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.     

Stimulation of astaxanthin formation in the yeast Xanthophyllomyces dendrorhous by the fungus Epicoccum nigrum

A fungal contaminant on an agar plate containing colonies of Xanthophyllomyces dendrorhous markedly increased carotenoid production by yeast colonies near to the fungal growth. Spent-culture filtrate from growth of the fungus in yeast-malt medium also stimulated carotenoid production by X. dendrorhous. Four X. dendrorhous strains including the wild-type UCD 67-385 (ATCC 24230), AF-1 (albino mutant, ATCC 96816), Yan-1 (beta-carotene mutant, ATCC 96815) and CAX (astaxanthin overproducer mutant) exposed to fungal concentrate extract enhanced astaxanthin up to approximately 40% per unit dry cell weight in the wild-type strain and in CAX. Interestingly, the fungal extract restored astaxanthin biosynthesis in non-astaxanthin-producing mutants previously isolated in our laboratory, including the albino and the beta-carotene mutant. The fungus was identified as Epicoccum nigrum by morphology of sporulating cultures, and the identity confirmed by genetic characterization including rDNA sequencing analysis of the large-subunit (LSU), the internal transcribed spacer, and the D1/D2 region of the LSU. These E. nigrum rDNA sequences were deposited in GenBank under accesssion numbers AF338443, AY093413 and AY093414. Systematic rDNA homology alignments were performed to identify fungi related to E. nigrum. Stimulation of carotenogenesis by E. nigrum and potentially other fungi could provide a novel method to enhance astaxanthin formation in industrial fermentations of X. dendrorhous and Phaffia rhodozyma.     

Improved growth of the red yeast, Phaffia rhodozyma (Xanthophyllomyces dendrorhous), in the presence of tricarboxylic acid cycle intermediates

Catabolites related to tricarboxylic acid cycle affected growth and carotenogenesis in Phaffia rhodozyma. Glutamate, glutamine, aspartate, asparagine and proline at 75 mM of N increased biomass from 2 g l^–1 to 2.9–4.7 g l^–1 but decreased carotenoid from 420 g g^–1 yeast to 200–260 g g^–1 yeast in strain 67-385. However, simple nitrogen sources did not decrease carotenoid formation. Tricarboxylic acid intermediates repressed carotenogenesis to a less degree than the corresponding amino acids. Carotenoid hyper-producing mutants were impaired in nitrogen utilization. These results indicated that nitrogen assimilation and the concentrations of tricarboxylic acid cycle intermediates are involved in regulation of carotenoid biosynthesis.     

Production and partial characterization of a beta-amylase by Xanthophyllomyces dendrorhous

AIMS: The characterization of a beta-amylase produced by Xanthophyllomyces dendrorhous. METHODS AND RESULTS: Growth in different culture media showed that X. dendrorhous produces an amylase whose synthesis is repressed by the carbon source and induced by starch and maltose. Enzymatic assays using substrates with different degrees of polymerization together with viscosity experiments revealed that the enzyme was beta-amylase. According to the biochemical characterization, the enzyme has a molecular weight of 240 kDa and a Km of 1.35 mg ml-1. The optimum pH and temperature were 5.5 and 50 degrees C, respectively. Using different inhibitors of the enzymatic activity it was shown that cysteine, tryptophan and serine are essential amino acids for catalysis. CONCLUSIONS: Xanthophyllomyces dendrorhous CECT1690 synthesizes and secretes beta-amylase that could be a by-product, in addition to carotenoid pigments, in the fermentation downstream. SIGNIFICANCE AND IMPACT OF THE STUDY: The beta-amylase produced by X. dendrorhous may have certain industrial applications.     

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.     

高产虾青素红法夫酵母的选育及代谢通量分析

以野生型红法夫酵母As2.1557为出发菌株,依次进行两轮紫外线诱变和亚硝基胍诱变,并以10-4与10-3mol/Lβ-紫罗酮作为筛选剂,得到突变株UV-N2-7,其虾青素产量和含量分别较出发菌株提高81.7%和2.25倍。采用Plackett-Burman设计法及响应面分析法对发酵培养基的组分及发酵条件进行了优化,突变株的虾青素产量由从初始的2.674 mg/L提高到了6.338 mg/L。建立了红法夫酵母的代谢网络,并对比了突变株和野生株间歇培养条件下对数生长期的代谢通量分布。结果表明:突变株与野生株相比,PP途径通量有所减小,而EMP途径和TCA循环有所增强,突变株用于菌体生长的通量有所减小;二者的丙酮酸脱氢酶的活性均较低,分泌至胞外的丙酮酸的代谢通量约为32%。因此预计通过遗传改造和发酵控制提高丙酮酸脱氢酶的活性可能会进一步提高虾青素的产率。     

Biology of the red yeastXanthophyllomyces dendrorhous (Phaffia rhodozyma)

Phaffia rhodozyma is a red-pigmented basidiomycetous yeast that produces astaxanthin. Because of this property, it is fermented commercially on a large scale. Astaxanthin is used as a food colorant for fish, and as an antioxidant, it is potentially useful in the human pharmaceutical industry. This review summarizes the published biology of this yeast: its morphology and ultrastructure, organization of the cytoskeleton, and the nuclear and extrachromosomal genomes. Alteration of sexual and vegetative phases in the life cycle and its biotechnological importance are also described.     

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.     

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.     

Efficient screening for astaxanthin-overproducing mutants of the yeast Xanthophyllomyces dendrorhous by flow cytometry

Astaxanthin possesses higher antioxidant activity than other carotenoids and, for this and other reasons, has great commercial potential for use in the aquaculture, pharmaceutical, and food industries. The basidiomycetous yeast Xanthophyllomyces dendrorhous is one of the best natural producers of astaxanthin, but wild-type cells accumulate only a small amount of astaxanthin. In this study, we developed an efficient flow cytometry method to screen for astaxanthin-overproducing mutants of X. dendrorhous. We first examined the relationship between cellular astaxanthin content and the intensity of fluorescence emitted from the cell. Although the fluorescence emission maximum of astaxanthin dissolved in acetone occurred at 570 nm, intracellular astaxanthin content correlated better with emission at around 675 nm in different X. dendrorhous strains. Using this emission wavelength, we screened cells mutagenized with ethyl methanesulfonate and successfully isolated mutants that produced 1.5-3.8-fold more astaxanthin than parent cells. This method enabled us to obtain overproducers five times more efficient than conventional screening from plate culture.     

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.     

红色酵母原生质体形成和再生条件的研究

了不同渗透压稳定剂、酶浓度、酶解时间,温度等对红色酵母原生质体的形成与再生的影响。实验结果表明：使用对数生长期早期的细胞,蜗牛酶浓度１％,３０℃处理５０－６ｍｉｎ,山梨醇为渗透压稳定剂,有利于原生质体制备和再生。     

红发夫酵母积累虾青素的代谢调控机理研究进展

由红发夫酵母合成的虾青素是一种非常有商业价值的类胡萝卜素.综述了近年来国内外在红发夫酵母中虾青素的生物合成途径、合成代谢调控机理等领域的相关研究进展,并提出了国内应开展的相关创新性研究.     

法夫酵母 JMU-MVP14菌体中类胡萝卜素成分分析

结合薄层色谱、柱色谱、以及高效液相色谱对虾青素高产菌株-法夫酵母JMU-MVP14中的类胡萝卜素成分进行初步研究。研究结果表明,硅胶柱层析和氧化镁柱层析相结合的方法对法夫酵母JMU-MVP14菌体中的类胡萝卜素成分有很好的分离效果。经过柱层析分离纯化后,各组分中类胡萝卜素的种类单一,有利于进一步通过各种波谱技术对其进行定性。此方法弥补了单纯依靠高效液相色谱（ODS 柱）对法夫酵母 JMU-MVP14菌体中类胡萝卜素分离效果不佳,可供选择的商业化类胡萝卜素标准品少,液相保留时间漂移等因素给法夫酵母JMU-MVP14菌体中类胡萝卜素定性带来的不足。     

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.     

Genetics and electrophoretic karyotyping of wild-type and astaxanthin mutant strains of Phaffia rhodozyma

In this work we establish the chromosomal composition of a wild-type, one astaxanthin and two -carotene overproducer strains of the red yeast Phaffia rhodozyma. The method used has been pulsed field gel electrophoresis, which has determined 9 DNA chromosomal bands in the yeast genome. The two largest bands are triplets and two other bands, VI and VIII, seem to be doublets. The size of the chromosomal bands varies between 0.35 and 2.5 Mb, suggesting a genome size of 25 Mb. The technique used, complemented with hybridization assays using specific DNA probes, provides direct information about the genomic organization of P. rhodozyma. We have also cloned and located in chromosomal bands different DNA sequences that code for the translation elongation factor 1 alpha (ef-1), a 7.6 kb BamHI fragment of repetitive DNA (possibly rDNA) and a randomly chosen fragment (named locus R2). Additionally, we have detected a chromosomal length polymorphism between wild-type strains and mutant strains affecting carotenogenesis obtained in our laboratory.     

Sucrose biotransformation by immobilized Phaffia rhodozyma and continuous neokestose production in a packed-bed reactor

The effects of important production variables on the biotransformation of sucrose by immobilized cells of Phaffia rhodozyma were investigated. Cell concentration had negative effect on the maximum concentration of neokestose and the optimal concentration was 16?g/l (calculated by dry weight). The concentration of sucrose had a positive effect on the maximum concentration of neokestose within 1.170?mol/l. Elevating the reaction temperature increased the reaction rate but decreased the maximum concentration of neokestose. Sugar cane juice could be used as an inexpensive substrate for neokestose production. Additionally, a 30-d continuous neokestose production was found feasible in a packed bed reactor, indicating that the cell immobilization with chitosan-coated alginate has the potential for industrial production.     

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.