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.

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.

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.

Towards commercial levels of astaxanthin production in Phaffia rhodozyma.

Astaxanthin (AX) is a potent antioxidant with increasing biotechnological and commercial potential as a feed supplement, and gives salmonids and crustaceans their attractive characteristic pink color. The red yeast Phaffia rhodozyma naturally produces AX as its main fermentation product but wild-type strains and those previously generated through classical random mutagenesis produce low yields of AX. Existing strains do not meet commercial economic requirements, fundamentally due to a lack of understanding of the underlying mechanisms and genotype-phenotype associations regarding AX production in P. rhodozyma. In the present study, screening of P. rhodozyma CBS 6938 mutant strains generated through chemical and ultra violet radiation mutagenesis delivered increased AX production yields that were then maximized using culture media optimization and fed-batch culture kinetic modeling. The whole genomes of the wild-type and eight increased production strains were sequenced to identify genomic changes. The selected strains produced 50-fold more AX than the wild-type strain with a total biomass of around 100 gDCW/L and a carotenoid production of 1 g/L. Genomic variant analyses found 368 conserved mutations across the selected strains with important mutations found in protein coding regions associated with regulators and catalysts of AX precursors in the mevalonate pathway, the electron transport chain, oxidative stress mechanisms, and carotenogenesis.

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.

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.

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, 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.

Biological effect of astaxanthin on alcohol-induced gut damage in Carassius auratus used as experimental model.

Alcohol and its metabolites are responsible for damage both within the gastrointestinal tract and other organs. Alcohol abuse promote intestinal inflammation, that may be the cause of multiple organ dysfunctions and chronic disorders. In this research, the effect of astaxanthin, a powerful antioxidant with several biological effects, on alcohol damage-induced in the intestine of Carassius auratus, was investigated. In the fishes exposed to ethanol, an increase of the intestinal epithelium mucous cells and circulating macrophages, with intestinal mucosa disorganization was observed. In contrast, in the fishes treated with astaxanthin intestinal morphology was restored. By immunohistochemical analysis, using α-Smooth Muscle Actin (α-SMA) and Vasoactive intestinal polypeptide (VIP) antibodies, a reduction of inflammatory states alcohol-induced was evident, with more regular muscularis submucosa and more organized intestinal mucosa without inflammatory cells. The results suggest that astaxanthin treatments can be a good candidate for preventing damage within the gastrointestinal associated with excessive alcohol consumption.

Enzymatic Processes Triggered by PEF for Astaxanthin Extraction From Xanthophyllomyces dendrorhous.

The aim of this study was to evaluate the potential of pulsed electric fields (PEF) to improve the extraction of the lipid-soluble astaxanthin from fresh biomass of a wild-type (CECT 11028) and mutant (ATCC 74219) Xanthophyllomyces dendrorhous strain using ethanol as solvent. Inactivation and propidium uptake studies revealed that inactivation is a good index for estimated the proportion of irreversible permeabilized cells when inactivation is higher than 70% in the two strains. Ethanol was ineffective for extracting carotenoids from the PEF-treated cells (20 kV/cm, 135 µs) of the two strains. However, after aqueous incubation of PEF-treated X. dendrorhous ATCC 74219 cells for 12 h, up to 2.4 ± 0.05 mg/g dried weight (d.w.) of carotenoids were extracted in ethanol. From total carotenoid extracted, around 84% corresponded to all-trans astaxanthin. The detection and quantification of esterase activity in the supernatant and the relationship between the percentage of esterase activity quantified and the amount of carotenoids extracted indicate that the extraction of astaxanthin was mediated by enzymatic esterase activity triggered by PEF during incubation. On the other hand, the formation of a large lipid globule into the cytoplasm of PEF-treated X. dendrorhous CECT 11028 cells during aqueous incubation prevented carotenoid extraction. The process developed in this investigation represents a more sustainable and greener method that those previously used for extracting astaxanthin from yeast.

Phenotypic Analysis of Mutants of Ergosterol Biosynthesis Genes (ERG3 and ERG4) in the Red Yeast Xanthophyllomyces dendrorhous.

Xanthophyllomyces dendrorhous synthesizes astaxanthin, a carotenoid used in aquaculture. Astaxanthin is synthesized from metabolites of the mevalonate pathway, which are also precursors for sterols biosynthesis. The interruption of the CYP61 gene, which is involved in the synthesis of ergosterol (mutant CBS.cyp61 -), resulted in a phenotype that overproduces carotenoids due to the activation of the SREBP pathway. In this work, we constructed other mutants of ergosterol biosynthesis in this yeast to evaluate whether they have the same phenotype as mutant CBS.cyp61 -. By bioinformatic analysis, the ERG3 and ERG4 genes of X. dendrorhous were identified, and each gene was deleted in the wild-type strain. Mutants CBS.Δerg3 and CBS.Δerg4 did not produce ergosterol; CBS.Δerg3 primarily accumulated episterol, and CBS.Δerg4 primarily accumulated ergosta-5,7,22,24(28)-tetraenol. The transcription levels of the HMGS gene of the mevalonate pathway were evaluated by RT-qPCR, which showed a slight increase in CBS.Δerg4, but the transcription levels were still 10-fold lower than in strain CBS.cyp61 -. Both CBS.Δerg3 and CBS.Δerg4 did not overproduce carotenoids, even though they do not produce ergosterol. Thus, the results of this study indicate that the absence of ergosterol does not activate the SREBP pathway in X. dendrorhous, but rather it depends on other alterations in sterol composition.

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.

Strategy and regulatory mechanisms of glutamate feeding to enhance astaxanthin yield in Xanthophyllomyces dendrorhous.

Xanthophyllomyces dendrorhous is an excellent industrial source for production of natural astaxanthin, but the yield of astaxanthin is relative low due to the contradiction between biomass weight and astaxanthin accumulation. Glutamate, a metabolite connecting nitrogen and carbon metabolisms, is probably a promising entry point to interfere cellular metabolisms. Thus, the effect of glutamate on cell growth and astaxanthin accumulation in X. dendrorhous was investigated. Results showed that glutamate feeding facilitated glucose consumption and further led to the increment of astaxanthin content with little influence of cell growth. A comparative proteomics study was applied to decipher the regulatory mechanisms of enhanced astaxanthin biosynthesis in X. dendrorhous as a response to the glutamate feeding. The expressions of proteins with the highest degree of fold change were involved in carbohydrate, amino acids, and carotenogenesis metabolisms as well as redox and stress-associated metabolisms. In addition, a possible regulatory model of enhanced astaxanthin accumulation in response to glutamate feeding in X. dendrorhous is also proposed.

Genetic modification of the carotenoid pathway in the red yeast Xanthophyllomyces dendrorhous: Engineering of a high-yield zeaxanthin strain.

The red yeast Xanthophyllomyces dendrorhous was genetically engineered for high-yield accumulation of the carotenoid zeaxanthin. Initially, an astaxanthin hyper-producing mutant was used to generate a ß-carotene synthesizing transformant by inactivation of the astaxanthin synthase gene. Subsequently, a bacterial ß-carotene hydroxylase gene was genome integrated to establish ß-carotene to zeaxanthin conversion. Crucial for efficient zeaxanthin formation was the rate of this hydroxylation which was related to the number of integrated gene copies. Two strategies were followed to get multiple integrations, either random integration into the ribosomal DNA which resulted in a maximum copy number of 10, or directly integration of a total of 8 copies into both alleles of the astaxanthin synthase gene. Combining both procedures with additional insertion of the gene to enhance expression of the carotenogenesis limiting phytoene synthase, a transformant reaching a high level of zeaxanthin of 5.2 mg/g dw was finally generated. The application of pentose sugars including xylose as substrates for X. dendrorhous which avoids the inhibitory Crab-tree effect of glucose is favorable for carotenogenesis allowing the replacement of glucose by a hydrolysate of the waste product hemicellulose which is rich in xylose demonstrating ithe effectiveness as a sustainable and cost-efficient alternative for high-yield zeaxanthin formation.

Extraction and Analysis of Carotenes and Xanthophylls Produced by Xanthophyllomyces dendrorhous.

Xanthophyllomyces dendrorhous, a heterobasidiomycetous yeast that represents the teleomorphic state of Phaffia rhodozyma, is used as a natural source of several carotenoids, such as the xanthophyll astaxanthin. Here, we describe the culture procedure for the production of carotenoids in X. dendrorhous and a simple and rapid analytical method for the optimized extraction and HPLC determination of intracellular ß-carotene, astaxanthin, canthaxanthin, and zeaxanthin.

Isolation and Characterization of Extrachromosomal Double-Stranded RNA Elements from Carotenogenic Yeasts.

Double-stranded RNA (dsRNA) molecules are widely found in yeasts and filamentous fungi. It has been suggested that these molecules may play an important role in the evolution of eukaryote genomes and could be a valuable tool in yeast typing. The characterization of these extrachromosomal genetic elements is usually a laborious process, especially when trying to analyze a large number of samples. In this chapter, we describe a simple method to isolate dsRNA elements from yeasts using low amounts of starting material and their application to different Xanthophyllomyces dendrorhous strains and other psychrotolerant carotenogenic yeasts. Furthermore, the methodologies for enzymatic and hybridization characterizations and quantification of relative dsRNA abundance are detailed.

The inhibition of Caco-2 proliferation by astaxanthin from Xanthophyllomyces dendrorhous.

PURPOSE: To investigate the efficiency of natural astaxanthin that has been extracted from Xanthophyllomyces dendrorhous in inhibiting the proliferation and viability of colorectal adenocarcinoma cell line (Caco-2; colon cancer cells). METHODOLOGY: Caco-2 cells and normal human oralkeratinocytes (NOKs) were treated with different concentrations of extracted astaxanthin, ranging from 0.075 to 10 mg ml-1, for 24, 48 and 72 h. The number of cells was determined via MTS assay and the proliferating cells were investigated by bromodeoxyuridine (BrdU) assay.Results/Key findings. Of the Caco-2 cells, 30-50 % remained viable, while the NOKs showed 110-120 % survival when treated with 5 mg ml-1 astaxanthin. The Caco-2 cells showed distinct structural shrinkage when treated with the same concentration of astaxanthin. Fluorescent labelling of the DNA of the proliferative cells with BrdU showed a significant decrease in the number of the proliferative Caco-2 cells when the concentration of astaxanthin was increased to 5 mg ml-1. CONCLUSION: The natural astaxanthin from X. dendrorhous, at an appropriate concentration, is effective in terminating the viability of, or retarding the proliferative activity of, Caco-2 cells, without harmful effects on NOKs.

Extending our tools and resources in the non-conventional industrial yeast Xanthophyllomyces dendrorhous through the application of metabolite profiling methodologies.

INTRODUCTION: Xanthophyllomyces dendrorhous is a non-conventional industrial yeast. It has the unique ability among yeasts to produce geranylgeranyl pyrophosphate derived terpenoids such as carotenoids and in particular the high value pigment astaxanthin. OBJECTIVE: In order to fully exploit the industrial potential of Xanthophyllomyces using modern industrial biotechnology approaches the further development of "omic" resources in this organism are required to build on the now sequenced and annotated genome. To contribute to this goal, the present study has developed and implemented an efficient metabolite profiling system comprised of, quenching, extraction and associated GC-MS and UPLC analysis. METHOD: Four quenching methods and five extraction methods compatible with GC-MS and UPLC profiling were tested and validated by analysing steady state metabolite changes of Xanthophyllomyces cultivated at laboratory scale in liquid shake culture at lag, exponential and early and late stationary phases. RESULTS: A customised Automated Mass Spectral Deconvolution and Identification System (AMDIS) library has been created for Xanthophyllomyces, over 400 compounds are present in the library of which 78 are detected and quantified routinely in polar and non-polar derived extracts. A preliminary biochemical network has been constructed. Over a standardised laboratory growth cycle, changes in metabolite levels have been determined to create reference point for future strain improvement approaches and the initial biochemical network construction. Correlation analysis has illustrated that astaxanthin formation correlates positively with different sectors of intermediary metabolism (e.g. the TCA cycle intermediates and amino acid formation), "short" saturated fatty acids and ß-carotene, while other metabolites are reduced in response to astaxanthin production. These sectors of intermediary metabolism offer potential future targets for the manipulation resulting in the generation of strains with improved titres of given terpenoids. DISCUSSION: In summary a robust metabolite profiling system for Xanthophyllomyces is in place to further our understanding and potential exploitation of this underutilised industrial yeast.

Production of astaxanthin at moderate temperature in Xanthophyllomyces dendrorhous using a two-step process.

In order to achieve continuous industrial production of astaxanthin in Xanthophyllomyces dendrorhous, a moderate temperature (25-37°C) fermentation process was needed. In this study, a two-step process with a 20°C pre-culture for 18 h and a 30°C culture for 30 h was performed to achieve the astaxanthin yields of 116.42 µg g-1 dry cell weight, which was lower than that in the normal process (20°C, 96 h). However, cell yield (YX/S) and product yield (YP/S) showed no significant differences between the two processes, suggesting that moderate temperature did not affect the productivity of astaxanthin. The transcriptional levels of genes involved in astaxanthin synthesis were compared in different culture times and a negative correlation between temperature and expression of carotenogenic genes was found. This work provided a potential method for continuous production of astaxanthin using X. dendrorhous at moderate temperature throughout the year.