-N′-nitro-N-nitrosoguanidine mutagenic treatment. Auxotrophic and carotene mutants were forced to revert to the wild-type phenotype. Pigment producing and prototroph revertants behaved as diploid except for adenine less mutants. These results are in agreement with the limited spectrum of auxotrophs obtained in this strain for the ADE1 locus. To analyze the genetic characteristic of the adenine genetic marker of X. dendrorhous, protoplast fusion experiments with several adenine less mutants were performed. The experiments presented in this work suggest that the ATCC 2430 (UDC 67-385) strain of X. dendrorhous is diploid and a heterozygous constitution is proposed for the ADE1 locus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Production of the UVB-absorbing compound mycosporine-glutaminol-glucoside by Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The ability of the basidiomycetous yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) to accumulate astaxanthin is responsible for the industrial use of this yeast as a microbial source of pigments for aquaculture. It is also hypothesized that astaxanthin accounts for its ability to thrive in highly oxidative and UV-exposed habitats. Here, we assessed the ability of this species to synthesize UV-absorbing compounds generally known as mycosporines, evaluated the effect of culture media in the production of these compounds and compared its UV growth resistance and tolerance with other yeasts. The 48 wild and collection strains screened were positive for mycosporines and a unique compound identified as mycosporine-glutaminol-glucoside (MGG) was detected. Thus, the ability of X. dendrorhous to produce MGG, as described here for the first time, is so far unique among the Cystofilobasidiales. The compound was synthesized constitutively, although growth under visible light and, to a greater extent, UVA radiation stimulated its production. Strains from UV-exposed habitats produced larger quantities and oligotrophic complex media seemed to favor MGG accumulation. UV tolerance and survival of X. dendrorhous was high and comparable to that of the polyextremophilic Rhodotorula mucilaginosa. The taxonomical and ecological implications of the production of MGG by X. dendrorhous are discussed.     

Homothallic life cycle in the diploid red yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

Sexual activity was induced in the basidiomyceteous Phaffia rhodozyma (Xanthophyllomyces dendrorhous) by depletion of nitrogen from the culture medium. This activity involved both mating between two yeast cells and the formation of basidiospores. Mating is possibly started by a G1 phase arrest of the cell cycle, as in other yeasts. The life cycle exhibited homothallic features. Crosses between genetically marked strains, and pulse-field gel electrophoresis of the chromosomal DNA of cells derived from individual spores revealed evidence of karyogamy, meiosis and even recombination. The segregation ratio in tetrads pointed to diploid vegetative cells, which formed tetraploid zygotes and the immediate meiosis then gave rise to diploid progenies again. Apart from the type strain Phaffia rhodozyma CBS 5905, all the examined strains were able to sporulate.     

Xanthophyllomyces dendrorhous for the industrial production of astaxanthin

Astaxanthin is a red xanthophyll (oxygenated carotenoid) with large importance in the aquaculture, pharmaceutical, and food industries. The green alga Haematococcus pluvialis and the heterobasidiomycetous yeast Xanthophyllomyces dendrorhous are currently known as the main microorganisms useful for astaxanthin production at the industrial scale. The improvement of astaxanthin titer by microbial fermentation is a requirement to be competitive with the synthetic manufacture by chemical procedures, which at present is the major source in the market. In this review, we show how the isolation of new strains of X. dendrorhous from the environment, the selection of mutants by the classical methods of random mutation and screening, and the rational metabolic engineering, have provided improved strains with higher astaxanthin productivity. To reduce production costs and enhance competitiveness from an industrial point of view, low-cost raw materials from industrial and agricultural origin have been adopted to get the maximal astaxanthin productivity. Finally, fermentation parameters have been studied in depth, both at flask and fermenter scales, to get maximal astaxanthin titers of 4.7 mg/g dry cell matter (420 mg/l) when X. dendrorhous was fermented under continuous white light. The industrial scale-up of this biotechnological process will provide a cost-effective method, alternative to synthetic astaxanthin, for the commercial exploitation of the expensive astaxanthin (about $2,500 per kilogram of pure astaxanthin).     

Isolation and Characterization of the Epoxide Hydrolase-Encoding Gene from Xanthophyllomyces dendrorhous

The epoxide hydrolase (EH)-encoding gene (EPH1) from the basidiomycetous yeast Xanthophyllomyces dendrorhous was isolated. The genomic sequence has a 1,236-bp open reading frame which is interrupted by eight introns that encode a 411-amino-acid polypeptide with a calculated molecular mass of 46.2 kDa. The amino acid sequence is similar to that of microsomal EH and belongs to the α/β hydrolase fold family. The EPH1 gene was not essential for growth of X. dendrorhous in rich medium under laboratory conditions. The Eph1-encoding cDNA was functionally expressed in Escherichia coli. A sixfold increase in specific activity was observed when we used resting cells rather than X. dendrorhous. The epoxides 1,2-epoxyhexane and 1-methylcyclohexene oxide were substrates for both native and recombinant Eph1. Isolation and characterization of the X. dendrorhous EH-encoding gene are essential steps in developing a yeast EH-based epoxide biotransformation system.     

Expression of the carotenoid biosynthesis genes in Xanthophyllomyces dendrorhous

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.     

Isolation and characterization of the epoxide hydrolase-encoding gene from Xanthophyllomyces dendrorhous

The epoxide hydrolase (EH)-encoding gene (EPH1) from the basidiomycetous yeast Xanthophyllomyces dendrorhous was isolated. The genomic sequence has a 1,236-bp open reading frame which is interrupted by eight introns that encode a 411-amino-acid polypeptide with a calculated molecular mass of 46.2 kDa. The amino acid sequence is similar to that of microsomal EH and belongs to the alpha/beta hydrolase fold family. The EPH1 gene was not essential for growth of X. dendrorhous in rich medium under laboratory conditions. The Eph1-encoding cDNA was functionally expressed in Escherichia coli. A sixfold increase in specific activity was observed when we used resting cells rather than X. dendrorhous. The epoxides 1,2-epoxyhexane and 1-methylcyclohexene oxide were substrates for both native and recombinant Eph1. Isolation and characterization of the X. dendrorhous EH-encoding gene are essential steps in developing a yeast EH-based epoxide biotransformation system.     

Topology of microtubules and actin in the life cycle of Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The morphology of budding and conjugating cells and associated changes in microtubules and actin distribution were studied in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) by phase-contrast and fluorescence microscopy. The non-budding interphase cell showed a nucleus situated in the central position and bundles of cytoplasmic microtubules either stretching parallel to the longitudinal cell axis or randomly distributed in the cell; none of these, however, had a character of astral microtubules. During mitosis, the nucleus divided in the daughter cell, cytoplasmic microtubules disappeared and were replaced by a spindle. The cytoplasmic microtubules reappeared after mitosis had finished. Actin patches were present both in the bud and the mother cell. Cells were induced to mate by transfer to ribitol- containing medium without nitrogen. Partner cells fused by conjugation projections where actin patches had been accumulated. Cell fusion resulted in a zygote that produced a basidium with parallel bundles of microtubules extended along its axis and with actin patches concentrated at the apex. The fused nucleus moved towards the tip of the basidium. During this movement, nuclear division was taking place; the nuclei were eventually distributed to basidiospores. Mitochondria appeared as vesicles of various sizes; their large amounts were found, often lying adjacent to microtubules, in the subcortical cytoplasm of both vegetative cells and zygotes.     

Metabolic engineering of the astaxanthin-biosynthetic pathway of Xanthophyllomyces dendrorhous

This review describes the different approaches that have been used to manipulate and improve carotenoid production in Xanthophyllomyces dendrorhous. The red yeast X. dendrorhous (formerly known as Phaffia rhodozyma) is one of the microbiological production systems for natural astaxanthin. Astaxanthin is applied in food and feed industry and can be used as a nutraceutical because of its strong antioxidant properties. However, the production levels of astaxanthin in wild-type isolates are rather low. To increase the astaxanthin content in X. dendrorhous, cultivation protocols have been optimized and astaxanthin-hyperproducing mutants have been obtained by screening of classically mutagenized X. dendrorhous strains. The knowledge about the regulation of carotenogenesis in X. dendrorhous is still limited in comparison to that in other carotenogenic fungi. The X. dendrorhous carotenogenic genes have been cloned and a X. dendrorhous transformation system has been developed. These tools allowed the directed genetic modification of the astaxanthin pathway in X. dendrorhous. The crtYB gene, encoding the bifunctional enzyme phytoene synthase/lycopene cyclase, was inactivated by insertion of a vector by single and double cross-over events, indicating that it is possible to generate specific carotenoid-biosynthetic mutants. Additionally, overexpression of crtYB resulted in the accumulation of beta-carotene and echinone, which indicates that the oxygenation reactions are rate-limiting in these recombinant strains. Furthermore, overexpression of the phytoene desaturase-encoding gene (crtI) showed an increase in monocyclic carotenoids such as torulene and HDCO (3-hydroxy-3',4'-didehydro-beta,-psi-carotene-4-one) and a decrease in bicyclic carotenoids such as echinone, beta-carotene and astaxanthin.     

Metabolic Engineering of the Carotenoid Biosynthetic Pathway in the Yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a β-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the β-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono- and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via β-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3′-4′-didehydro-β-ψ-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.     

Metabolic engineering of the carotenoid biosynthetic pathway in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a beta-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the beta-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono- and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via beta-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3'-4'-didehydro-beta-psi-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.     

Effects of oxidative stress on the production of carotenoid pigments byPhaffia rhodozyma (Xanthophyllomyces dendrorhous)

The resistance to killing by free radicals of two mutants ofPhaffia rhodozyma was determined. Mutant 5–7 did not produce astaxanthin but produced β-carotene, while mutant 3–4 did not produce any carotenoid pigments. The resistance of mutant 5–7 was the same as that of the wild type but mutant 3–4 was rapidly killed. Carotenoid pigments increased the resistance to killing by free radicals. We investigated the effects of free radicals, generated by H₂O₂ and Fe²⁺ added to the medium, on wild-type cells and mutants ofP. rhodozyma. Unpigmented mutants of basidiomycetous yeasts (Rhodotorula spp. and others) are more susceptible to killing by UV-irradiation than the pigmented, wild-type strains. Therefore, we investigated the effect of free radicals on a similar basidiomycetous yeast,P. rhodozyma, a species of economic importance, in the biological production of astaxanthin.