Screening of astaxanthin-overproducing Xanthophyllomyces dendrorhous strains via iterative ARTP mutagenesis and cell sorting by flow cytometry.

AIMS: The astaxanthin-producing yeast Xanthophyllomyces dendrorhous is widely used in aquaculture. Due to the production of carotenoid, this yeast shows visible color; however, high-throughput approaches for identification of astaxanthin-overproducing strains remain rare. METHODS AND RESULTS: This study verified an effective approach to identify astaxanthin-overproducing mutants of X. dendrorhous by flow cytometry (FCM) and cell sorting. First, the mutant libraries were generated by atmospheric and room-temperature plasma (ARTP) mutagenesis. Second, a highly direct correlation between the concentrations of intracellular astaxanthin and the levels of emitting fluorescence was constructed by testing a variety of astaxanthin-contained populations via FCM and cell sorting. Third, iterative cell sorting efficiently improves the identification of astaxanthin-overproducing strains. Finally, two mutants producing 4.96 mg astaxanthin g-1 DCW (dry cell weight) and 5.30 mg astaxanthin g-1 DCW were obtained, which were 25.3% and 33.8% higher than that of the original strain, respectively. CONCLUSIONS: This study demonstrated that iterative ARTP mutagenesis along with cell sorting by FCM is effective for identifying astaxanthin-overproduction strains.

An enhanced electron transport chain improved astaxanthin production in Phaffia rhodozyma.

Astaxanthin (AX) is a carotenoid pigment with antioxidant properties widely used as a feed supplement. Wild-type strains of Phaffia rhodozyma naturally produce low AX yields, but we increased AX yields 50-fold in previous research using random mutagenesis of P. rhodozyma CBS6938 and fermentation optimization. On that study, genome changes were linked with phenotype, but relevant metabolic changes were not resolved. In this study, the wild-type and the superior P. rhodozyma mutant strains were grown in chemically defined media and instrumented fermenters. Differential kinetic, metabolomics, and transcriptomics data were collected. Our results suggest that carotenoid production was mainly associated with cell growth and had a positive regulation of central carbon metabolism metabolites, amino acids, and fatty acids. In the stationary phase, amino acids associated with the TCA cycle increased, but most of the fatty acids and central carbon metabolism metabolites decreased. TCA cycle metabolites were in abundance and media supplementation of citrate, malate, α-ketoglutarate, succinate, or fumarate increased AX production in the mutant strain. Transcriptomic data correlated with the metabolic and genomic data and found a positive regulation of genes associated with the electron transport chain suggesting this to be the main driver for improved AX production in the mutant strain.

Effect of ethanol on astaxanthin and fatty acid production in the red yeast Xanthophyllomyces dendrorhous.

AIM: The effects of detergent, ethanol and ethanol with plant meadowfoam oil on the growth of the red heterobasidomycete Xanthophyllomyces dendrorhous and on the production of astaxanthin (3,3'-dihydroxy-ß,ß-carotene-4,4'-dione) and fatty acids in this red yeast were investigated. METHODS AND RESULTS: Ethanol supplementation at a final concentration of 0.8% (v/v) caused an increase in the growth, astaxanthin production and fatty acid production of treated X. dendrorhous compared with untreated X. dendrorhous. Supplementation of meadowfoam oil with 0.8% ethanol further improved the growth and astaxanthin production of X. dendrorhous. Fatty acid compositions following supplementation with various concentrations of ethanol and oil were also analysed. With 0.8% ethanol supplementation, the ratio of linoleic acid (C18:2) and α-linolenic acid (C18:3ω3, ALA) decreased. Conversely, with 1.8% ethanol supplementation, the ALA ratio increased. CONCLUSIONS: Ethanol can serve as a promoting factor for coproduction of astaxanthin and fatty acids in X. dendrorhous, whereas simultaneous supplementation of ethanol and meadowfoam oil can cause further astaxanthin production. SIGNIFICANCE AND IMPACT OF STUDY: Astaxanthin is widely used in various functional products because of its antioxidant activity. This study shows that X. dendrorhous can coproduce astaxanthin and functional fatty acids at high levels following supplementation with ethanol.

Enhancing astaxanthin yield in Phaffia rhodozyma: current trends and potential of phytohormones.

Astaxanthin is an important ketocarotenoid with remarkable biological activities and high economic value. In recent times, natural astaxanthin production by microorganisms has attracted much attention particularly in pharmaceuticals, nutraceuticals, cosmetics, and food and feed industries. Though, currently, productivity is still low and has restricted scale-up application in the commercial market, microbial production of astaxanthin has enormous prospects as it is a greener alternative to the predominating chemical synthesis. Over the years, Phaffia rhodozyma has attracted immense interest particularly in the field of biovalorization and sustainable production of natural nutraceuticals as a promising source of natural astaxanthin since it is able to use agro-food waste as inexpensive nutrient source. Many research works have, thus, been devoted to improving the astaxanthin yield from this yeast. Considering that the yeast was first isolated from tree exudates, the use of phytohormones and plant growth stimulators as prospective stimulants of astaxanthin production in the yeast is promising. Besides, it has been shown in several studies that phytohormones could improve cell growth and astaxanthin production of algae. Nevertheless, this option is less explored for P. rhodozyma. The few studies that have examined the effect of phytohormones on the yeast and its astaxanthin productivity reported positive results, with phytohormones such as 6-benzylaminopurin and gibberellic acid resulting in increased expression of carotenogenesis genes. Although the evidence available is scanty, the results are promising. KEY POINTS: • Phaffia rhodozyma is a promising source of natural astaxanthin • For industrialization, astaxanthin productivity of P. rhodozyma still needs optimization • Phytohormones could potentially augment astaxanthin yield of P. rhodozyma.

Metabolic engineering for high yield synthesis of astaxanthin in Xanthophyllomyces dendrorhous.

Astaxanthin is a carotenoid with a number of assets useful for the food, cosmetic and pharmaceutical industries. Nowadays, it is mainly produced by chemical synthesis. However, the process leads to an enantiomeric mixture where the biologically assimilable forms (3R, 3'R or 3S, 3'S) are a minority. Microbial production of (3R, 3'R) astaxanthin by Xanthophyllomyces dendrorhous is an appealing alternative due to its fast growth rate and easy large-scale production. In order to increase X. dendrorhous astaxanthin yields, random mutant strains able to produce from 6 to 10 mg/g dry mass have been generated; nevertheless, they often are unstable. On the other hand, site-directed mutant strains have also been obtained, but they increase only the yield of non-astaxanthin carotenoids. In this review, we insightfully analyze the metabolic carbon flow converging in astaxanthin biosynthesis and, by integrating the biological features of X. dendrorhous with available metabolic, genomic, transcriptomic, and proteomic data, as well as the knowledge gained with random and site-directed mutants that lead to increased carotenoids yield, we propose new metabolic engineering targets to increase astaxanthin biosynthesis.

Xanthophyllomyces dendrorhous, a Versatile Platform for the Production of Carotenoids and Other Acetyl-CoA-Derived Compounds.

Xanthophyllomyces dendrorhous (with Phaffia rhodozyma as its anamorphic state) is a basidiomycetous, moderately psychrophilic, red yeast belonging to the Cystofilobasidiales. Its red pigmentation is caused by the accumulation of astaxanthin, which is a unique feature among fungi. The present chapter reviews astaxanthin biosynthesis and acetyl-CoA metabolism in X. dendrorhous and describes the construction of a versatile platform for the production of carotenoids, such as astaxanthin, and other acetyl-CoA-derived compounds including fatty acids by using this fungus.

Astaxanthin for the Food Industry.

Xanthophyll astaxanthin, which is commonly used in aquaculture, is one of the most expensive and important industrial pigments. It is responsible for the pink and red color of salmonid meat and shrimp. Due to having the strongest anti-oxidative properties among carotenoids and other health benefits, natural astaxanthin is used in nutraceuticals and cosmetics, and in some countries, occasionally, to fortify foods and beverages. Its use in food technology is limited due to the unknown effects of long-term consumption of synthetic astaxanthin on human health as well as few sources and the high cost of natural astaxanthin. The article characterizes the structure, health-promoting properties, commercial sources and industrial use of astaxanthin. It presents the possibilities and limitations of the use of astaxanthin in food technology, considering its costs and food safety. It also presents the possibilities of stabilizing astaxanthin and improving its bioavailability by means of micro- and nanoencapsulation.

Xanthophyllomyces dendrorhous-Derived Astaxanthin Regulates Lipid Metabolism and Gut Microbiota in Obese Mice Induced by A High-Fat Diet.

Astaxanthin is an important antioxidant with many biological activities such as anti-tumor, anti-obesity, cardioprotective, and immuno-modulatory activities. Most of these biological activities are derived from (3S,3'S)-astaxanthin, while the activities of (3R,3'R)-astaxanthin are rarely reported. The purpose of this study was to investigate the effect of (3R,3'R)-astaxanthin on lipid metabolism and gut microbiota in mice fed with a high-fat diet. In this work, 40 male C57BL/6 mice were divided into 8 groups fed a high-fat diet supplemented or not with (3R,3'R)-astaxanthin or Xanthophyllomyces dendrorhous for 8 weeks. The weight gain, energy intake, fat index, plasma triacylglycerol and cholesterol, liver triacylglycerol and cholesterol, and gut microbiota were determined. The results showed that the addition of (3R,3'R)-astaxanthin/X. dendrorhous to the high-fat diet as a supplement prevented weight gain, reduced plasma and liver triacylglycerol, and decreased plasma and liver total cholesterol. The addition of (3R,3'R)-astaxanthin/X. dendrorhous also regulated the gut microbiota of the mice, which optimized the ratio of Bacteroides to Firmicutes and increased the content of Verrucomicrobia, especially Akkermansia. The changes in the gut microflora achieved a healthier structure, thus reducing the incidence of obesity. Thus (3R,3'R)-Astaxanthin has the function of regulating lipid metabolism and gut microbiota to prevent obesity caused by a high-fat diet. The production strain of (3R,3'R)-astaxanthin, X. dendrorhous, has the same function as astaxanthin in preventing obesity caused by a high-fat diet, which reflects its potential ability as a probiotic drug.

Characterization of the cytochrome P450 monooxygenase genes (P450ome) from the carotenogenic yeast Xanthophyllomyces dendrorhous.

BACKGROUND: The cytochromes P450 (P450s) are a large superfamily of heme-containing monooxygenases involved in the oxidative metabolism of an enormous diversity of substrates. These enzymes require electrons for their activity, and the electrons are supplied by NAD(P)H through a P450 electron donor system, which is generally a cytochrome P450 reductase (CPR). The yeast Xanthophyllomyces dendrorhous has evolved an exclusive P450-CPR system that specializes in the synthesis of astaxanthin, a carotenoid with commercial potential. For this reason, the aim of this work was to identify and characterize other potential P450 genes in the genome of this yeast using a bioinformatic approach. RESULTS: Thirteen potential P450-encoding genes were identified, and the analysis of their deduced proteins allowed them to be classified in ten different families: CYP51, CYP61, CYP5139 (with three members), CYP549A, CYP5491, CYP5492 (with two members), CYP5493, CYP53, CYP5494 and CYP5495. Structural analyses of the X. dendrorhous P450 proteins showed that all of them have a predicted transmembrane region at their N-terminus and have the conserved domains characteristic of the P450s, including the heme-binding region (FxxGxRxCxG); the PER domain, with the characteristic signature for fungi (PxRW); the ExxR motif in the K-helix region and the oxygen-binding domain (OBD) (AGxDTT); also, the characteristic secondary structure elements of all the P450 proteins were identified. The possible functions of these P450s include primary, secondary and xenobiotic metabolism reactions such as sterol biosynthesis, carotenoid synthesis and aromatic compound degradation. CONCLUSIONS: The carotenogenic yeast X. dendrorhous has thirteen P450-encoding genes having potential functions in primary, secondary and xenobiotic metabolism reactions, including some genes of great interest for fatty acid hydroxylation and aromatic compound degradation. These findings established a basis for future studies about the role of P450s in the carotenogenic yeast X. dendrorhous and their potential biotechnological applications.

Engineering of the carotenoid pathway in Xanthophyllomyces dendrorhous leading to the synthesis of zeaxanthin.

Zeaxanthin is an essential nutrient for prevention of macular degeneration. However, it is limited in our diet. For the production of zeaxanthin, we have engineered zeaxanthin synthesis into a carotenoid mutant of Xanthophyllomyces dendrorhous which is blocked in astaxanthin synthesis and accumulates ß-carotene instead. Two strategies were followed to reach high-yield zeaxanthin synthesis. Total carotenoid synthesis was increased by over-expression of genes HMGR, crtE, and crtYB encoding for limiting enzymes in the pathway leading to and into carotenoid biosynthesis. Then bacterial genes crtZ were used to extend the pathway from ß-carotene to zeaxanthin in this mutant. The increase of total carotenoids and the formation of zeaxanthin is dependent on the number of gene copies of crtYB and crtZ integrated into the X. dendrorhous upon transformation. The highest zeaxanthin content around 500 µg/g dw was reached by shaking flask cultures after codon optimization of crtZ for Xanthophyllomyces. Stabilization of carotenoid and zeaxanthin formation in the final transformant in the absence of selection agents was achieved after passing through a sexual cycle and germination of basidiospores. The values for the transformant before and after stabilization were very similar resembling about 70 % of total carotenoids and corresponding to a conversion rate of 80 % for hydroxylation of ß-carotene to zeaxanthin. The stabilized transformant allowed experimental small-scale fermentation yielding X. dendrorhous cells with a zeaxanthin content similar to the shaking flask cultures. Our result demonstrates the potential of X. dendrorhous for its development as a zeaxanthin producer and its suitability for large-scale fermentation.

Comparative genomics provides new insights into the diversity, physiology, and sexuality of the only industrially exploited tremellomycete: Phaffia rhodozyma.

BACKGROUND: The class Tremellomycete (Agaricomycotina) encompasses more than 380 fungi. Although there are a few edible Tremella spp., the only species with current biotechnological use is the astaxanthin-producing yeast Phaffia rhodozyma (Cystofilobasidiales). Besides astaxanthin, a carotenoid pigment with potent antioxidant activity and great value for aquaculture and pharmaceutical industries, P. rhodozyma possesses multiple exceptional traits of fundamental and applied interest. The aim of this study was to obtain, and analyze two new genome sequences of representative strains from the northern (CBS 7918T, the type strain) and southern hemispheres (CRUB 1149) and compre them to a previously published genome sequence (strain CBS 6938). Photoprotection and antioxidant related genes, as well as genes involved in sexual reproduction were analyzed. RESULTS: Both genomes had ca. 19 Mb and 6000 protein coding genes, similar to CBS 6938. Compared to other fungal genomes P. rhodozyma strains and other Cystofilobasidiales have the highest number of intron-containing genes and highest number of introns per gene. The Patagonian strain showed 4.4 % of nucleotide sequence divergence compared to the European strains which differed from each other by only 0.073 %. All known genes related to the synthesis of astaxanthin were annotated. A hitherto unknown gene cluster potentially responsible for photoprotection (mycosporines) was found in the newly sequenced P. rhodozyma strains but was absent in the non-mycosporinogenic strain CBS 6938. A broad battery of enzymes that act as scavengers of free radical oxygen species were detected, including catalases and superoxide dismutases (SODs). Additionally, genes involved in sexual reproduction were found and annotated. CONCLUSIONS: A draft genome sequence of the type strain of P. rhodozyma is now available, and comparison with that of the Patagonian population suggests the latter deserves to be assigned to a distinct variety. An unexpected genetic trait regarding high occurrence of introns in P. rhodozyma and other Cystofilobasidiales was revealed. New genomic insights into fungal homothallism were also provided. The genetic basis of several additional photoprotective and antioxidant strategies were described, indicating that P. rhodozyma is one of the fungi most well-equipped to cope with environmental oxidative stress, a factor that has probably contributed to shaping its genome.

Enhancement of astaxanthin production in Xanthophyllomyces dendrorhous by efficient method for the complete deletion of genes.

BACKGROUND: Red yeast, Xanthophyllomyces dendrorhous is the only yeast known to produce astaxanthin, an anti-oxidant isoprenoid (carotenoid) widely used in the aquaculture, food, pharmaceutical and cosmetic industries. The potential of this microorganism as a platform cell factory for isoprenoid production has been recognized because of high flux through its native terpene pathway. Recently, we developed a multiple gene expression system in X. dendrorhous and enhanced the mevalonate synthetic pathway to increase astaxanthin production. In contrast, the mevalonate synthetic pathway is suppressed by ergosterol through feedback inhibition. Therefore, releasing the mevalonate synthetic pathway from this inhibition through the deletion of genes involved in ergosterol synthesis is a promising strategy to improve isoprenoid production. An efficient method for deleting diploid genes in X. dendrorhous, however, has not yet been developed. RESULTS: Xanthophyllomyces dendrorhous was cultivated under gradually increasing concentrations of antibiotics following the introduction of antibiotic resistant genes to be replaced with target genes. Using this method, double CYP61 genes encoding C-22 sterol desaturases relating to ergosterol biosynthesis were deleted sequentially. This double CYP61 deleted strain showed decreased ergosterol biosynthesis compared with the parental strain and single CYP61 disrupted strain. Additionally, this double deletion of CYP61 genes showed increased astaxanthin production compared with the parental strain and the single CYP61 knockout strain. Finally, astaxanthin production was enhanced by 1.4-fold compared with the parental strain, although astaxanthin production was not affected in the single CYP61 knockout strain. CONCLUSIONS: In this study, we developed a system to completely delete target diploid genes in X. dendrorhous. Using this method, we deleted diploid CYP61 genes involved in the synthesis of ergosterol that inhibits the pathway for mevalonate, which is a common substrate for isoprenoid biosynthesis. The resulting decrease in ergosterol biosynthesis increased astaxanthin production. The efficient method for deleting diploid genes developed in this study has the potential to improve industrial production of various isoprenoids in X. dendrorhous.

Regulation of carotenogenesis in the red yeast Xanthophyllomyces dendrorhous: the role of the transcriptional co-repressor complex Cyc8-Tup1 involved in catabolic repression.

BACKGROUND: The yeast Xanthophyllomyces dendrorhous produces carotenoids of commercial interest, including astaxanthin and ß-carotene. Although carotenogenesis in this yeast and the expression profiles of the genes controlling this pathway are known, the mechanisms regulating this process remain poorly understood. Several studies have demonstrated that glucose represses carotenogenesis in X. dendrorhous, suggesting that this pathway could be regulated by catabolic repression. Catabolic repression is a highly conserved regulatory mechanism in eukaryotes and has been widely studied in Saccharomyces cerevisiae. Glucose-dependent repression is mainly observed at the transcriptional level and depends on the DNA-binding regulator Mig1, which recruits the co-repressor complex Cyc8-Tup1, which then represses the expression of target genes. In this work, we studied the regulation of carotenogenesis by catabolic repression in X. dendrorhous, focusing on the role of the co-repressor complex Cyc8-Tup1. RESULTS: The X. dendrorhous CYC8 and TUP1 genes were identified, and their functions were demonstrated by heterologous complementation in S. cerevisiae. In addition, cyc8 - and tup1 - mutant strains of X. dendrorhous were obtained, and both mutations were shown to prevent the glucose-dependent repression of carotenogenesis in X. dendrorhous, increasing the carotenoid production in both mutant strains. Furthermore, the effects of glucose on the transcript levels of genes involved in carotenogenesis differed between the mutant strains and wild-type X. dendrorhous, particularly for genes involved in the synthesis of carotenoid precursors, such as HMGR, idi and FPS. Additionally, transcriptomic analyses showed that cyc8 - and tup1 - mutations affected the expression of over 250 genes in X. dendrorhous. CONCLUSIONS: The CYC8 and TUP1 genes are functional in X. dendrorhous, and their gene products are involved in catabolic repression and carotenogenesis regulation. This study presents the first report involving the participation of Cyc8 and Tup1 in carotenogenesis regulation in yeast.

Molecular characterization and heterologous expression of a Xanthophyllomyces dendrorhous α-glucosidase with potential for prebiotics production.

Basidiomycetous yeast Xanthophyllomyces dendrorhous expresses an α-glucosidase with strong transglycosylation activity producing prebiotic sugars such as panose and an unusual tetrasaccharides mixture including α-(1-6) bonds as major products, which makes it of biotechnological interest. Initial analysis pointed to a homodimeric protein of 60 kDa subunit as responsible for this activity. In this study, the gene Xd-AlphaGlu was characterized. The 4131-bp-long gene is interrupted by 13 short introns and encodes a protein of 990 amino acids (Xd-AlphaGlu). The N-terminal sequence of the previously detected 60 kDa protein resides in this larger protein at residues 583-602. Functionality of the gene was proved in Saccharomyces cerevisiae, which produced a protein of about 130 kDa containing Xd-AlphaGlu sequences. All properties of the heterologously expressed protein, including thermal and pH profiles, activity on different substrates, and ability to produce prebiotic sugars were similar to that of the α-glucosidase produced in X. dendrorhous. No activity was detected in S. cerevisiae containing exclusively the 1256-bp from gene Xd-AlphaGlu that would encode synthesis of the 60 kDa protein previously detected. Data were compatible with an active monomeric α-glucosidase of 990 amino acids and an inactive hydrolysis product of 60 kDa. Protein Xd-AlphaGlu contained most of the elements characteristic of α-glucosidases included in the glycoside hydrolases family GH31 and its structural model based on the homologous human maltase-glucoamylase was obtained. Remarkably, the Xd-AlphaGlu C-terminal domain presents an unusually long 115-residue insertion that could be involved in this enzyme's activity against long-size substrates such as maltoheptaose and soluble starch.

Molecular cloning and characterization of the ATP citrate lyase from carotenogenic yeast Phaffia rhodozyma.

ATP citrate lyase (ACL), is a key cytosolic source of acetyl-CoA for fatty acid and sterol biosynthesis and appear to be involved in carotenoid biosynthesis in yeasts. Three homologous DNA sequences encoding ACLs in Phaffia rhodozyma were isolated i.e two genes and one cDNA. The two genes were multi-intronic, with 3450-bp-coding sequences and both genes, as the cDNA, encoded identical 120.1-kDa polypeptides. Full-length amino acid sequences of these ACLs showed the two multidomains, PLN02235 and PLN02522, which are necessary for activity. The ACLs showed 82-87% similarity to putative ACLs from other basidiomycetes and 71% similarity to human ACL. The acl cDNA was used to express the heterologous ACL 6XHis-tagged which was identified using MALDI-TOF-MS. The sequenced peptides with 42.2% coverage showed 100% identity to the amino acid sequence generated in silico. The recombinant ACL purified to homogeneity showed an activity of 2 U. This is the first study to characterize a recombinant ACL from a carotenogenic yeast. The present study provides a key foundation for future studies to assess (a) the possible occurrence of alternative splicing, (b) identify the promoter(s) sequence(s) and (c) the involvement of ACL in the differential regulation of fatty acid and carotenoid biosynthesis in yeasts.

Carotenoid production and gene expression in an astaxanthin-overproducing Xanthophyllomyces dendrorhous mutant strain.

The primary carotenoid synthesized by Xanthophyllomyces dendrorhous is astaxanthin, which is used as a feed additive in aquaculture. Cell growth kinetics and carotenoid production were correlated with the mRNA levels of the idi, crtE, crtYB, crtI, crtS and crtR genes, and the changes in gene sequence between the wild-type and a carotenoid overproducer XR4 mutant strain were identified. At the late stationary phase, the total carotenoid content in XR4 was fivefold higher than that of the wild-type strain. Additionally, the mRNA levels of crtE and crtS increased during the XR4 growth and were three times higher than the wild-type strain in the late stationary phase. Moreover, the nucleotide sequences of crtYB, crtI and crtR exhibited differences between the strains. Both the higher crtE and crtS transcript levels and the crtYB, crtI and crtR mutations can, at least in part, act to up-regulate the carotenoid biosynthesis pathway in the XR4 strain.

Advances in Metabolic Engineering for the Accumulation of Astaxanthin Biosynthesis.

Astaxanthin, a lipophilic carotenoid renowned for its strong antioxidant activity, holds significant commercial value across industries such as feed, food, and cosmetics. Although astaxanthin can be synthesized through chemical methods, it may contain toxic by-products in the synthesized astaxanthin, limiting its application in medicine or functional food. Natural astaxanthin can be extracted from algae, however, the cultivation cycle of algae is relatively longer compared to microorganisms. With the advancement of synthetic biology and metabolic engineering, the method of microbial fermentation has emerged as a promising strategy for the large-scale production of astaxanthin. This article provides a comprehensive overview of the research progress in astaxanthin biosynthesis, highlighting the use of the natural host Xanthophyllomyces dendrorhous, and the heterologous hosts Yarrowia lipolytica and Saccharomyces cerevisiae. Additionally, future research prospects are also discussed.

Favored isolation and rapid identification of the astaxanthin-producing yeast Xanthophyllomyces dendrorhous(Phaffia rhodozyma) from environmental samples.

Xanthophyllomyces dendrorhous (Phaffia rhodozyma) yeasts are biotechnologically exploited as a natural source of astaxanthin for aquaculture. Based on results of recent studies, it has become clear that this species possesses a greater genetic variability generating the necessity to uncover it and assess its potential for the astaxanthin industry. However, difficulties for the isolation of the X. dendrorhous hinder extensive environmental surveys which need to be carried out to better understand the habitat, distribution and genetic diversity of this species. We extensively searched for distinctive physiological traits of X. dendrorhours by testing phenotypic properties simultaneously with a panel of common sympatric fungi. As a result we obtained a new and innovative strategy for improving X. dendrorhous recovery rate and identification from environmental samples. This strategy involved the use of trehalose-based media, and a rapid X. dendrorhous identification method based on the simultaneous spectrophotometric detection of astaxanthin and UV-absorbing compounds (mycosporines). The proposed procedures proved effective in field trials conducted in natural environments of Patagonia (Argentina) and thus represent an important tool for the discovery of new astaxanthin-producing strains of X. dendrorhous useful for the aquaculture industry.

Metabolic mechanism of astaxanthin biosynthesis in Xanthophyllomyces dendrorhous in response to sodium citrate treatment.

Astaxanthin is an important ketocarotenoid widely used in industries. However, its application is limited because of its low yield. Sodium citrate (Na-citrate), one of the major carbon sources for microorganisms, can promote cell growth and product accumulation. The basidiomycetous red yeast Xanthophyllomyces dendrorhous was thus used to study the effect of Na-citrate on cell growth and astaxanthin synthesis. The highest biomass and astaxanthin yield (6.0 g/L and 22.5 mg/L) were obtained in shake-flask when 3 g/L Na-citrate was added at 24 h and were 1.8 and 2.0 times higher than those of the control group, respectively. Furthermore, metabolomics and real-time reverse transcription PCR (qRT-PCR) analysis were conducted to study the metabolic pathways of X. dendrorhous in response to Na-citrate. The qRT-PCR assay revealed that Na-citrate facilitated glucose consumption, promoted the metabolic flux from glycolysis, and regulated the tricarboxylic acid (TCA) cycle, providing more energy and substrates for the synthesis of astaxanthin. The gene analysis revealed that adding Na-citrate significantly upregulated the expression of six key genes (ICL, HMGS, crtE, crtYB, crtI, and crtS) involved in pathways related to astaxanthin biosynthesis. These results suggest that exogenous Na-citrate treatment is a potentially valuable strategy to stimulate astaxanthin production in X. dendrorhous.

Astaxanthin Synthesis by Yeast Xanthophyllomyces dendrorhous and its Mutants on Media Based on Plant Extracts.

The study evaluated the effect of media based on plant extracts: potato, carrot and barley malt broth, on growth and astaxanthin synthesis by yeast Xanthophyllomyces dendrorhous DSM 5626 and its mutants. The carrot medium promoted carotenogenesis most effectively. In cultures on this medium the highest volumetric and cellular concentrations of astaxanthin were recorded for four out of five tested strains. Also the share of astaxanthin in the total carotenoids produced by the tested strains was the highest.