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1.  Functional Characterization of the Xanthophyllomyces dendrorhous Farnesyl Pyrophosphate Synthase and Geranylgeranyl Pyrophosphate Synthase Encoding Genes That Are Involved in the Synthesis of Isoprenoid Precursors 
PLoS ONE  2014;9(5):e96626.
The yeast Xanthophyllomyces dendrorhous synthesizes the carotenoid astaxanthin, which has applications in biotechnology because of its antioxidant and pigmentation properties. However, wild-type strains produce too low amounts of carotenoids to be industrially competitive. Considering this background, it is indispensable to understand how the synthesis of astaxanthin is controlled and regulated in this yeast. In this work, the steps leading to the synthesis of the carotenoid precursor geranylgeranyl pyrophosphate (GGPP, C20) in X. dendrorhous from isopentenyl pyrophosphate (IPP, C5) and dimethylallyl pyrophosphate (DMAPP, C5) was characterized. Two prenyl transferase encoding genes, FPS and crtE, were expressed in E. coli. The enzymatic assays using recombinant E. coli protein extracts demonstrated that FPS and crtE encode a farnesyl pyrophosphate (FPP, C15) synthase and a GGPP-synthase, respectively. X. dendrorhous FPP-synthase produces geranyl pyrophosphate (GPP, C10) from IPP and DMAPP and FPP from IPP and GPP, while the X. dendrorhous GGPP-synthase utilizes only FPP and IPP as substrates to produce GGPP. Additionally, the FPS and crtE genes were over-expressed in X. dendrorhous, resulting in an increase of the total carotenoid production. Because the parental strain is diploid, the deletion of one of the alleles of these genes did not affect the total carotenoid production, but the composition was significantly altered. These results suggest that the over-expression of these genes might provoke a higher carbon flux towards carotenogenesis, most likely involving an earlier formation of a carotenogenic enzyme complex. Conversely, the lower carbon flux towards carotenogenesis in the deletion mutants might delay or lead to a partial formation of a carotenogenic enzyme complex, which could explain the accumulation of astaxanthin carotenoid precursors in these mutants. In conclusion, the FPS and the crtE genes represent good candidates to manipulate to favor carotenoid biosynthesis in X. dendrorhous.
PMCID: PMC4010515  PMID: 24796858
2.  Increase in the astaxanthin synthase gene (crtS) dose by in vivo DNA fragment assembly in Xanthophyllomyces dendrorhous 
BMC Biotechnology  2013;13:84.
Xanthophyllomyces dendrorhous is a basidiomycetous yeast that is relevant to biotechnology, as it can synthesize the carotenoid astaxanthin. However, the astaxanthin levels produced by wild-type strains are low. Although different approaches for promoting increased astaxanthin production have been attempted, no commercially competitive results have been obtained thus far. A promising alternative to facilitate the production of carotenoids in this yeast involves the use of genetic modification. However, a major limitation is the few available molecular tools to manipulate X. dendrorhous.
In this work, the DNA assembler methodology that was previously described in Saccharomyces cerevisiae was successfully applied to assemble DNA fragments in vivo and integrate these fragments into the genome of X. dendrorhous by homologous recombination in only one transformation event. Using this method, the gene encoding astaxanthin synthase (crtS) was overexpressed in X. dendrorhous and a higher level of astaxanthin was produced.
This methodology could be used to easily and rapidly overexpress individual genes or combinations of genes simultaneously in X. dendrorhous, eliminating numerous steps involved in conventional cloning methods.
PMCID: PMC3852557  PMID: 24103677
Xanthophyllomyces dendrorhous; Astaxanthin synthase; DNA assembler
3.  Diversity and extracellular enzymatic activities of yeasts isolated from King George Island, the sub-Antarctic region 
BMC Microbiology  2012;12:251.
Antarctica has been successfully colonized by microorganisms despite presenting adverse conditions for life such as low temperatures, high solar radiation, low nutrient availability and dryness. Although these “cold-loving” microorganisms are recognized as primarily responsible for nutrient and organic matter recycling/mineralization, the yeasts, in particular, remain poorly characterized and understood. The aim of this work was to study the yeast microbiota in soil and water samples collected on King George Island.
A high number of yeast isolates was obtained from 34 soil and 14 water samples. Molecular analyses based on rDNA sequences revealed 22 yeast species belonging to 12 genera, with Mrakia and Cryptococcus genera containing the highest species diversity. The species Sporidiobolus salmonicolor was by far the most ubiquitous, being identified in 24 isolates from 13 different samples. Most of the yeasts were psychrotolerant and ranged widely in their ability to assimilate carbon sources (consuming from 1 to 27 of the 29 carbon sources tested). All species displayed at least 1 of the 8 extracellular enzyme activities tested. Lipase, amylase and esterase activity dominated, while chitinase and xylanase were less common. Two yeasts identified as Leuconeurospora sp. and Dioszegia fristingensis displayed 6 enzyme activities.
A high diversity of yeasts was isolated in this work including undescribed species and species not previously isolated from the Antarctic region, including Wickerhamomyces anomalus, which has not been isolated from cold regions in general. The diversity of extracellular enzyme activities, and hence the variety of compounds that the yeasts may degrade or transform, suggests an important nutrient recycling role of microorganisms in this region. These yeasts are of potential use in industrial applications requiring high enzyme activities at low temperatures.
PMCID: PMC3499239  PMID: 23131126
Antarctic yeasts; Psychrophilic-psychrotolerant yeasts; Extracellular enzyme activities; rDNA yeast identification
4.  Enhancement of carotenoid production by disrupting the C22-sterol desaturase gene (CYP61) in Xanthophyllomyces dendrorhous 
BMC Microbiology  2012;12:235.
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.
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.
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.
PMCID: PMC3552872  PMID: 23075035
Xanthophyllomyces dendrorhous; Astaxanthin; Ergosterol; Sterol C22-sterol desaturase; Cytochrome P450
5.  Molecular characterization of totiviruses in Xanthophyllomyces dendrorhous 
Virology Journal  2012;9:140.
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 in 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 dsRNAs 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.
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 Totiviridae 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.
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.
PMCID: PMC3561658  PMID: 22838956
X. dendrorhous; dsRNA; Totivirus; Mycovirus
6.  "Glucose and ethanol-dependent transcriptional regulation of the astaxanthin biosynthesis pathway in Xanthophyllomyces dendrorhous" 
BMC Microbiology  2011;11:190.
The yeast Xanthophyllomyces dendrorhous is one of the most promising and economically attractive natural sources of astaxanthin. The biosynthesis of this valuable carotenoid is a complex process for which the regulatory mechanisms remain mostly unknown. Several studies have shown a strong correlation between the carbon source present in the medium and the amount of pigments synthesized. Carotenoid production is especially low when high glucose concentrations are used in the medium, while a significant increase is observed with non-fermentable carbon sources. However, the molecular basis of this phenomenon has not been established.
In this work, we showed that glucose caused transcriptional repression of the three genes involved in the synthesis of astaxanthin from geranylgeranyl pyrophosphate in X. dendrorhous, which correlates with a complete inhibition of pigment synthesis. Strikingly, this regulatory response was completely altered in mutant strains that are incapable of synthesizing astaxanthin. However, we found that addition of ethanol caused the induction of crtYB and crtS gene expression and promoted de novo synthesis of carotenoids. The induction of carotenogenesis was noticeable as early as 24 h after ethanol addition.
For the first time, we demonstrated that carbon source-dependent regulation of astaxanthin biosynthesis in X. dendrorhous involves changes at the transcriptional level. Such regulatory mechanism provides an explanation for the strong and early inhibitory effect of glucose on the biosynthesis of this carotenoid.
PMCID: PMC3184065  PMID: 21861883
7.  Proteomic analysis of the carotenogenic yeast Xanthophyllomyces dendrorhous 
BMC Microbiology  2011;11:131.
The yeast Xanthophyllomyces dendrorhous is used for the microbiological production of the antioxidant carotenoid astaxanthin. In this study, we established an optimal protocol for protein extraction and performed the first proteomic analysis of the strain ATCC 24230. Protein profiles before and during the induction of carotenogenesis were determined by two-dimensional polyacrylamide gel electrophoresis and proteins were identified by mass spectrometry.
Among the approximately 600 observed protein spots, 131 non-redundant proteins were identified. Proteomic analyses allowed us to identify 50 differentially expressed proteins that fall into several classes with distinct expression patterns. These analyses demonstrated that enzymes related to acetyl-CoA synthesis were more abundant prior to carotenogenesis. Later, redox- and stress-related proteins were up-regulated during the induction of carotenogenesis. For the carotenoid biosynthetic enzymes mevalonate kinase and phytoene/squalene synthase, we observed higher abundance during induction and/or accumulation of carotenoids. In addition, classical antioxidant enzymes, such as catalase, glutathione peroxidase and the cytosolic superoxide dismutases, were not identified.
Our results provide an overview of potentially important carotenogenesis-related proteins, among which are proteins involved in carbohydrate and lipid biosynthetic pathways as well as several redox- and stress-related proteins. In addition, these results might indicate that X. dendrorhous accumulates astaxanthin under aerobic conditions to scavenge the reactive oxygen species (ROS) generated during metabolism.
PMCID: PMC3224108  PMID: 21669001
8.  Polymorphism of viral dsRNA in Xanthophyllomyces dendrorhous strains isolated from different geographic areas 
Virology Journal  2009;6:160.
Strains of the astaxanthin producing yeast Xanthophyllomyces dendrorhous have been isolated from different cold regions around the earth, and the presence of double stranded RNA (dsRNA) elements was described in some isolates. This kind of viruses is widely distributed among yeasts and filamentous fungi and, although generally are cryptic in function, their studies have been a key factor in the knowledge of important fungi. In this work, the characterization and genetic relationships among dsRNA elements were determined in strains representatives of almost all regions of the earth where X. dendrorhous have been isolated.
Almost all strains of X. dendrorhous analyzed carry one, two or four dsRNA elements, of molecular sizes in the range from 0.8 to 5.0 kb. Different dsRNA-patterns were observed in strains with different geographic origin, being L1 (5.0 kb) the common dsRNA element. By hybridization assays a high genomic polymorphism was observed among L1 dsRNAs of different X. dendrorhous strains. Contrary, hybridization was observed between L1 and L2 dsRNAs of strains from same or different regions, while the dsRNA elements of minor sizes (M, S1, and S2) present in several strains did not show hybridization with neither L1 or L2 dsRNAs. Along the growth curve of UCD 67-385 (harboring four dsRNAs) an increase of L2 relative to L1 dsRNA was observed, whiles the S1/L1 ratio remains constant, as well as the M/L1 ratio of Patagonian strain. Strains cured of S2 dsRNA were obtained by treatment with anisomycin, and comparison of its dsRNA contents with uncured strain, revealed an increase of L1 dsRNA while the L2 and S1 dsRNA remain unaltered.
The dsRNA elements of X. dendrorhous are highly variable in size and sequence, and the dsRNA pattern is specific to the geographic region of isolation. Each L1 and L2 dsRNA are viral elements able to self replicate and to coexist into a cell, and L1 and S2 dsRNAs elements could be part of a helper/satellite virus system in X. dendrorhous.
PMCID: PMC2764699  PMID: 19814805
9.  Cloning of the cytochrome p450 reductase (crtR) gene and its involvement in the astaxanthin biosynthesis of Xanthophyllomyces dendrorhous 
BMC Microbiology  2008;8:169.
The yeast Xanthophyllomyces dendrorhous synthesizes astaxanthin, a carotenoid with high commercial interest. The proposed biosynthetic route in this organism is isopentenyl-pyrophosphate (IPP) → geranyleranyl pyrophosphate (GGPP) → phytoene → lycopene → β-carotene → astaxanthin. Recently, it has been published that the conversion of β-carotene into astaxanthin requires only one enzyme, astaxanthin synthase or CrtS, encoded by crtS gene. This enzyme belongs to the cytochrome P450 protein family.
In this work, a crtR gene was isolated from X. dendrorhous yeast, which encodes a cytochrome P450 reductase (CPR) that provides CrtS with the necessary electrons for substrate oxygenation. We determined the structural organization of the crtR gene and its location in the yeast electrophoretic karyotype. Two transformants, CBSTr and T13, were obtained by deleting the crtR gene and inserting a hygromycin B resistance cassette. The carotenoid composition of the transformants was altered in relation to the wild type strain. CBSTr forms yellow colonies because it is unable to produce astaxanthin, hence accumulating β-carotene. T13 forms pale colonies because its astaxanthin content is reduced and its β-carotene content is increased.
In addition to the crtS gene, X. dendrorhous requires a novel gene, crtR, for the conversion of β-carotene to astaxanthin.
PMCID: PMC2575211  PMID: 18837978
10.  Cooperative Uptake of Microcin E492 by Receptors FepA, Fiu, and Cir and Inhibition by the Siderophore Enterochelin and Its Dimeric and Trimeric Hydrolysis Products 
Microcin E492 uptake by FepA, Fiu, and Cir is cooperative, with FepA being the main receptor. No TonB-mediated interaction with the ferric catecholate receptors is needed for microcin to exert action at the cytoplasmic membrane. Microcin E492 uptake by the receptors is inhibited by the dimer and trimer of dihydroxybenzoylserine.
PMCID: PMC1168642  PMID: 15980406

Results 1-10 (10)