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.     

Increased carotenoid production in Xanthophyllomyces dendrorhous G276 using plant extracts

The red yeast Xanthophyllomyces dendrorhous (previously named Phaffia rhodozyma) produces astaxanthin pigment among many carotenoids. The mutant X. dendrorhous G276 was isolated by chemical mutagenesis. The mutant produced about 2.0 mg of carotenoid per g of yeast cell dry weight and 8.0 mg/L of carotenoid after 5 days batch culture with YM media; in comparison, the parent strain produced 0.66 mg/g of yeast cell dry weight and a carotenoid concentration of 4.5 mg/L. We characterized the utilization of carbon sources by the mutant strain and screened various edible plant extracts to enhance the carotenoid production. The addition of Perilla frutescens (final concentration, 5%) or Allium fistulosum extracts (final concentration, 1%) enhanced the pigment production to about 32 mg/L. In a batch fermentor, addition of Perilla frutescens extract reduced the cultivation time by two days compared to control (no extract), which usually required five-day incubation to fully produce astaxanthin. The results suggest that plant extracts such as Perilla frutescens can effectively enhance astaxanthin production.     

Biogeography, host specificity, and molecular phylogeny of the basidiomycetous yeast Phaffia rhodozyma and its sexual form, Xanthophyllomyces dendrorhous

Phaffia rhodozyma (sexual form, Xanthophyllomyces dendrorhous) is a basidiomycetous yeast that has been found in tree exudates in the Northern Hemisphere at high altitudes and latitudes. This yeast produces astaxanthin, a carotenoid pigment with biotechnological importance because it is used in aquaculture for fish pigmentation. We isolated X. dendrorhous from the Southern Hemisphere (Patagonia, Argentina), where it was associated with fruiting bodies of Cyttaria hariotii, an ascomycetous parasite of Nothofagus trees. We compared internal transcribed spacer (ITS)-based phylogenies of P. rhodozyma and its tree host (Betulaceae, Corneaceae, Fagaceae, and Nothofagaceae) and found them to be generally concordant, suggesting that different yeast lineages colonize different trees and providing an explanation for the phylogenetic distance observed between the type strains of P. rhodozyma and X. dendrorhous. We hypothesize that the association of Xanthophyllomyces with Cyttaria derives from a previous association of the yeast with Nothofagus, and the sister relationship between Nothofagaceae and Betulaceae plus Fagaceae correlates with the phylogeny of X. dendrorhous strains originating from these three plant families. The two most basal strains of X. dendrorhous are those isolated from Cornus, an ancestral genus in the phylogenetic analysis of the host trees. Thus, we question previous conclusions that P. rhodozyma and X. dendrorhous represent different species since the polymorphisms detected in the ITS and intergenic spacer sequences can be attributed to intraspecific variation associated with host specificity. Our study provides a deeper understanding of Phaffia biogeography, ecology, and molecular phylogeny. Such knowledge is essential for the comprehension of many aspects of the biology of this organism and will facilitate the study of astaxanthin production within an evolutionary and ecological framework.     

基于动力学模型的法夫酵母发酵生产虾青素的补料策略优化

对法夫酵母的不同补料发酵方式进行了研究.基于底物抑制模型,提出了一种优化的两阶段补料策略,用于法夫酵母产虾青素的高密度发酵.在发酵的延迟期和对数生长期早期,糖浓度控制在25 g/L左右,在此条件下,生物量可以达到最大,且时间缩短.在对数生长期后期及稳定期,糖浓度控制在5 g/L,虾青素的合成时间可以有效延长.与传统的补料方式相比,采用此补料策略取得了较好的发酵效果.发酵终点细胞干重达到23.8g/L,虾青素产量达到29.05 mg/L,分别比分批发酵提高了52.8%和109%.     

Heterologous expression of pentalenene synthase (PSS) from Streptomyces UC5319 in Xanthophyllomyces dendrorhous

For the first time, the pentalenene synthase (PSS) gene from Streptomyces UC5319 was expressed in Xanthophyllomyces dendrorhous, a native producer of astaxanthin. For the expression of the gene and the concurrent knock out of the native crtE or crtYB genes, two new vectors were engineered and used for the transformation of the wild-type strain of X. dendrorhous. The transformations resulted in white colonies, showing a complete shutdown of the carotenoid production. Furthermore, an additional vector was constructed for the insertion of the PSS gene in the rDNA of the yeast. All the mutant strains produce the sesquiterpene pentalenene and show no difference in growth when compared to the wild-type strain. In this report, we demonstrate that X. dendrorhous is a suitable host for the expression of heterologous terpene cyclases and for the production of foreign terpene compounds.     

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.     

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

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

High-level production of astaxanthin by Xanthophyllomyces dendrorhous mutant JH1 using statistical experimental designs

Medium composition was optimized for high-level production of astaxanthin by Xanthophyllomyces dendrorhous mutant JH1 using statistical experimental designs. Glucose and yeast extract were the most important factors affecting astaxanthin production. Glucose 3.89%, yeast extract 0.29%, KH2PO4 0.25%, MgSO4 0.05%, MnSO4 0.02%, and CaCl2 0.01% were optimum for high-level production of astaxanthin. Under optimized conditions, the maximum concentration of astaxanthin obtained after 7 d of cultivation was 36.06 mg/l. The concentration of astaxanthin predicted by a polynomial model was 36.16 mg/l.     

Effect of polar and non-polar carotenoids on Xanthophylomyces dendrorhous membranes by EPR

The red yeast Xanthophyllomyces dendrorhous is one of the microbiological production systems for natural carotenoids. High-performance liquid chromatography (HPLC) and electron paramagnetic resonance spectroscopy (EPR) experiments were performed on X. dendrorhous membranes in order to study the effect of incorporation rates of different type of carotenoids. In the case of fluid-phase membranes, it was found that polar carotenoids, such as astaxanthin and cis-astaxanthin, increased the EPR order parameter and decreased the motional freedom and phase-transition temperature. In contrast the non-polar carotenoids beta-cryptoxanthin and beta-carotene decreased the EPR order parameter and increased motional freedom and phase-transition temperature. A noteworthy coherence was observed between the polarities of the strains and the phase-transition temperatures.     

Engineering of geranylgeranyl pyrophosphate synthase levels and physiological conditions for enhanced carotenoid and astaxanthin synthesis in <Emphasis Type="Italic">Xanthophyllomyces dendrorhous</Emphasis>

The basidiomycetous yeast, Xanthophyllomyces dendrorhous, is one of the very few organisms which can be used for biological production of the carotenoid astaxanthin. crtE cDNA has been cloned from this fungus for engineering of the terpenoid pathway. The function of its gene product as a geranylgeranyl pyrophosphate synthase was established. X. dendrorhous was transformed with the crtE cDNA to divert metabolite flow from the sterol pathway towards carotenoid biosynthesis. Transformants were obtained with increased levels of geranylgeranyl pyrophosphate synthase leading to higher carotenoid levels including astaxanthin. Physiological conditions for maximum carotenoid synthesis for wild type and the CrtE transformant were dim light and extra air supply of the shaking culture. These conditions and the transformation with crtE had additive effects and resulted in an 8-fold higher astaxanthin formation as compared to the initial wild type culture without illumination and extra air supply yielding 451 μg/g dry wt within 4 days of growth.     

The crtS gene of Xanthophyllomyces dendrorhous encodes a novel cytochrome-P450 hydroxylase involved in the conversion of beta-carotene into astaxanthin and other xanthophylls

The conversion of beta-carotene into xanthophylls is a subject of great scientific and industrial interest. We cloned the crtS gene involved in astaxanthin biosynthesis from two astaxanthin producing strains of Xanthophyllomyces dendrorhous: VKPM Y2410, an astaxanthin overproducing strain, and the wild type ATCC 24203. In both cases, the ORF has a length of 3166 bp, including 17 introns, and codes for a protein of 62.6 kDa with similarity to cytochrome-P450 hydroxylases. crtS gene sequences from strains VKPM Y2410, ATCC 24203, ATCC 96594, and ATCC 96815 show several nucleotide changes, but none of them causes any amino acid substitution, except a G2268 insertion in the 13th exon of ATCC 96815 which causes a change in the reading frame. A G1470 --> A change in the 5' splicing region of intron 8 was also found in ATCC 96815. Both point mutations explain astaxanthin idiotrophy and beta-carotene accumulation in ATCC 96815. Mutants accumulating precursors of the astaxanthin biosynthetic pathway were selected from the parental strain VKPM Y2410 (red) showing different colors depending on the compound accumulated. Two of them were blocked in the biosynthesis of astaxanthin, M6 (orange; 1% astaxanthin, 71 times more beta-carotene) and M7 (orange; 1% astaxanthin, 58 times more beta-carotene, 135% canthaxanthin), whereas the rest produced lower levels of astaxanthin (5-66%) than the parental strain. When the crtS gene was expressed in M7, canthaxanthin accumulation disappeared and astaxanthin production was partially restored. Moreover, astaxanthin biosynthesis was restored when X. dendrorhous ATCC 96815 was transformed with the crtS gene. The crtS gene was heterologously expressed in Mucor circinelloides conferring to this fungus an improved capacity to synthesize beta-cryptoxanthin and zeaxanthin, two hydroxylated compounds from beta-carotene. These results show that the crtS gene is involved in the conversion of beta-carotene into xanthophylls, being potentially useful to engineer carotenoid pathways.     

Citrus residues isolates improve astaxanthin production by Xanthophyllomyces dendrorhous

The wild strain and two astaxanthin-overproducing mutant strains, W618 and GNG274, of Xanthophyllomyces dendrorhous were analyzed in order to assess their ability to grow and synthesize astaxanthin in a minimal medium containing (per liter): 2 g KH2PO4, 0.5 g MgSO4, 2 g KNO3, and 1 g yeast extract, and supplemented with citrus residues isolates as a carbon source (citrus medium). The selected strain W618 was evaluated under various contents of citrus juice. At the content of 20% (v/v), the highest astaxanthin production reached 22.63 mg L(-1), which was two-fold more than that observed in yeast malt medium. Addition of 8% (v/v) n-hexadecane to the citrus medium was found to be optimal, increasing the astaxanthin yield by 21.7%.     

Perfusion culture process plus H2O2 stimulation for efficient astaxanthin production by Xanthophyllomyces dendrorhous

A semicontinuous perfusion culture process (repeated medium renewal with cell retention) was evaluated together with batch and repeated fed-batch processes for astaxanthin production in shake-flask cultures of Xanthophyllomyces dendrorhous. The perfusion process with 25% medium renewal every 12 h for 10 days achieved a biomass density of 65.6 g/L, a volumetric astaxanthin yield of 52.5 mg/L, and an astaxanthin productivity of 4.38 mg/L-d, which were 8.4-fold, 5.6-fold, and 2.3-fold of those in the batch process, 7.8 g/L, 9.4 mg/L, and 1.88 mg/L-d, respectively. The incorporation of hydrogen peroxide (H(2)O(2)) stimulation of astaxanthin biosynthesis into the perfusion process further increased the astaxanthin yield to 58.3 mg/L and the productivity to 4.86 mg/L-d. The repeated fed-batch process with 8 g/L glucose and 4 g/L corn steep liquor fed every 12 h achieved 42.2 g/L biomass density, 36.5 mg/L astaxanthin yield, and 3.04 mg/L-d astaxanthin productivity. The lower biomass and astaxanthin productivity in the repeated fed-batch than in the perfusion process may be mostly attributed to the accumulation of inhibitory metabolites such as ethanol and acetic acid in the culture. The study shows that perfusion process plus H(2)O(2) stimulation is an effective strategy for enhanced astaxanthin production in X. dendrorhous cultures.     

Yeasts from high-altitude lakes: influence of UV radiation

Mountain lakes located at a high elevation are typically exposed to high UV radiation (UVR). Little is known about the ecology and diversity of yeasts inhabiting these extreme environments. We studied yeast occurrence (with special emphasis on those producing carotenoid pigments) at five high-altitude (>1400 m a.s.l.) water bodies located in the Nahuel Huapi National Park (Bariloche, Argentina). Isolates were identified using a polyphasic approach. Production of photoprotective compounds (carotenoids and mycosporines) by yeast isolates, and UVB resistance of selected species were studied. All water samples contained viable yeast cells in variable numbers, generally ranging from 49 to 209 cells L⁻¹. A total of 24 yeast species was found; at least four represented novel species. Carotenogenic yeasts prevailed in lakes with low water conductivity and higher transparency and chlorophyll a levels. Apparently, the ability to produce photoprotective compounds in yeasts was related to the transparency of mountain lake waters, and strains from more transparent waters developed increased UVB resistance. Our results indicate that UVR is an important environmental factor affecting the yeast community structure in aquatic habitats.     

An efficient method for the extraction of astaxanthin from the red yeast Xanthophyllomyces dendrorhous

This study investigated an efficient method for the extraction of astaxanthin from the red yeast Xanthophyllomyces dendrorhous. The extraction process comprised three steps: (1) cultivating the yeast; (2) treating the yeast culture suspension with microwaves to destroy the cell walls and microbodies; and (3) drying the yeast and extracting the astaxanthin pigment using ethanol, methanol, acetone, or a mixture of the three as the extraction solvent. Ultimately, various treatment tests were performed to determine the conditions for optimal pigment extraction, and the total carotenoid and astaxanthin contents were quantified. A frequency of 2,450 MHz, an output of 500 watts, and irradiation time of 60 s were the most optimum conditions for yeast cell wall destruction. Furthermore, optimal pigment extraction occurred when using a cell density of 10 g/l at 30 C over 24 h, with a 10% volume of ethanol.     

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.     

Utilization of mustard waste isolates for improved production of astaxanthin by Xanthophyllomyces dendrorhous

Astaxanthin production in the wild strain Xanthophyllomyces dendrorhous TISTR 5730 was investigated using different mustard waste media, including mustard waste residue extract (MRE), mustard waste residue hydrolysate (MRH), mustard waste precipitated extract (MPE), and mustard waste precipitated hydrolysate (MPH). The growth of X. dendrorhous and the production of astaxanthin were dependent on the type and initial concentrations of mustard waste media. The MPH medium was the best substrate resulting in yields of biomass and astaxanthin of 19.6 g/L and 25.8 mg/L, respectively, under optimal conditions. MPH medium improved astaxanthin production 11-fold compared to the commonly used commercial yeast malt medium, and 1.3–2.1-fold compared to other mustard waste media.