DE102014210308A1 - Yeast strain for the production of carotenoids - Google Patents

Abstract

Yeast strain of the genus Saccharomyces with established carotenoid biosynthesis pathway, characterized in that it can overexpress a PDR1 gene or PDR1 homologue.

Description

The invention relates to a yeast strain for the production of carotenoids, processes for its preparation and its use for the production of carotenoids.

The term "carotenoid" refers to a structurally diverse class of pigments derived from the isoprenoid biosynthetic pathway. The first important step in carotenoid biosynthesis is the generation of phytoene from geranylgeranyl pyrophosphate. Carotenoids may be cyclic or acyclic and may or may not contain oxygen, so the term carotenoids includes both carotenes and xanthophylls.

In general, carotenoids are hydrocarbon compounds that have a conjugated polyene carbon backbone that is formed from isopentenyl pyrosophosphate phosphate. Included herein are triterpenes and tetraterpenes, as well as their oxygenated derivatives and other compounds of lengths of e.g. C35, C50, C60, C70, C80. Many carotenoids have strong light-absorbing properties and can be greater in length C200. C30 diapocarotenoids are typically composed of six isoprene units connected such that the array of isoprene units in the center of the molecule is inverted and that the two central methyl groups are in a 1,6-positional relationship and the remaining non-terminal methyl groups are in a 1 , 5-positional relationship stand. Such C30 carotenoids may be derived from an acyclic C30H42 structure having a long central chain of conjugated double bonds, e.g. by hydrogenation, dehydrogenation, cyclization, oxidation, esterification, glycosylation or a combination of these processes. C40 carotenoids typically consist of eight isoprene units connected in such a way that the arrangement of isoprene units in the center of the molecule is inverted and that the two central methyl groups are in a 1,6-positional relationship and the remaining nonterminal methyl groups in a 1, 5-positional relationship stand. Such C40 carotenoids may have arisen from an acyclic C40H42 structure having a long central chain of conjugated double bonds, e.g. by hydrogenation, dehydrogenation, cyclization, oxidation, esterification, glycosylation or a combination of these processes. The class of C40 carotenoids also contains certain compounds that result from rearrangement of the carbon backbone or from removal of parts of that structure. More than 600 different carotenoids have been identified in nature; Carotenoids include: antheraxanthin, adonirubin, adonixanthin, astaxanthin, canthaxanthin, capsorubrin, [beta] -cryptoxanthin, [alpha] -carotene, [beta] -carotene, [beta], [psi] -carotene, [delta] -carotene , [epsilon] carotene, echinenone, 3-hydroxyechinenone, 3'-hydroxyechinenone, [gamma] carotene, [psi] carotene, 4-keto- [gamma] -carotene, [zeta] -carotene, [alpha] - Cryptoxanthin, Deoxyflexixanthin, Diatoxanthin, 7,8-Didehydroastaxanthin, Didehydrolycopene, Fucoxanthin, Fucoxanthinol, Isorenieratene, [beta] -Isorenieratene, Lactucaxanthin, Lutein, Lycopene, Myxobactone, Neoxanthin, Neurospores, Hydroxyneurospores, Peridinin, Phytoene, Rhodopin, Rhodopin glucoside, 4 Keto-rubixanthin, siphonaxanthin, spheroidene, spheroidenone, spirilloxanthin, torulene, 4-keto-torulenes, 3-hydroxy-4-keto-torulenes, uriolides, uriolide acetate, violaxanthin, zeaxanthin [beta] -diglucoside, zeaxanthin and C30 carotenoids , In addition, carotenoids contain derivatives of these molecules which may contain hydroxy, methoxy, oxo, epoxy, carboxy, or aldehyde groups. Also included are esters (e.g., glycoside esters, fatty acid esters) and sulfate derivatives (e.g., esterified xanthophylls). They serve as antioxidants for membrane protection by trapping oxygen and peroxide radicals. Their antioxidant properties are attributable to their structure. Carotenoids are one of the most important ingredients in food. They are natural dyes, e.g. Yellow or red, which has a major impact on the acceptance of many foods. In addition, some carotenoids are precursors of vitamin A. Carotenoids often have a health-promoting effect by being able to prevent against diseases such as cancer, cardiovascular diseases, macular degeneration or cataracts.

The interest in carotenoids has increased significantly in recent years, mainly due to their health benefits, but also due to the growth of different markets or sectors. These include agriculture, especially aquaculture and the poultry industry, the dietary supplement industry, the food industry, where carotenoids are used as colorants for sausages, soft drinks or baked goods, as well as the pharmaceutical or cosmetics industry.

Carotenoids are synthesized from isoprene precursors, some of which are involved in the production of steroids or sterols. The best-known isoprene biosynthetic pathway is the so-called "mevalonate way". Acetyl-CoA is converted to mevalonate via hydroxymethylglutaryl-CoA (HMG-CoA). Mevalonate is then phosphorylated and converted to isopentenyl pyrophosphate (IPP). After isomerization of IPP to dimethyl allyl pyrophosphate (DMAPP), after three consecutive isomerization reactions with IPP, the 10-carbon molecule geranyl pyrophosphate (GPP), followed by the 15-carbon molecule, becomes Farneyl pyrophosphate (FPP) and finally the 20-carbon molecule geranylgeranyl pyrophosphate (GGPP) formed. Enzymes involved in carotenoid biosynthesis include GGPP synthase (CrtE), phytoene synthase (CrtB), phytoene or desaturase (CrtI), lycopene cyclase (crtY), carotenoid ketolase (crtW), carotenoid hydroxylase (CrtZ ), Astaxanthin synthase (CrtS).

The yeast Saccharomyces cerevisiae is a robust, industrially well-established production organism that has good growth characteristics, a broad substrate spectrum and exceptional salt and osmotolerance. It is therefore particularly suitable for biotechnological production on an industrial scale. S. cerevisiae is fully sequenced, well characterized and all tools for genetic optimization are established. In the production of lipids, S. cerevisiae offers a great advantage because it is able to deposit large quantities of lipidic components in an inert form intracellularly in lipid particles, whereby the physiology of the yeast cell remains largely unaffected. S. cerevisiae has no native carotenoid biosynthetic pathways, but produces via the mevalonate biosynthetic pathway all the terpenes that are the building blocks of carotenoids, such as isopentenyl diphosphate, dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate (see US Pat 1 ).

In Ukibe, K. et al, 2009, Appl Environ Microbiol 75: 7205-11 or Verwaal, R. et al, Yeast 27: 983-98 It has already been shown that the yeast S. cerevisiae is in principle able to synthesize carotenoids when genes (or enzymes) of the carotenoid biosynthetic pathway are heterologously introduced.

In the case of phytoene, the enzymes GGPP synthase and phytoene synthase must be introduced, for which the corresponding genes are called crtE and crtB. For the production of beta-carotene, the expression of additional genes with the names crtI and crtY is necessary, which encode the enzymes phytoene desaturase and lycopene cyclase ( Ukibe, K. et al, Applied and Environmental Microbiology. 2009, 75 (22), pp. 7205-7211 ; Verwaal, R. et al, Applied and Environmental Microbiology. 2007, 73 (13), pp. 4342-4350) , The literature also discloses a bifunctional enzyme which catalyzes two enzymatic steps. This is the phytoene synthase / lycopene cyclase from Xanthophyllomyces dendrorhous, which is encoded by the gene crtYB. This enzyme combines the activities of the genes crtY and crtB, so that in addition to the gene crtYB, only the genes crtE and crtI must be expressed for the synthesis of beta-carotene. For the synthesis of zeaxanthin and beta-cryptoxanthin additionally the expression of the gene crtZ is necessary. The synthesis of astaxanthin is achieved by the additional expression of the gene crtW (see 4 ).

In these studies, these genes were introduced from the basidiomycete yeast Xanthophyllomyces dendrorhous into S. cerevisiae, which led to the synthesis of ß-carotene, zeaxanthin, astaxanthin and other carotenoids, but still in amounts that seem too small for industrial production.

Verwaal, R. et al, Yeast 27: 983-98 showed that S. cerevisiae is able to secrete carotenoids. Responsible for this were transport proteins, which are involved in the "Pleiotropic Drug Resistance" (PDR) network. However, PDR genes and their influence on the carotenoid content of the cells were not investigated in this study.

"Multiple drug resistance", which in yeast corresponds to the "pleiotropic drug response", describes the phenomenon that cells have or develop resistance to potentially toxic molecules from the environment. This is the responsibility of transporters, who send these substances out of the cell. The PDR1 gene encodes a transcription factor containing the "Pleiotropic drug response" in yeast (yeastgenome.org) regulated. PDR1 encodes the zinc finger transcription factor Pdr1p, which is the regulator of "pleitropic drug responses" in yeast. Pdr1p serves as a transcriptional activator and repressor by binding to Pleitropic drug response (PDRE) elements found in the promoter regions of target genes.

The object of the present invention was to provide a yeast strain which produces carotenoids in larger quantities than known yeast strains and stores them intracellularly.

The object is achieved by a yeast strain of the genus Saccharomyces with an established carotenoid biosynthesis pathway, characterized in that it can overexpress a PDR1 gene or PDR1 homologue.

The PDR1 gene is preferably characterized by the nucleic acid sequence SEQ ID NO: 6 and the PDR1 gene product Pdr1p, preferably by the amino acid sequence SEQ ID NO: 7. In the context of the present invention, as PDR1 homologs, those genes are to be understood which are suitable for proteins with Pdr1p Function encoded in an analysis using the algorithm BLAST 2.2.1 ( Old school, Stephen; Gish, Warren; Miller, Webb; Myers, Eugene; Lipman, David (1990). "Basic local alignment search tool". Journal of Molecular Biology 215 (3): 403-410 ) have a sequence identity of greater than 80%, preferably greater than 90%, particularly preferably greater than 95%, to SEQ ID NO: 7.

Thus, PDR1 homologs are also allelic variants of the PDR1 gene, in particular functional variants which are derived by deletion, insertion or substitution of nucleotides from SEQ ID NO: 6, but the Pdr1p activity of the respective gene product is retained. The activity of the PDR1 homologs, as well as the activity of the PDR1 gene, is determined by determining the increase in carotenoid biosynthesis in strains in which carotenoid biosynthesis has been established.

Particularly preferably, the PDR1 gene or the PDR1 homologs to the yeast codon optimized gene PDR1 (SEQ ID NO: 6).

For the purposes of the present invention, a gene is considered overexpressed if the level of protein activity of the encoded gene product is increased by at least 5% compared to a yeast strain of the same genetic background that has not been genetically modified to express the protein.

The increased activity of the gene product is evident from a correspondingly increased carotenoid production compared to a strain which is unmodified in its PDR1 expression.

Preferably, in the yeast strain of the invention, the carotenoid content is increased at least 2-fold compared to a carotenoid-producing yeast strain which does not overexpress the PDR1 gene.

The carotenoid biosynthesis pathway is preferably established in the yeast strain according to the invention by expressing the genes crtYB, crtE and crtI.

Particularly preferred are the yeast codon optimized gene crtE (SEQ ID NO: 3), the yeast codon optimized gene crtYB, (SEQ ID NO: 4) and the yeast codon optimized gene crtI (SEQ ID NO: 5 ) from X. dendrorhous.

Preferably, the yeast strain of the invention is a strain of the species Saccharomyces cerevisiae. Again, it is preferably a strain of the species S. cerevisiae of the enzymes involved in carotenoid biosynthesis and overexpresses at least one PDR1 gene or PDR1 homologue.

Particular preference is given to a strain of the species Saccharomyces which expresses the genes GGPP synthase and phytoene synthase, and / or phytoene desaturase and / or lycopene cyclase and overexpresses at least one PDR1 gene or PDR1 homologue.

In particular, it is preferably the strain AH22th3ura8 which additionally overexpresses at least one PDR1 gene or PDR1 homolog.

The GGPP synthases are preferably an enzyme having one of the following Genbank ACCESSION # AAT92871, XP_447025, NP_984623, XP_390273, XP_404791, XP_368486, Q92236, AAO85432, XP_572774, XP_502923, AAK11525, XP_326920, CAF32032, BAD29965, XP_384767, BAD29970 , CAB89115, CAG09545, CAI13753, AAH69913, AAH67768, XP_455003, P56966, NP_001007, AAT65717, NP_956329, BAA90525, XP_405729, AAK11531, XP_412280, AAC05273, NP_523958, XP_402074, EAL30191, XP_536340, XP_424685, AAH06798, AAP06018, XP_460338, AAC05595, EAK92197 , XP_535573, AAH83212, XP_486466, CAH18006, CAA75568, XP_397455, XP_410947, XP_381914, XP_364478, XP_360889, XP_369218, XP_406544, XP_367595, XP_363775, XP_368486, XP_390273, Q92236, AAK11525, CAF32032, XP_404791, AAO85432, BAD29965, BAD29970, BAA90525, AAT65717 , XP_384767, CAB89115, XP_572774, AAK11531, XP_502923, CAI13753, CAG09545, XP_412280, P56966, NP_001007, AAH69913, AAH67768, NP_956329, EAL30191, XP_424685, XP_536340, NP_523958, AAC05273, XP_405729, AAC0 5595, XP_402074, AAP06018, AAH06798, XP_535573, AAH83212, AAP21085, NP_984623, XP_447025, AAT92871, XP_486466, XP_410947, XP_397455, XP_455003, EAK92197, XP_381914, XP_460338, CAH18006, XP_360889, XP_406544, XP_364478, XP_363775, XP_367595, XP_369218, C39273, BAB79600, BAA14124, AAN85596, AAA32797, Q08291, S52584, S53722, AAC44848, BAA19583, S71230, BAA23157, AAC77874, CAB38744, BAA78047, BAA82613, CAB56064, BAA86284, T11021, AAF78199, AAG10424, CAC10561, T50879, BAB01343, Q42698, Q43133, P54976, BAB50600, BAB60678, BAB60820, NP_189589, NP_188651, NP_188069, NP_188073, AAL01997, AAL01998, NP_252732, NP_245470, NP_390308, NP_440010, NP_440010, AAL17614, NP_520343, AAL76349, AAM21638, AAM21639, NP_622916, AAM48650, NP_659794, AAM64496, AAM65107, NP_680811, ZP_000474, ZP_001252, NP_698760, E84566, F85434, AC1245, E83997, G84566, AH2910, D87505, A89932, F97685, AI3285, BAC42571, NP_785195, NP_790546, AAO63392, AAO93113, NP_833891, AAP59037, ZP_001374, NP_864766, NP_875521, NP_881399, NP_884694, NP_888456, NP_893187, NP_894940, NP_896835, NP_896835, AAQ65086, NP_945877, NP_946867, NP_952815, AAR37805, AAR37858, AAR98495, AAR99082, NP_965349, NP_980544, EAA96348, EAB36506, EAB36642, EAC39208, EAD26007, EAE43084, EAE70061, EAF70308, EAG88494, EAH52060, EAH78354, EAH84117, EAI11762, EAI49391, EAI54846, EAI68356, EAI68713, EAI69401, EAI73873, EAJ73634, EAJ77351, EAK70639, ZP_001751, AAS76253, ZP_001957, 1RTRB, ZP_001863, ZP_0 02002, ZP_001711, ZP_002073, ZP_002074, AAS82860, ZP_002108, YP_010568, BAD18313, ZP_002315, ZP_002335, AAT35222, ZP_002401, ZP_002435, ZP_002626, ZP_002702, ZP_002705, ZP_002732, ZP_002914, ZP_003024, ZP_003129, ZP_003177, ZP_003225, AAT51323, ZP_003301, YP_034222, YP_040995, YP_043579, AAT71982, AAT90315, YP_066435, YP_075673, YP_085511, YP_092166, ZP_001298, YP_105136, YP_111769, ZP_003630, YP_129021, AAV74395, AAV74396, YP_148246, YP_156518, YP_162590, YP_171470, YP_175959, YP_186407, YP_190690, AAW66658, YP_194187, YP_197469, YP_201938, YP_196510.

The phytoene dehydrogenases are preferably enzymes having one of the following genebank accession numbers: 1613414B, 1613414F, 1904206A, 2121278A, A86203, A96612, A99470, AAA24820, AAA34001, AAA50313, AAA64981, AAA91161, AAA99519, AAC44798, AAC44850, AAC48983, AAF78201, AAG10426, AAG14399, AAG28700, AAG50743, AAH85048, AAK51545, AAK51557, AAK64299, AAL02000, AAL15300, AAL38046, AAL73986, AAL80005, AAL91366, AAM45380, AAM48646, AAM63349, AAM94364, AAN75037, AAN85599, AAO24235, AAO46892, AAO46894, AAO53257, AAO53258, AAO64750, AAO93135, AAP59036, AAP79175, AAQ04224, AAQ04225, AAQ65246, AAQ65246, AAQ88931, AAR37797, AAR37802, AAR37850, AAR37855, AAR86105, AAR98491, AAR98494, AAR98733, AAS17750, AAT01639, AAT35222, AAT74579, AAT74580, AAT76050, AAT76434, AAT90316, AAU34019, AAW23161, AB2035, AB2064, AC2446, AF1557, AF2029, AG2103, AG2509, AH1199, AI2185, AI2273, B55548, B84327, B90061, BAA14127, BAA20276, BAA76534, BAB10768, BAB50520, BAB51896, BAB68552, BAB79603, BAB82461, B AB82462, BAB98016, BAC75676, BAC77668, BAC77671, BAD07279, BAD07280, BAD07287, BAD07288, CAA52098, CAA60479, CAA66626, CAB38739, CAB38743, CAB40843, CAB56041, CAB56062, CAB59726, CAB65434, CAB94794, CAC85667, CAD19989, CAD27442, CAD55814, CAE00192, CA 0283576, CAF19330, CAF21094, CAF21337, CAH91165, E90061, EAA90383, EAA98598, EAB09790, EAB14136, EAB18725, EAB29729, EAB30992, EAB41377, EAB54727, EAB76679, EAB87028, EAB92587, EAB94459, EAB96864, EAC01884, EAC38895, EAC60360, EAD05874, EAD05999, EAD20520, EAE06978, EAE70773, EAF04985, EAF51354, EAF62819, EAF75453, EAG09111, EAG19412, EAG23070, EAG25053, EAG25054, EAG29279, EAG39845, EAG56100, EAG63013, EAG68633, EAG71574, EAG89835, EAH04928, EAH04936, EAH08586, EAH22597, EAH22853, EAH31648, EAH55579, EAH68071, EAH73302, EAH79041, EAH86965, EAH97108, EAH99977, EAI01660, EAI03576, EAI06784, EAI11087, EAI15261, EAI15547, EAI17521, EAI21398, EAI29728, EAI38468, EAI43591, EAI51589, EAI58453, EAI72974, EAI77885, EAI78272, EAI80262, EAI83937, EAI 86664, EAJ00517, EAJ05570, EAJ08238, EAJ15524, EAJ18144, EAJ20649, EAJ21683, EAJ24413, EAJ28774, EAJ30522, EAJ35157, EAJ37407, EAJ39929, EAJ54356, EAJ54959, EAJ56207, EAJ58447, EAJ59958, EAJ63347, EAJ66054, EAJ67637, EAJ69812, EAJ74441, EAJ76472, EAJ76473, EAJ80355, EAJ80839, EAJ81408, EAJ86174, EAJ87600, EAJ88203, EAJ88682, EAJ92341, EAJ94774, EAJ97555, EAJ97958, EAK07654, EAK08513, EAK08529, EAK10609, EAK10614, EAK12902, EAK13034, EAK15092, EAK22483, EAK23222, EAK24187, EAK24674, EAK28785, EAK34731, EAK34742, EAK36883, EAK37522, EAK42705, EAK43213, EAK52580, EAK53452, EAK58759, EAK62665, EAK63558, F84187, F90272, G87635, G90413, H83880, H84320, JC7723, NP_060220, NP_080435, NP_193157, NP_214383, NP_276913, NP_293819, NP_294534, NP_294585, NP_295972, NP_338490, NP_376437, NP_377056, NP_388895, NP_441167, NP_441254, NP_442491, NP_442727, NP_562475, NP_568712, NP_601630, NP_601630, NP_616426, NP_624522, NP_626360, NP_630834, NP_643053, NP_647302, NP_659552, NP_661086, NP_661546, NP_6617 01, NP_662300, NP_681023, NP_681127, NP_682351, NP_693380, NP_693382, NP_737250, NP_763380, NP_786524, NP_822198, NP_822828, NP_827278, NP_851528, NP_857496, NP_868798, NP_869339, NP_870237, NP_874530, NP_874561, NP_874977, NP_892236, NP_892265, NP_892458, NP_893232, NP_894882, NP_895385, NP_895793, NP_895829, NP_896854, NP_896994, NP_898304, NP_898346, NP_902647, NP_923340, NP_923639, NP_923813, NP_925079, NP_931515, NP_936379, NP_940208, NP_945754, NP_946860, NP_946866, NP_948851, NP_962004, NP_968600, NP_974222, NP_974545, O49901, P17059, P54971, P54978, P54979, P54981, P54982, P74306, Q01671, Q02861, Q38893, Q40406, Q9FV46, Q9SE20, Q9SMJ3, Q9ZTN9, Q9ZTP4, S29314, S32171, S49624, S52586, S65060, T10701, T31463, T46822, T48646 T50745, T50749, T50893, T50910, T51119, T51123, XP_324732, XP_383241, XP_401825, XP_470568, XP_473486, XP_477063, XP_525801, XP_540198, YP_006049, YP_013621, YP_024310, YP_041986, YP_041988, YP_044561, YP_044564, YP_062471, YP_117947, YP_120612, YP_135077, YP_136483, YP_145331, YP_145348, YP_171014, YP_172823, YP_173078, YP_173207, YP_184572, YP_187368, YP_187371, YP_187371, YP_187371, ZP_000490, ZP_000509, ZP_000518, ZP_000566, ZP_000627, ZP_000627, ZP_001073, ZP_001081, ZP_001091, ZP_001116, ZP_001117, ZP_001119, ZP_001124, ZP_001510, ZP_001591, ZP_001593, ZP_001602, ZP_001614, ZP_001645, ZP_001650, ZP_001722, ZP_001746, ZP_001752, ZP_001770, ZP_001777, ZP_001787, ZP_001837, ZP_001867, ZP_002073, ZP_002077, ZP_002339, ZP_002680, ZP_002705, ZP_002771, ZP_002892, ZP_002916, ZP_002963, ZP_003022, ZP_003036, ZP_003107, ZP_003202, ZP_003258, ZP_003268, ZP_003269, ZP_003276, ZP_003283, ZP_003557, ZP_003559, ZP_003565, ZP_003577, ZP_003593, ZP_003595, ZP_003685.

The synthases and lycopene cyclases are preferably enzymes having one of the following genebank accession numbers: 1613414C, A49558, AAA19428, AAA32836, AAA64982, AAB87738, AAC44849, AAD38051, AAF78202, AAF82616, AAG10427, AAG28701, AAK07734, AAK07735, AAK15621, AAL02001, AAL76346, AAL82578, AAM45379, AAM48647, AAM62787, AAM94363, AAN85600, AAO24767, AAO39835, AAO46895, AAO47570, AAO73816, AAP22038, AAP55451, AAP55453, AAP55461, AAP55471, AAP55484, AAP55486, AAP56083, AAP56124, AAP56127, AAP56136, AAP56148, AAP56155, AAP56156, AAP56157, AAP56158, AAP79176, AAQ91837, AAR08445, AAR31885, AAR37803, AAR37856, AAR86104, AAR87868, AAR98492, AAS02284, AAS17009, AAS18307, AAT28184, AAT35222, AAT38473, AAT46069, AAT74581, AAT90319, AAV74394, AAW23162, AC2035, AC2035, BAB18514, BAB79604, BAD07278, BAD07286, BAD62106, BAD62107, C90061, CAA47625, CAA68575, CAB07958, CAB38740, CAB51949, CAB56063, CAB86388, CAB93661, CAB94795, CAC19567, CAC27383, CAD19988, CAD29284, CAE76609, E37802, E No. 84320, EAA98758, EAB01965, EAB04170, EAB07138, EAB09791, EAB19826, EAB35029, EAB41375, EAB78706, EAB92586, EAC06949, EAC18360, EAC25793, EAC29883, EAC32813, EAC33105, EAC38486, EAC52233, EAC60029, EAC68026, EAC96197, EAD08701, EAD20866, EAD32755 EAD38008, EAD50152, EAD50402, EAD81123 EAD93882, EAE12860, EAE16121, EAE31084, EAE35665, EAE44717, EAE46627, EAE47846, EAE72264, EAE76009, EAE86335, EAE89581, EAF18881, EAF64277, EAF67931, EAF84745, EAF94004, EAG06083, EAG21950, EAG43625, EAG50171, EAG57517 , EAG62787, EAG65580, EAG68110, EAG72283, EAG78750, EAG80445, EAG93220, EAH04927, EAH08972, EAH10377, EAH22151, EAH31461, EAH50033, EAH64480, EAH79040, EAH99976, EAI02786, EAI02787, EAI03575, EAI05900, EAI61004, EAI70669, EAI83938, EAJ05110, EAJ05569 EAJ08876, EAJ35156, EAJ38900, EAJ49645, EAJ54357, EAJ60475, EAJ64125, EAJ67499, EAJ76471, EAJ76950, EAJ78637, EAJ78787, EAJ79616, EAJ80356, EAJ81914, EAJ87417, EAK08514, EAK08523, EAK12901, EAK22180, EAK24859, EAK28345, EAK34732, EAK34736, EAK3 7296, EAK37521, EAK56335, G84363, NP_274195, NP_284085, NP_294586, NP_388961, NP_441168, NP_443763, NP_624523, NP_630832, NP_662273, NP_682350, NP_693381, NP_786525, NP_822199, NP_822829, NP_851527, NP_868799, NP_874560, NP_879992, NP_884101, NP_889809, NP_892264, NP_895828, NP_898345, NP_902648, NP_902649, NP_924690, NP_931516, NP_946861, NP_949079, NP_962005, NP_968601, O07333, P08196, P21683, P37269, P37271, P37272, P53797, P54975, P54977, P65860, Q9SSU8, Q9UUQ6, S22474, S32170, S52587, S56668, S68307, T10702, T46594, T50746, T50895, XP_324765, XP_383242, XP_403902, YP_006040, YP_103126, YP_112342, YP_117945, YP_120611, YP_136628, YP_136629, YP_145340, YP_145343, YP_160917, YP_160918, YP_162605, YP_172822, YP_187369, YP_192648, ZP_000044, ZP_001091, ZP_001591, ZP_001657, ZP_001690, ZP_001746, ZP_001837, ZP_001867, ZP_002096, ZP_002248, ZP_002450, ZP_002680, ZP_002710, ZP_002791, ZP_002892, ZP_002916, ZP_003036, ZP_003269, ZP_003351, ZP_003487, ZP_003501, ZP_003591, ZP_003628.

The carotenoid ketolases are preferably enzymes having one of the following genebank accession numbers: AAA99932, AAB48668, AAC25611, AAF78203, AAH16427, AAN03484, AAN85497, AAN86030, AAO64399, AAQ23139, AAT35222, AAT35555, AAV41372, AB2307, AF2204, BAB54999, BAB58879, BAC98366, CAA60478, CAB56059, D87673, EAA79304, EAA80363, EAA81403, EAA84711, EAB82380, EAB86624, EAC05755, EAD12219, EAD71182, EAD94927, EAF11582, EAF98072, EAG19345, EAG38273, EAG79800, EAG96474, EAH00349, EAH36448, EAH40683, EAH53180, EAH96648, EAI05260, EAI17468, EAI53009, EAI54054, EAI67818, EAI68153, EAI89684, EAJ27674, EAJ45589, EAJ45589, EAJ67118, EAJ74221, EAJ74653, EAJ88396, EAJ88887, EAK06069, EAK11467, EAK16824, EAK28828, EAK28828, EAK31112, EAK42591, NP_045063, NP_081575, NP_338204, NP_440788, NP_441220, NP_682690, NP_770721, NP_848964, NP_857223, NP_881760, NP_882469, NP_886657, NP_895643, NP_896386, NP_897461, NP_924674, NP_927525, NP_947075, P54972, Q39982, Q44261, T31123, XP_330780, XP_368 852, XP_380194, XP_383758, XP_405100, XP_409222, YP_102417, YP_108945, YP_132414, YP_154670, YP_ 166682, YP_168846, YP_172377, ZP_001068, ZP_001112, ZP_001607, ZP_001757, ZP_001787, ZP_002218, ZP_002456, ZP_003028, ZP_003107, ZP_003264, ZP_003458, ZP_003513.

The carotenoid hydroxylases are preferably enzymes having one of the following genebank accession numbers: AAC44852, AAC49443, AAD54243, AAG10430, AAG10793, AAG33636, AAL80006, AAM44971, AAM51300, AAM77007, AAN85601, AAO53295, AAS48097, AAS55552, AAS88426, AAT48741, AAT84408, AAV85452, AAV85453, BAA14129, BAB79605, BAC77670, BAD07283, BAD07291, CAA70427, CAA70888, CAB55625, CAB55626, CAB56060, CAC06712, CAC95130, EAB30128, EAC49462, EAC86129, EAD61089, EAD76156, EAD88640, EAE27903, EAE28203, EAE78743, EAF12173, EAH29370, EAH44202, EAI00766, EAI29017, EAJ30844, EAJ72524, EAK10611, EAK53455, EAK63955, H90469, NP_745389, NP_922503, P54973, Q44262, S52982, XP_473611, YP_024309, ZP_003055, ZP_003107.

The astaxanthin synthases and putative astaxanthin synthases are preferably enzymes having one of the following Genbank accession numbers: AAM56288, XP_571276, EAL20013, XP_401804, XP_397817, XP_399595, XP_403279, XP_382294, XP_406021, XP_381224, XP_391479, XP_569261, EAL22841, XP_359866.

The lycopene cyclases, beta and epsilon subunits are preferably enzymes having one of the following genebank accession numbers: AAK07431, , Q38932, AAB53336, AAG10428, AAK07434, AAM45382, O65837, AAL69394, BAE79549, XP_463351, AAS48096, AAX92679, AAL92114, AAK07433, AAL47019, AAT46065, BAD07293, BAD07285, BAD07277, EAJ62839, BAE43547, BAE43550, BAE43557, BAE43558, BAE43553, BAE43545 , BAE43556, BAE43552, BAE43560, BAE43554, BAE43551, BAE43519, BAE43535, BAE43541, BAE43542, BAE43517, BAE43534, BAE43537, BAE43533, BAD02774, BAD02766, BAE43540, BAE43514, BAE43544, BAE43538, BAE43528, BAE43546, BAE43526, BAE43543, BAD02742, BAD02770 , BAE43522, BAE43559, BAE43527, BAE43548, AAF44700, BAE43555, BAE43549, AAU14144, AAN86060, AAR89632, AAM21152, AAD38049, AAU05146, AAU05145, AAK07430, , BAE79544, BAE78471, Q43415, AAF23013, , AAW88382, AAG10429, AAM45381, AAM14335, AAO18661, AAA81880, Q43503, S66350, XP_464409, CAD70565, Q43578, AAL92175, AAX54906, S66349, AAG21133, CAB92977, CAB93342, Q9SEA0, Q42435, AAO64977, Q40424, , AAQ02668, CAA54961, EAJ62838, YP_401079, YP_172741, ZP_011, EAK50052, NP_892751, NP_875182, YP_382237, YP_397130, NP_896821, YP_397570, ZP_010, EAK17149, YP_291882, NP_875528, NP_893181, NP_895600, EAI47456, YP_291268, ZP_010, AAF34191, ZP_010, YP_376736 , ZP_003, NP_894954, AAT76051, EAK22047, NP_294525.

Saccharomyces strains, especially S. cerevisiae strains are available from the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstraße 7B, 38124 Braunschweig.

Particularly preferably, the strain according to the invention additionally comprises a transcriptionally deregulated HMG-CoA reductase. The HMG-CoA reductase is preferably deregulated in such a way that the enzyme formed no longer has an N-terminal membrane domain but only the catalytic subunit. As a result, there is no feedback regulation due to intermediates of the isoprenoid metabolism.

Most preferably, the strain of the invention is AH22tH3ura8-GK4-GK6.

A yeast strain according to the invention is prepared, for example, by introducing and expressing in Saccharomyces, preferably in S. cerevisiae, the genes mentioned, preferably the heterologous Ge genes GGPP synthase and phytoene synthase, and / or phytoene desaturase and / or lycopene cyclase and overexpressing the gene PDR1 or a PDR1 homolog is introduced and overexpressed. For yeast codon optimized variants of the genes are preferably used, as they have been mentioned, for example, above. The expression or overexpression of said genes can be done both episomally and chromosomally.

The transformation of the yeast cells with said genes is preferably carried out in the usual way, such as in Schiestl RH, et al., Curr Genet. Dec, 16 (5-6): 339-346 (1989), or Manivasakam P., et al., Nucleic Acids Res. Sep 11, 21 (18): 4414-4415 (1993). , or Morgan RJ, Experientia Suppl., 46: 155-166 (1983) described.

For the expression of the heterologous genes or the overexpression of native genes it is preferred to use promoters which are not subject to catabolite repression and are not specific Need carbon source for induction. The skilled worker is aware of such promoters. These include, for example, promoters of the genes phosphofructokinase (PPK, triose phosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GPD, TDH3 or GAPDH), pyruvate kinase (PYK), phosphoglycerate kinase (PGK), glucose-6-phosphate isomerase promoter (PGI1) Other suitable promoters can be found in the application WO 93/03159 be removed. Suitable promoters are also ribosomal protein-encoding gene promoters (TEFI), the lactase gene promoter (LAC4), the alcohol dehydrogenase promoter (ADH1), preferably an altered (constitutive) version of the ADH1 promoter (SEQ ID NO: 8), ADH4 and the like ), the enolase promoter (ENO1) and the hexose (glucose) transporter promoter (HXT7). However, other promoters, both constitutive and inducible, and enhancer or upstream activating sequences are known in the art.

Suitable terminator sequences are e.g. available from the cytochrome c1 (CYC1) gene or the enolase 1 (ENO1) gene.

Preferred promoters are the TEF1 promoter, TDH3 promoter, constitutive version of the ADH1 promoter, ENO1 promoter

Preferably, the heterologous genes, more preferably the genes GGPP synthase and phytoene synthase, and / or phytoene desaturase and / or lycopene cyclase are introduced by chromosomal integration, for example in the form of a gene cassette in S. cerevisiae. The gene PDR1 or a PDR1 homolog is preferably also introduced in the form of a gene cassette. Such a method is described in the examples.

The sequence of the genes phytoene synthase / lycopene cyclase, GGPP synthase and phytoene desaturase is known from the databases of the National Center for Biotechnology Information (NCBI).

PDR1 encodes a yeast transcription factor. Its gene sequence is known from the databases of the National Center for Biotechnology Information (NCBI).

Preferably, the yeast strain according to the invention can be prepared by the fact that in a S. cerevisiae strain which can produce carotenoids, since it contains the heterologous genes coding for the enzymes phytoene synthase / lycopene cyclase, GGPP synthase and phytoene desaturase of X. dendrorhous, and in in which the HMG-CoA reductase gene is deregulated, the PDR1 gene or a homolog of the PDR1 gene is introduced and overexpressed.

Preferably, a yeast strain according to the invention can be produced by using a yeast strain with a deregulated terpene biosynthesis pathway as starting strain. The preparation of such strains is for example in WO2011023298 described.

Such strains show a strong overproduction of triterpene squalene and all precursors relevant to carotenoid production (see 1 ). A strong overproduction is preferably to be understood as meaning that after 72 hours of cultivation under standard conditions in a shaking flask> 10% of the dry biomass is squalene or one of the precursors mentioned. Preferably, a carotenoid selected from the group of phytoene, beta-carotene, beta-cryptoxanthin, zeaxanthin can be produced with a strain according to the invention.

The deregulation of the terpene biosynthesis pathway is preferably achieved by transcriptionally deregulating HMG-CoA reductase and removing it from the negative regulation at the protein level. HMG-CoA reductase is the key enzyme for terpene overproduction, as it is known as a pacemaker enzyme in the sterol biosynthetic pathway and represents a "bottleneck" in the biosynthetic pathway from acetyl-CoA to squalene (see 1 ). In these strains a PDR1 gene is introduced in a manner known per se. The strain must express at least the two enzymes GGPP synthase and phytoene synthase and may optionally express any number of other carotenoid synthesis genes.

These yeast strains are cultured in a manner known per se and the carotene isolated after cell disruption. The production of carotenoids with yeasts of the genus Saccharomyces is, for example, in Ukibe, K., Hashida, K., Yoshida, N., Takagi, H., Applied and Environmental Microbiology. 2009, 75 (22), pp. 7205-7211 ; Verwaal, R., Jiang, Y., Wang, J., Yeast. 2010, 27 (12), pp. 983-998 ; Verwaal, R., Wang, J., Meijnen, J.-P., Applied and Environmental Microbiology. 2007, 73 (13), pp. 4342-4350 ; Misawa, N., Shimada, H., Journal of Biotechnology. 1997, 59 (3), pp. 169-181 or Schmidt-Dannert, C., Curr Opin Biotechnol. 2000 11 (39), pp. 255-61 described. The invention thus also relates to a process for the production of carotenoids in which a yeast strain is fermented in a manner known per se and the carotenoids are isolated after cell disruption, characterized in that the yeast strain is a yeast strain according to the invention.

Surprisingly, there is a significantly increased amount of intracellular carotenoids after 120 to 144 hours cultivation of the strain in the shake flask. The content of intracellular carotenoids was determined after cell disruption by HPLC and compared with the value of a control strain which expresses only the carotenoid synthesis genes and does not overexpress the PDR1 gene.

1 shows exemplarily the biosynthetic pathway of squalene in the yeast Saccharomyces cerevisiae.

2 shows gene cassette 4, which is prepared and used in Example 3.

3 Figure 1 shows the gene cassette 6 prepared and used in Example 4.

4 shows by way of example the carotenoid biosynthesis pathway in the yeast Saccharomyces cerevisiae

The following examples serve to further illustrate the invention.

Example 1: Cultivation for the strain evaluation (growth and productivity)

• 1) Vorkultur: 20 ml WMVIII Medium wurde in einem 100 ml Schüttelkolben mit 20 µl der entsprechenden Glycerinkultur angeimpft und für 48 h bei 30 °C und 150 rpm auf einem Inkubationsschüttler geschüttelt.
• 2) Hauptkultur: 20 ml WMVIII Medium wurde in einem 100 ml Schüttelkolben mit 1 % (v/v) der Vorkultur angeimpft und für 120 h bei 30 °C und 150 rpm auf einem Inkubationsschüttler geschüttelt.

The standard culture for the strains of the yeast Saccharomyces cerevisiae was as follows:

• 1) Preculture: 20 ml of WMVIII medium were inoculated in a 100 ml shake flask with 20 μl of the corresponding glycerol culture and shaken for 48 h at 30 ° C. and 150 rpm on an incubation shaker.
• 2) Main culture: 20 ml WMVIII medium was inoculated in a 100 ml shake flask with 1% (v / v) of the preculture and shaken for 120 h at 30 ° C and 150 rpm on a incubation shaker.

Genetically AH22tH3ura8-GK4 strains (see example 2 and 3 for preparation) are auxotrophic for leucine. Therefore, the medium was supplemented with leucine (400 mg / L)

Genetically AH22ura3 or AH22tH3ura8 strains are auxotrophic for leucine and uracil. Therefore, the medium was supplemented with leucine (400 mg / L) and uracil (100 mg / L)).

Composition of the WMVIII medium (1 L): 50 g sucrose, 250 mg NH ₄ H ₂ PO ₄ , 2.8 g NH ₄ Cl, 250 mg MgCl ₂ × 6H ₂ O, 100 mg CaCl ₂ × 2H ₂ O, 2 g KH ₂ PO ₄ , 550 mg MgSO ₄ × 7H ₂ O, 75 mg meso-inositol and 10 g Na-glutamate. The described substances were weighed and made up with 1 L of distilled water. After autoclaving, sterile-filtered (0.22 μm) trace elements, vitamins and amino acids were added: 1 ml 1000 × trace elements, 4 ml 250 × vitamin solution (stock solutions).

Trace elements: 1000 times concentrated, for one liter: 1.75 g ZnSO ₄ .7H ₂ O; 0.5 g of Fe ₂ SO ₄ .7H ₂ O; 0.1 g CuSO ₄ × 5 H ₂ O; 0.1 g MnCl ₂ × 4 H ₂ O; 0.1 g NaMoO ₄ × 2 H ₂ O.

Vitamin solution: concentrated 250 times, for one liter: 2.5 g nicotinic acid; 6.25 g of pyridoxine; 2.5 g of thiamine; 0.625 g of biotin; 12.5 g of Ca-pantothenic acid.

Both solutions were sterile filtered (0.22 μm). Leucine was supplemented if necessary (400 mg / L). Amino acid stock solutions were prepared at a concentration of 20 mg / ml and sterile filtered.

Example 2 Construction of the strain AH22tH3ura8

The DNA sequence of t-HMG1 ( Basson et al. (Mol. Cell Biol. 8 (1988), 3793-3808 ) was isolated by PCR from genomic DNA of Saccharomyces cerevisiae S288C available from the American Type Culture Collection ( ATCC 204508 ) using standard methods ( Mortimer and Johnston (Genetics 113 (1986), 35-43 ). The primers used in this case were the DNA oligomer tHMG-5 '(SEQ ID NO: 1) and tHMG-3' (SEQ ID NO: 2). The DNA fragment that was obtained was introduced into the cloning vector pUC19 after Klenow treatment, available from New England Biolabs Inc., USA, Ipswich and resulted in the vector pUC19-tHMG.

After plasmid isolation and restriction of pUC19-tHMG with the endonucleases EcoRI and BamHI, the fragment obtained was inserted into the yeast expression vector pPT2b ( Lang and Looman (Appl. Microbiol. Biotechnol. 44 (1995), 147-156 ), which was also treated with EcoRI and BamHI. The plasmid pPT2b-tHMG thus prepared contains the truncated ADH1 promoter ( Bennetzen and Hall (Yeast 7 (1982), 475-477 ) and TRP1 terminator ( Chumper G, Carbon J. Sequence of a yeast DNA fragment containing a chromosomal replicator and the TRP1 gene. Genes. 1980 Jul; 10 (2): 157-166 ) between which the tHMG1 DNA fragment can be found.

A DNA region was isolated from the vector pPT2b-tHMG with the endonucleases EcoRV and NruI. This area carries the so-called medium-length ADH1 promoter, the t-HMG1 gene and the TRP1 terminator.

This DNA piece was obtained in the yeast vector YEp13 from the American Type Culture Collection ( ATCC 37115 ) brought in ( Fischhoff et al. (Gene 27 (1984), 239-251) ) treated with the endonucleases SphI and a DNA polymerase. The resulting plasmid YEp13-tHMG was transformed into S. cerevisiae AH22URA3 ( Polakowski et al 1999 ; AH22 is commercially available; Preparation of AH22ura3 is beschrie-described) introduced.

Chromosomal integration of t-HMG1 into AH22ura3 using the chromosomal integrative plasmid YDpUHK3

The vector YEpH2 ( US2004235088 A1 ) was treated with the endonucleases EcoRV and NruI. This produced a DNA fragment having the following regions: a transcriptionally activating region of the tetracycline resistance gene ( Sidhu and Bollon (10 (1990) 157-166) ), the medium-length ADH1 promoter, the t-HMG1 and TRP1 terminator (expression cassette). This DNA fragment was introduced into the vector YDpU ( Berben et al. 1991 Berben G., Dumont J., Gilliquet V, Bolle PA. and Hilger F. (1991), Yeast 7, 475-477 ) who was treated with StuI. The vector YDpUH2 / 12 which was produced therewith was treated with the endonucleases SmaI and ligated with a DNA sequence coding for kanamycin resistant ( Webster, TD, Dickson, RC (1983), Gene 26: 243-252 ). The construct that was prepared (YDpUHK3) was treated with EcoRV. The yeast strain Saccharomyces cerevisiae AH22 ( ATCC 38626; ATCC Manassas, Virginia ) was transformed with this construct.

The transformation of the yeast with the linearized vector results in the chromosomal integration of the entire vector in the URA3 gene locus. To eliminate the regions of the integrated vector that are not part of the expression cassette ( E. coli origin, E. coli ampicillin resistance gene, TEF promoter and kanamycin resistance genes ), the transformed yeasts were subjected to selection pressure by FOA selection ( Boeke et al. (Methods in Enzymology 154 (1987), 164-175) ), which favors uracil auxotrophy in yeast.

The uracil auxotrophic strain thus obtained bears the name AH22tH3ura8 ( Polakowski 1998 ) and has integrated the t-HMG1 expression cassette chromosomally in the URA3 gene.

Example 3:

Construction of the strain AH22tH3ura8-GK4

The genes crtE (optimized for yeast codon, SEQ ID NO: 3), crtYB (optimized for yeast codon, SEQ ID NO: 4) and crtI (optimized for yeast codon SEQ ID NO: 5) of X. dendrorhous were used in GeneArt ( GenArt Regensburg) in a cluster (gene cassette 4, GK4). The three genes were arranged as in 2 with the URA3 marker gene for selection in yeast (URA3 coding region including 400 bp upstream and 200 bg downstream; http://www.yeastgenome.org/cgibin/getSeq?query=YEL021W&flankl=400&flankr=200&format=fasta ) and flanking regions homologous to the native HO locus (the first and last 40 bp of the HO coding region http://www.yeastgenome.org/cgibin/getSeq?query=YDL227C&seqtype=ORF%20Genomic%20DNA&format=fas ta ) for the genomic integration of the gene cassette into the HO locus. For expression of the crtE genes, a modified (constitutive) version of the ADH1 promoter (SEQ ID NO: 8) and TRP1 terminator is used (300 bp downstream of the TRP1 coding region; http://www.yeastgenome.org/cgibin/getSeq?query=YDR007W&flankl=1000&flankr=1000&format=fasta ), whereas for the crtYB gene the ENO1 promoter and ENO1 terminator are used (600 bp upstream and 300 bp downstream of the ENO1 coding region; http://www.yeastgenome.org/cgibin/getSeq?query=YGR254W&flankl=600&flankr=300&format=fasta ) and for the crtI gene the TDH3 promoter and TDH3 terminator are used (600 bp upstream and 300 bp downstream of the TDH3 coding region, see sequence http://www.yeastgenome.org/cgibin/getSeq?query=YGR192C&flankl=600&flankr=300&format=fasta ).

The strain AH22tH3ura8 from example 2 is used for the transformation with this gene cassette ( 2 ). After transformation of the corresponding yeast strain with the gene cassette via the lithium acetate method ( Gietz et al., 1992 ) he was on WMVIII ( Lang and Looman, 1995 ) Agar plates without uracil are cultured for selection on uracil prototrophic colonies (transformants carrying the expression cassette in place of the HO coding region in the genome). The strain AH22tH3ura8-GK4 was obtained.

Example 4: Overexpression of the S. cerevisiae PDR1 gene

The gene PDR1 (optimized for yeast codon, SEQ ID NO: 6) was in GeneArt (GeneArt, Regensburg, Germany) in a cluster (gene cassette 6, GK6) with the LEU2 marker for the selection of positive transformants (the LEU2 coding region incl 400 bp upstream and 200 bp downstream, sequence see http://www.yeastgenome.org/cgibin/getSeq?query=YCL018W&flankl=4 00 & flankr = 200 & format = fasta and with flanking regions homologous to the native YHRCΔ14 locus (the first and last 40 bp of the YHRCΔ14 coding region, see sequence http://www.yeastgenome.org/cgibin/getSeq?query=YHRCdelta14&seqt ype = ORF% 20Genomic% 20DNA & format = fasta ). For expression of the PDR1 gene, the ENO1 promoter and ENO1 terminator was used (600 bp upstream and 300 bp downstream of the ENO1 coding region, see Sequence http://www.yeastgenome.org/cgibin/getSeq?query=YGR254W&flankl=600&flankr=300&format=fasta )

The strain AH22tH3ura8-GK4 from Example 3 (expressing crtE, crtYB and crtI) was transformed with this gene cassette. The transformation was carried out with the lithium acetate method ( Gietz et al., 1992 ). The strain was on WMVIII ( Lang and Looman, 1995 ) Agar plates cultured without uracil and leucine for selection on uracil and leucine prototrophic colonies.

The dry matter (BTS) and the concentration of carotenoids produced was determined for the thus prepared inventive strain AH22tH3ura8-GK4-GK6 and for the known from the prior art starting strain AH22tH3ura8-GK4 according to the prior art, after each of the strains for 120 h in Shake flasks were cultivated in WMVIII medium at 28 ° C on a incubation shaker.

For the extraction of the carotenoids, 1.8 ml of culture were added to an Eppendorf tube. The cells were pelleted and washed twice with 1 ml H ₂ O. After the second wash, the resuspended cells were placed in a pre-weighed vessel into which a hole had been punched in the lid. The cells were lyophilized overnight. After complete drying, the vessel was weighed again to calculate the cell dry weight. 0.25 ml of the same culture was added to a 2 ml vessel for carotenoid extraction. The cells were pelleted and the supernatant was removed. The pelleted cells were frozen at -80 ° C and stored.

The same volume of cubic zirconia beads was added to the cell pellets, along with 1 ml of ice-cold extraction solution (a 50/50 v / v mixture of hexane and ethyl acetate containing 0.01% butylhydroxytoluene (BHT)). The mixture was shaken (Mini-BeadBeater-8, BioSpec Products, Inc.) at the highest level for 5 minutes at 4 ° C. The mixture was then centrifuged for one minute (max speed) and the supernatant was collected and placed in a chilled 16 ml glass vial. The cell debris was again extracted at least three times without the addition of zirconia beads. All supernatants were pooled in a 16 ml glass vial. After extraction, the glass vials were centrifuged at 4 ° C for 5 minutes at 2000 rpm in a Sorvall benchtop centrifuge and the supernatant transferred to a new 16 ml glass vial. The supernatants were concentrated with a Speed Vac (room temperature in the dark) and the samples were stored at -20 ° C or -80 ° C until just before HPLC analysis. Throughout the implementation, care was taken to avoid contact with oxygen, light, heat and acid.

For carotenoid analysis, the samples were resuspended in ice-cold extraction solvent. An Alliance HPLC 2795 (Waters) equipped with a Waters XBridge C18 column (3.5 μm, 2.1 x 50 mm) and a Thermo Basic 8 guard column (2.1 x 10 mm) was used. Reliable carotenoid samples were used as standards. The mobile phases and flow rates are shown below (solvent A = ethyl acetate, solvent B = water, solvent C = methanol, solvent D = acetonitrile). The injection volume was 10 μl. The detector was a Waters 996 photodiode array detector. The retention times for lipophilic molecules are astaxanthin (1,159 min), zeaxanthin (1,335), β-apo-8'-carotenal (2.86 min), ergosterol (3.11 min), lycopene (3.69 min), beta-carotene (4.02 min) and phytoene (4.13 min). Astaxanthin, zeaxanthin, β-apo-8'-carotenal, lycopene and β-carotene were detected at 475 nm, phytoene at 286 nm. Table 1: Retention times for lipophilic molecules Time (min) Flow rate (mL / min) % A % B % C % D 12:50 0.0 20.0 0.0 80.0 3:00 1:00 20.0 0.0 0.0 80.0 4:50 1:00 80.0 0.0 20.0 0.0 5:50 1:00 0.0 0.0 60.0 40.0 6:50 1:00 0.0 0.0 80.0 20.0 7:50 1:00 0.0 0.0 100.0 0.0 8:50 1:00 0.0 0.0 100.0 0.0 9:50 1:00 0.0 20.0 0.0 80.0 10:50 12:50 0.0 20.0 0.0 80.0

The results are shown in Table 2. Table 2: Carotenoid production of the S. cerevisiae strain AH22tH3ura8-GK4 with and without additional expression of the PDR1 gene (on GK6). tribe Cultivation time [h] BTS [g / l] Phytoene [μg / g _BTS ] Beta carotene [μg / g _BTS ] AH22tH3ura8-GK4-GK6 AH22tH3ura8-GK4 120 120 10.5 10.8 11017 2661 842 294 BTS: bio dry substance

It was found that the strain according to the invention which carries the PDR1 gene (line 1 of Tab. 2) produces significantly higher amounts of carotenoids than the strain according to the prior art. SEQUENCE LISTING

This is followed by a sequence protocol according to WIPO St. 25. This can be downloaded from the official publication platform of the DPMA.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• WO 93/03159 [0037]
• WO 2011023298 [0044]
• US 2004235088 A1 [0065]

Cited non-patent literature

• Ukibe, K. et al, 2009, Appl Environ Microbiol 75: 7205-11 or Verwaal, R. et al., Yeast 27: 983-98 [0007]
• Ukibe, K. et al, Applied and Environmental Microbiology. 2009, 75 (22), pp. 7205-7211 [0008]
• R. et al, Applied and Environmental Microbiology. 2007, 73 (13), pp. 4342-4350) [0008]
• R. et al, Yeast 27: 983-98 [0010]
• "Pleiotropic drug response" in yeast (yeastgenome.org) [0011]
• Old school, Stephen; Gish, Warren; Miller, Webb; Myers, Eugene; Lipman, David (1990). "Basic local alignment search tool". Journal of Molecular Biology 215 (3): 403-410 [0014]
• Schiestl RH, et al., Curr Genet. Dec, 16 (5-6): 339-346 (1989), or Manivasakam P., et al., Nucleic Acids Res. Sep 11, 21 (18): 4414-4415 (1993) [0036]
• Morgan RJ, Experientia Suppl., 46: 155-166 (1983) [0036]
• Ukibe, K., Hashida, K., Yoshida, N., Takagi, H., Applied and Environmental Microbiology. 2009, 75 (22), pp. 7205-7211 [0047]
• Verwaal, R., Jiang, Y., Wang, J., Yeast. 2010, 27 (12), pp. 983-998 [0047]
• Verwaal, R., Wang, J., Meijnen, J.-P., Applied and Environmental Microbiology. 2007, 73 (13), pp. 4342-4350 [0047]
• Misawa, N., Shimada, H., Journal of Biotechnology. 1997, 59 (3), pp. 169-181 [0047]
• Schmidt-Dannert, C., Curr Opin Biotechnol. 2000, 11 (39), pp. 255-61 [0047]
• Basson et al. (Mol. Cell Biol. 8 (1988), 3793-3808 [0061]
• ATCC 204508 [0061]
• Mortimer and Johnston (Genetics 113 (1986), 35-43 [0061]
• Lang and Looman (Appl.Microbiol.Biotechnol 44 (1995), 147-156 [0062]
• Bennetzen and Hall (Yeast 7 (1982), 475-477 [0062]
• Chumper G, Carbon J. Sequence of a yeast DNA fragment containing a chromosomal replicator and the TRP1 gene. Genes. 1980 Jul; 10 (2): 157-166 [0062]
• ATCC 37115 [0064]
• Fischhoff et al. (Gene 27 (1984), 239-251) [0064]
• Polakowski et al 1999 [0064]
• Sidhu and Bollon (10 (1990) 157-166) [0065]
• Berben et al. 1991 Berben G., Dumont J., Gilliquet V, Bolle PA. and Hilger F. (1991), Yeast 7, 475-477 [0065]
• Webster, TD, Dickson, RC (1983), Gene 26: 243-252 [0065]
• ATCC 38626; ATCC Manassas, Virginia [0065]
• E. coli origin, E. coli ampicillin resistance gene, TEF promoter and kanamycin resistance genes [0066]
• Boeke et al. (Methods in Enzymology 154 (1987), 164-175) [0066]
• Polakowski 1998 [0067]
• http://www.yeastgenome.org/cgibin/getSeq?query=YEL021W&flankl=400&flankr=200&format=fasta [0068]
• http://www.yeastgenome.org/cgibin/getSeq?query=YDL227C&seqtype=ORF%20Genomic%20DNA&format=fas ta [0068]
• http://www.yeastgenome.org/cgibin/getSeq?query=YDR007W&flankl=1000&flankr=1000&format=fasta [0068]
• http://www.yeastgenome.org/cgibin/getSeq?query=YGR254W&flankl=600&flankr=300&format=fasta [0068]
• http://www.yeastgenome.org/cgibin/getSeq?query=YGR192C&flankl=600&flankr=300&format=fasta [0068]
• Gietz et al., 1992 [0069]
• Lang and Looman, 1995 [0069]
• http://www.yeastgenome.org/cgibin/getSeq?query=YCL018W&flankl=4 00 & flankr = 200 & format = fasta [0070]
• http://www.yeastgenome.org/cgibin/getSeq?query=YHRCdelta14&seqt ype = ORF% 20Genomic% 20DNA & format = fasta [0070]
• http://www.yeastgenome.org/cgibin/getSeq?query=YGR254W&flankl=600&flankr=300&format=fasta [0070]
• Gietz et al., 1992 [0071]
• Lang and Looman, 1995 [0071]

Claims (8)

Yeast strain of the genus Saccharomyces with established carotenoid biosynthesis pathway, characterized in that it can overexpress a PDR1 gene or PDR1 homologue. Yeast strain according to claim 1, characterized in that the PDR1 or PDR1 homologue is the yeast codon-optimized gene PDR1 of SEQ ID NO: 6. Yeast strain according to claim 1 or 2, characterized in that it is a strain of the species Saccharomyces cerevisiae. Yeast strain according to claim 1, 2 or 3, characterized in that it expresses the genes GGPP synthase and phytoene synthase, and / or phytoene desaturase and / or lycopene cyclase and overexpressed at least one PDR1 gene or PDR1 homologue. Yeast strain according to one of claims 1 to 4, characterized in that it comprises a transcriptionally deregulated HMG-CoA reductase. A method for producing a yeast strain according to any one of claims 1 to 5, characterized in that introduced and expressed in Saccharomyces the genes GGPP synthase and / or phytoene desaturase and / or lycopene cyclase and overexpressing the gene PDR1 or introduced a PDR1 homolog and overexpressed. Process for the preparation of carotenoids in which a yeast strain is fermented in a manner known per se and the carotene is isolated after cell disruption, characterized in that the yeast strain is a yeast strain according to one of claims 1 to 5. A method according to claim 7, characterized in that a carotenoid selected from the group phytoene, beta-carotene, beta-cryptoxanthin, zeaxanthin is produced.

Images