WO1992022648A1 - Procede servant a engendrer des microorganismes a production elevee de carotenoide, microorganismes obtenus selon ce procede et procede servant a produire des cellules ou des parties de cellules contenant du carotenoide, ou du carotenoide purifie - Google Patents

Procede servant a engendrer des microorganismes a production elevee de carotenoide, microorganismes obtenus selon ce procede et procede servant a produire des cellules ou des parties de cellules contenant du carotenoide, ou du carotenoide purifie

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Publication number
WO1992022648A1
WO1992022648A1 PCT/DK1992/000186 DK9200186W WO9222648A1 WO 1992022648 A1 WO1992022648 A1 WO 1992022648A1 DK 9200186 W DK9200186 W DK 9200186W WO 9222648 A1 WO9222648 A1 WO 9222648A1
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WIPO (PCT)
Prior art keywords
carotenoid
cells
producing
astaxanthin
microorganism
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PCT/DK1992/000186
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English (en)
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Ingrid Stampe Villadsen
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Ingrid Stampe Villadsen
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Application filed by Ingrid Stampe Villadsen filed Critical Ingrid Stampe Villadsen
Publication of WO1992022648A1 publication Critical patent/WO1992022648A1/fr
Priority to NO934613A priority Critical patent/NO934613L/no

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • This invention concerns a method of generating high carotenoid-producing, in particular astaxanthin-producing, microorganisms by treating cells of a carotenoid-producing microorganism strain with a mutagen, culturing the mutagenized cells and selecting a mutant colony showing increased content of carotenoid.
  • the invention also concerns the high carotenoid-producing, in particular astaxanthin-producing microorganisms generated by this method, and especially some deposited strains of the yeast Phaffia rhodozyma.
  • the invention concerns a process for producing carotenoid-containing, in particular astaxanthin- containing, microbial cells or cell parts or purified carotenoid, in particular astaxanthin, by cultivation of the high carotenoid-producing microorganisms of the invention.
  • red colour of the meat of anadromous fish such as salmon or sea trout is due to red pigments such as astaxanthin which is present in the feed consumed by the fish.
  • red pigments such as astaxanthin which is present in the feed consumed by the fish.
  • the fish obtain their red colour from crustaceans or other astaxanthin- containing organisms, but when being bred in fish farms, the fish do not have access to these natural pigmentation sources and therefore do not obtain the attractive red colour unless red pigments are supplied in the feed.
  • astaxanthin isolated from crustacean wastes or produced synthetically as well as other synthetic red pigments such as cantaxanthin have been used as consti ⁇ tuents in fish feed.
  • cantaxanthin in animal feed is prohibited in certain countries, and the synthetic astaxanthin production as well as the process for isolating natural astaxanthin are rather expensive and often also subject to seasonal variations.
  • the carotenoids are a group of pigments, yellow to red in color, which are widely distributed in the plant and animal kingdoms. The color varies dependent on the lenghts of the chromophore and the type of oxygen-containing groups attached. Pigment formation is seldom in yeasts but is a characteristics of various species of the yeast genera Rhodotorula, Cryptococcus, Sporobolomyces and
  • Phaffia Also certain bacteria, other fungi and unicellu ⁇ lar algae produce carotenoids.
  • Rhodotorula rubra produces torulene and torularrhodin
  • Rhodotorula aurea and some species from the Mucorales produce 0-carotene
  • yeast Phaffia rhodo- zyma the bacteria Mycobacterium lacticola and Brevibacte- rium 103
  • the green alga Haematococcus pluvialis produce astaxanthin.
  • the carotenoids like all terpenoids, are synthesized from hydroxymethylglutaryl-coenzyme A, which is first converted to mevalonic acid.
  • the specific part of the pathway begins with the condensation of two molecules of geranylgeranyl pyrophosphate to form phytoene, a colourless carotene.
  • Four dehydrogenations transform phytoene into lycopene, the pigment of red tomatoes.
  • Two cyclisations convert lycopene to ⁇ -carotene.
  • these reactions are carried out by an enzyme aggregate that contains four copies of a dehydrogenase and two copies of a cyclase.
  • Astaxanthin was found to be the major pigment synthesized by this yeast; in naturally occurring yeast it comprised approximately 85% of the carotenoid mixture.
  • carotenoid pigments The most important function of carotenoid pigments, is to protect the organisms against oxidative damage, e.g. the deleterious effect of singlet oxygen and free radicals (Ref. 14, 17).
  • the protection of _>-carotene against radiation occurs by the absorption of energy in the blue region of the spectrum.
  • Recent epidemiological and oncogenical studies suggest that normal and high levels of 0-carotene in the body may protect against cancer (Ref. 18, 19).
  • 0-carotene also functions as a precursor of vitamin A in mammals.
  • Phaffia rhodozyma The yeast Phaffia rhodozyma was found in Japan in 1967, in the exodeous fluids excreted from beach trees and named after H. Phaff who isolated this organism (Ref. 1). Phaffia rhodozyma was shown to be different from other pigmented yeasts in the ability to produce the carotenoid pigment, astaxanthin (3,3'-dihydroxy-0,0-carotene-4,4'- dione) and was, in addition, ascribed to a new genus (Ref. 6).
  • the genus, Phaffia was placed in the Deuteromycotina described as having the unique characteristics of being a carotenoid-producing, fermentative yeast. Its ability to ferment sugars is in contrast to other carotenoid- synthesizing yeasts which are all strictly aerobic. Its special metabolic properties such as the ability to split urea by urease and its ability to assimiliate inositol are rare among ascomycetous yeasts and so is its ability to produce starchlike components. Another characteristic is the content of 48,3 mol-% G+C, which is the upper limit for ascomycetous yeasts, but lower than what is found in basidiomyceteous yeasts (Ref. 9).
  • Phaffia reproduces by enteroblastic budding leading to a multilayered cell wall.
  • the polysaccharides in the laminar cell wall contain a high proportion of ⁇ -l,3-glucan indicative of both ascomyceteous and basidiomyceteous yeasts (Ref. 8), but the nature of the cell wall has lead to the belief that Phaffia is an imperfect species of basidiomyceteous origin.
  • P. rhodozyma the only known species in this genus, was hitherto thought to reproduce exclusively by budding, and attempts to identify a sexual cycle have up to this moment been unsuccesfull (Ref. 6).
  • Phaffia Other characteristics include the absence of ballistoconidia, but the presence of chlamydospores. In connection with the present invention it has been determined that Phaffia also produces haploid spores of sexual origin.
  • Inhibitors of the redox pathway (Ref. 2 and the interna ⁇ tional (PCT) patent application WO 90/01552) and the end ring analog of 0-carotene, 0-ionone (Ref. 3) have been reported to be af great value in selecting yeasts with increased astaxanthin content; in both cases from wild type levels 150-350 ⁇ g/g dry weight up to 1000 ⁇ g/g dry weight.
  • mutant strains with carotenoid content up to about 1200 ⁇ g/g dry weight can be done by visual inspection.
  • one aspect of the present invention is a method of generating carotenoid-producing, in particular astaxanthin-producing, microorganisms with a carotenoid content of at least 1600 ⁇ g/g dry weight, preferably at least 1900 ⁇ g/g dry weight, when cultured in shake flask to the stationary phase, by treating cells of a carotenoid-producing microorganism strain with a mutagen, culturing the mutagenized cells, and selecting a mutant colony showing increased content of carotenoid, said method comprising the following steps: (a) treating the cells with the mutagen,
  • Phaffia the combination of 1 mM 0-ionone and 0,1 mM thymol kills more than 10 s cells.
  • Incuba ⁇ tion of a mixture of white mutants of Phaffia and wild type astaxanthin containing cells in 0,1 mM 0-ionone results in a survival primarily of the astaxanthin containing cells.
  • the reason for the unstability remains unknown; many factors, some of which are discovered in connection with this invention may influence the stability.
  • mutagenizations have been performed in the exponential growth phase, where the cells are diploid, and therefore it is difficult to isolate mutants at all - another is that the astaxanthin biosynthetic enzymes may reside in a plasmid, unstable by nature, or in the genome of the mitochondria, the number of copies of which vary with the growth phase, or that the astaxanthin in the cells are bound to mitochondria or to peroxisomes, and therefore depends on their synthesis for its location.
  • Phaffia When Phaffia is grown in shake flasks containing conven ⁇ tional YM medium at 21 °C, two to three types of cells can be distinguished, depending on the stage of growth. In the early exponential growth phase, cells are reproduced by the formation of buds with a relatively broad basis, like the cells seen in the description of the genus (Ref. 9).
  • the sizes of the two types of cells were: Big cells 10.7 X 10 ⁇ m and the small cells 7.4-5.0 X 4.1 ⁇ m, corresponding to volumes of 460 ⁇ m 3 and 98-66 ⁇ m 3 , respectively.
  • a defined complete medium that contains urea and not ammonia as the easily accessible nitrogen source was used.
  • urea nitrogen
  • conjugation will occur followed by a sporulation (Example 4).
  • ammonia-containing medium sporulation is less prominent, mainly diploid cells are observed.
  • Another requirement for sporulation is the presence of all amino acids and the bases uracil and adenine. Without these latter bases the cells may conjugate, but not sporulate.
  • the final proof of a sexual cycle came from an analysis on a SKATRON flow cytometer on populations of cells fixed in different stages af the whole life cycle or growth deve ⁇ lopment of Phaffia.
  • the cells were stained with DAPI, a conventional stain for DNA (Ref. 10) and the cell size (rather the scatter of individual cells) as well as the content of DNA was determined in each single cell. Due to the very thick cell wall found in Phaffia 10 x normal amounts of DAPI were used.
  • Fig. 1 demonstrates the analysis of a sample taken in the early stationary growth phase. Some cells with a DNA content of N, as well as cells with a DNA content of 4N can be seen. Treating the cells with mutanase, as demonstrated in the following, to open the cells, resulted in the appearence of more cells with a content of N, together with the appearence of a new type of cells detected with the forward scatter.
  • the small cells are contained within the big cells, they have a DNA content of 4N and must be haploid, the big cells are diploid (2N) and have replicated the DNA in their genome to 4N before they undergo a meiosis and each cell then gives rise to four external spores, each with a haploid DNA content.
  • 2N diploid
  • Phaffia rhodozyma most obviously belongs to the basidiomyceteous yeasts, due to the appearence of external spores liberated from sexual cells undergoing meiosis, in accordance with previous beliefs.
  • the cells are grown to the haploid phase before they are treated with the mutagen.
  • this haploid phase occurs in the late stationary phase of growth in defined medium.
  • the production of astaxanthin in Phaffia rhodozyma is dependent on the sporulation.
  • a method of removing the thick cell wall of Phaffia rhodozyma has been found and used to identify the particulate nature of astaxanthin-containing structures in the cell.
  • Mutanase was used to prove the existence of a sexual cycle as well as to fractionate the subcellular components of Phaffia in order to determine the subcellular location of the pigment astaxanthin.
  • the method used for purification of nuclei was used with several modifications: Gram amounts of cells were used, the cells were broken with mutanase, (50 g of cells and 2.3 g of mutanase), and the protoplasts were disintegrated with a Potter-Elvehjelm homogenisator at maximum speed for 3 min. No absolute purification of the astaxanthin-containing subcellular particles were obtained using this method, but an immense enrichment of the particles containing the pigment was obtained in the supernatant after the precipitation of the nuclei. The fraction heavily enriched for astaxanthin contained raspberrylike structures, that could be pelleted easily, thus establishing the particulate nature of astaxanthin- containing structures in Phaffia.
  • peroxisome-inducing medium (Ref. 13), as the sole carbon source when using Phaffia rhodozyma for a commercial production of astaxanthin.
  • the peroxisome-inducing substance for use in such medium may for example be selected from the group consisting of unsaturated fatty acids having at least 14 carbon atoms, D-alanine, alkylamines and mixtures thereof, optionelly in combination with lower alcohols.
  • Figure 1 is a graph showing the content of DNA in cells of Phaffia rhodozyma at 0D.,_ 0 18.5 after 4 days of growth in YM medium.
  • DNA was stained as in Ref. 10 except that 10 ⁇ g of DAPI/ml was used.
  • N indicates the genomic DNA content (haploid), whereas 4N indicates the presence of cells with 4 x genomic DNA content.
  • Figure 2 is a graph illustrating the growth of Phaffia rhodozyma in a minimal salt medium with glucose as the carbon source and urea as the nitrogen source. The optical density was measured in a 1 cm cuvette using a Zeiss PMQII spectrophotometer.
  • Figure 3 shows photos of: (a) Two haploid cells having conjugated and budded to form af bigger diploid cell, (b) One cell containing (c) three nuclei when stained with DAPI. Magnifications, a: 1400x; b,c: lOOOx.
  • Figure 4 shows photos of diploid cells in sporulation: (a) A diploid cell simultaneously producing two haploid spores, (b) A diploid cell producing four haploid spores. Magnification, 3500x.
  • Figure 5 is a schematic illustration of the sexual life cycle of Phaffia rhodozyma. In the early exponential growth phase the cells are budded and haploid (N). Cells of opposite mating types make contact and conjugate to form diploid cells (2N). The conjugated pairs form diploid buds (2N) that eventually sporulate to form four haploid external spores (N).
  • EMS mutagenesis The strain to be mutagenized was grown in 50 ml of YM medium, in shake flasks for two days. From the exponential growing culture, around 3 X 10 ⁇ cells were harvested by centrifugation 5000 RPM for 5 min. The pellet was resuspended in 5 ml 10 mM sodium phosphate pH 7.0, 30 ⁇ l of EMS was added, and the cells were incubated for 10- 30 minutes at room temperature while shaking. The reaction was stopped by adding 25 ml 1% NaCl. The cells were washed twice in the above buffer and resuspended in YM medium. Further selection was made as described below.
  • ICR 170 mutagenesis: The strain to be mutagenized was grown in 50 ml of YM medium, in shake flasks for two days. From the exponentially growing culture, around 3 X 10' cells were harvested by centrifugation 5000 RPM for 5 min. The pellet was resuspended in 5 ml freshly prepared solution of 100 ⁇ g/ml "ICT 170" in 0.1 M sodium phosphate pH 7.0, and the cells were incubated for one hour at room temperature while shaking. The reaction was stopped by adding 25 ml of 1% NaCl, the cells were washed twice in the above buffer and resuspended in YM medium. Further selection was made as described below. For survival measurements treated as well as untreated cells were diluted and plated on YM plates to ensure a survival of about 50% in each case.
  • Phaffia rhodozyma strain CBS 5908 and employing EMS treatment to a survival of about 50% the content of carotenoids was increased from 350 ⁇ g/g to 1529 ⁇ g/g dry weight by visual screening, obtaining a strain termed IVP 104.
  • This strain was treated twice with "ICR 170" and a strain termed IVP 106 having 1710 ⁇ g/g dry weight was selected.
  • the strain IVP 106 was finally treated again with EMS, the mutagenized cells were plated on thymol- containing plates, and a strain termed IVP 107 was selected containing 1952 ⁇ g/g dry weight when cultured in shake flasks.
  • IVP 107 is deposited in Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg IB, D-3300 Braunschweig, Germany, under the accession number: DSM 6560.
  • strain IVP 104 was mutagenized with "ICR 170" as above, but higher producing strains were selected directly on thymol-containing plates. As a result a strain termed IVP 112 containing 2050 ⁇ g carotenoids/g dry weight was obtained. IVP 112 is deposited in Deutsche Sammlung von Mikroorganismen und Zellkulturen under the accession number: DSM 6561.
  • the strain DBT 415 obtained from Danisco Bioteknologi A/S, Raffinaderivej, 2300 Copenhagen S, Denmark, and origina ⁇ ting from Phaffia rhodozyma strain ATCC 24261 was mutage- nized with "ICR 170", and higher producing colonies were selected on thymol-containing plates.
  • a strain termed IVP 14 was selected as containing 1962 ⁇ g/g carotenoids dry weight. IVP 14 is deposited in Deutsche Sammlung von Mikroorganismen und Zellkulturen under the accession number: DSM 6559.
  • Phaffia occurs almost synchronously when the cells are grown in a defined medium supplemented with amino acids and bases, with glucose as the C-source and urea as the N-source.
  • DCMP Phaffia rhodozyma
  • a small inoculum of cells of DSM strain 6559 grown in a defined medium is inoculated into 50 ml of the defined complete medium described above in a 250 ml shake flask so that a minium of 6 generations of growth is allowed before
  • OD 4 ⁇ 5 cn 0 nm reaches 0,1 (measured in a Zeiss PMQII spectrophotometer).
  • the cells are grown at 21 °C while shaking at 170 RPM. In exponentially growing cultures till a cell concentration of around 2 x 10 cells single cells budding in the typical manner described for Phaffia are seen. From OD 0.5 to 1.0 the cells pair, only to conjugate later. After fusion a larger diploid bud appears around OD 1.5 (FIG. 3a). In stationary phase occuring from OD 3 to 6 the diploid cells sporulate to excrete four external haploid spores mainly formed at one end of the cell (FIG. 4). After OD 6 the culture contains predominantly single haploid cells. Staining the DNA in a sporulating culture with DAPI revealed the occurrence of three or four nuclei in one cell seen in FIG. 3b,c, one nucleus here being present in the bud.
  • Phaff, H.J., et al. "A comparative study of the yeast flora associated with trees on the Japanese islands and on the west coast of North America", pp. 759-774 in Proc. IV IFS Fermentation Technology Today, Kyoto, Society of Fermentation Technology Osaka (1972).

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Abstract

On crée des microorganismes à production élevée de caroténoïde, en particulier à production d'astaxanthine, en traitant les cellules d'une souche de microorganisme produisant du caroténoïde avec un mutagène, en cultivant les cellules à mutagenèse en présence d'un inhibiteur de phytoène déshydrogénase afin de réduire le niveau de production de caroténoïde dans toutes les cellules, et en choisissant une colonie ou une cellule mutante présentant une coloration de caroténoïde accrue comparée aux cellules de fond. En particulier, le microorganisme produisant le caroténoïde est le Phaffia rhodozyma; on laisse croître les cellules jusqu'à ce qu'elles atteignent la phase stationnaire avancée avant de les traiter avec le mutagène. On laisse grandir les microorganismes obtenus jusqu'à ce qu'ils présentent une biomasse contenant du caroténoïde en une teneur d'au moins 1600, de préférence au moins 1900 νg/g en poids sec. Lorsqu'on cultive un microorganisme produisant du caroténoïde pour produire des cellules microbiennes contenant du caroténoïde ou du caroténoïde purifié, des rendements particulièrement élevés en caroténoïde peuvent être obtenus si on laisse grandir les cellules jusqu'à la phase haploïde avant qu'elles ne soient récoltées ou soumises à un traitement ultérieur. De rendements encore plus élevées peuvent être obtenus si l'on utilise un bouillon de culture contenant des substances produisant le peroxysome.
PCT/DK1992/000186 1991-06-14 1992-06-15 Procede servant a engendrer des microorganismes a production elevee de carotenoide, microorganismes obtenus selon ce procede et procede servant a produire des cellules ou des parties de cellules contenant du carotenoide, ou du carotenoide purifie WO1992022648A1 (fr)

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Application Number Priority Date Filing Date Title
NO934613A NO934613L (no) 1991-06-14 1993-12-14 Fremgangsmåte for fremst av höytytende karotenoidproduserende mikroorganismer, mikroorganismer oppnådd ved fremgangsmåten og en fremg for fremst av karotenoidinneholdende celler eller cellebestanddeler eller renset karotenoid

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DK911151A DK115191D0 (da) 1991-06-14 1991-06-14 Fremgangsmaade til frembringelse af carotenoidproducerende, isaer astaxanthinproducerende, mikroorganismer, mikroorganismer opnaaet ved fremgangsmaaden og fremgangsmaade til fremstilling af carotenoidholdige mikroorganismeceller eller -celledele eller oprenset carotenoid
DK1151/91 1991-06-14

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US5466599A (en) * 1993-04-19 1995-11-14 Universal Foods Corporation Astaxanthin over-producing strains of phaffia rhodozyma
US5599711A (en) * 1987-04-15 1997-02-04 Gist-Brocades, N.V. Astaxanthin-producing yeast cells, methods for their preparation and their use
WO1997023633A1 (fr) * 1995-12-22 1997-07-03 Gist-Brocades B.V. Ameliorations apportees a des methodes pour transformer des souches de phaffia, souches de phaffia ainsi obtenues et adn de recombinaison obtenu selon lesdites methodes
US5972642A (en) * 1987-04-15 1999-10-26 Dsm N.V. Astaxanthin-producing yeast cells, methods for their preparation and their use
EP1058108A2 (fr) 1999-05-31 2000-12-06 AVL List GmbH Méthode et appareil pour le diagnositic et la commande d'un moteur à combustion interne
US8404468B2 (en) 2007-05-23 2013-03-26 Cognis Ip Management Gmbh Efficient astaxanthin production strains derived from Haematococcus pluvialis
CN111868228A (zh) * 2017-11-28 2020-10-30 国立研究开发法人科学技术振兴机构 新型微藻类及其用途
CN114350532A (zh) * 2022-01-12 2022-04-15 威海东巽生物科技有限公司 一种基于高产虾青素红法夫酵母的复壮方法
CN115044527A (zh) * 2022-02-15 2022-09-13 昆明理工大学 一种肌醇在促进雨生红球藻生产虾青素中的应用

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WO1990001552A1 (fr) * 1988-08-08 1990-02-22 Igene Biotechnology, Inc. Procedes de production in vivo d'astaxanthine et de levure phaffia rhodozyma a teneur en astaxanthine superieure
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EP0427405A1 (fr) * 1989-10-27 1991-05-15 Enzymatix Ltd. Levures et leurs utilisation dans la production d'astaxanthin
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WO1990001552A1 (fr) * 1988-08-08 1990-02-22 Igene Biotechnology, Inc. Procedes de production in vivo d'astaxanthine et de levure phaffia rhodozyma a teneur en astaxanthine superieure
WO1991002060A1 (fr) * 1989-07-28 1991-02-21 Igene Biotechnology, Inc. Procedes de production in vivo d'astaxanthine et de levure de phaffia rhodozyma a teneur accrue en astaxanthine
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EP0438182A1 (fr) * 1990-01-19 1991-07-24 Phillips Petroleum Company Nouvelles souches de Phaffia rhodozyma contenant de grandes quantités d'astaxanthine

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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5599711A (en) * 1987-04-15 1997-02-04 Gist-Brocades, N.V. Astaxanthin-producing yeast cells, methods for their preparation and their use
US5679567A (en) * 1987-04-15 1997-10-21 Gist-Brocades, B.V. Astaxanthin-producing yeast cells, methods for their preparation and their use
US5709856A (en) * 1987-04-15 1998-01-20 Gist-Brocades N.V. Astaxanthin-producing yeast cells, methods for their preparation and their use
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