WO2006136311A1 - Metabolic engineering of q10 production in yeasts of the genus sporidiobolus - Google Patents

Abstract

The invention relates to an expression system comprised of a host organism of the species Sporidiobolus and of a DNA vector of the one selection marker gene, which codes for a protein that, after transformation of the host organism, enables a selection of positive transformants and is selected from the group consisting of antibiotic-resistant genes, the genes, which complement an auxotrophy of the host organism, and the genes, which code for a protein, which are capable of entering a chromophoric reaction, whereby the expression of the selection marker gene is controlled by at least one genetic regulation element active in the host organism. The invention is characterized in that the DNA vector contains a gene to be expressed, which codes for a protein, whereby the gene to be expressed is a gene from the Q-10 biosynthetic pathway.

Description

Metabolism engineering of QlO production in yeasts of the genus Sporidiobolus

The invention relates to a system for the expression of genes from the Q10 biosynthesis pathway in yeasts of the genus Sporidiobolus.

Strains of the species Sporidiobolus ruineniae are known to be capable of naturally producing large quantities of the economically valuable natural product coenzyme Q10 (also called ubiquinone, hereinafter referred to as Q10) (see, for example, US Pat. No. 4,070,244, EP 1469078 A). However, the achieved Q10 yields are still too low for industrial use.

One way to increase Q10 yields is metabolic engineering of strains of the genus Sporidiobolus.

In this case, metabolite engineering is defined in a general form such that a genetic expression system is used to purposefully increase the yield of a desired metabolite in a host organism used for production. This can be done in one embodiment by the fact that enzymes of a metabolic pathway in recombinant form increasingly produced and thereby increased yields of the product are obtained from the metabolic pathway. In another embodiment, the production of individual enzymes of a metabolic pathway (for example negative regulators) or of enzymes of competing metabolic pathways is purposefully reduced or suppressed in order to increase the production yield of the desired metabolic product.

The host organism used for the metabolization engineering will usually be selected from among those from which the desired metabolite can be obtained in the correct form and already with naturally high yields. Strains of the genus Sporidiobolus are suitable for the metabolism engineering of the Q10 metabolism. A prerequisite for the metabolism engineering of the Q10 metabolic pathway in strains of the genus Sporidiobolus is thus a genetic expression system.

Genetic expression systems are known for various prokaryotic and eukaryotic microorganisms. These gene expression systems are characterized by the fact that they can be used to produce individual proteins in recombinant form. However, as already mentioned, metabolic engineering preferably selects a production host with, of course, already relatively high yields, and as a rule it is not possible to resort to an already available genetic expression system. Instead, a genetic expression system for strain improvement by metabolism engineering has to be newly developed.

Examples of known prokaryotic expression systems are Escherichia coli and Bacillus subtilis. The methods for the genetic manipulation of these organisms are well established. However, specific disadvantages of these expression systems are the often low production rates of enzymatically active protein due to the folding of the produced proteins in non-active form or the lack of post-translational modification of the expressed proteins (e.g., lack of incorporation of prosthetic groups or lack of glycosylation).

These disadvantages of prokaryotic expression systems can be circumvented by using eukaryotic expression systems. Common eukaryotic expression systems include cell culture systems of both mammalian cells and insect cells, as well as eukaryotic microorganisms such as yeasts or filamentous fungi. While in these expression systems the protein to be expressed is generally formed in active form, the production rate is in many cases too low, in particular in the expression of heterologous proteins. As an example, the expression in the yeasts Saccharomyces cerevisiae or Pichia pastoris serve, or in filamentous fungi of the genus Aspergillus. Filamentous mushrooms often cause problems in fermentative protein production.

For the reasons mentioned above, the established prokaryotic and eukaryotic expression systems are often not suitable for the genetic optimization of a metabolic pathway by metabolic engineering. Specifically, as far as the production of Q10 is concerned, this ubiquinone z. B. in E. coli (Q8), baker's yeast (Q6), Pichia pastoris (Q8) or Bacillus (Q8) is not produced in the correct form, so that a genetic optimization with great effort connected, since only the metabolic pathway to the Q10 has to be established.

Basidiomycete yeasts of the genus Sporidiobolus have been described early as good producers of Q10 (US 4070244). By contrast, no technical use was reported. EP 1469078 (corresponding to US Application Serial Number 10/803841) discloses a method for improving Q10 production in Sporidiobolus by classical strain optimization by means of mutagenesis and selection and subsequent fermentation. This method is suitable for producing Sporidiobolus strains with a Q10 productivity of 1.38 mg / g dry biomass or more.

Specifically, the good fermentation properties reported there make S. ruineniae not only an interesting host organism for Q10 production, but generally for metabolite engineering and also for recombinant protein expression.

Yeasts of the genus Sporidiobolus belong to the higher developed fungi of the genus Basidiomycetes. The sporidiobolus expression system according to the invention is therefore suitable not only for metabolism engineering but also for the production of proteins which can not be produced with other expression systems or only with poor yields. Little is known about the genetic engineering of fungi and yeasts of the Basidiomycete genus. For a sporidobolysis expression system, the prior art does not provide instructions for technical action. Wery et al. (Biotechnology Techniques (1998) 12 ₁ 399-405) z. B. report an expression system for the yeast Phaffia rhodozyma (synonymously also called Xanthophyllomyces dendrorhous), which is discussed for the microbial production of astaxanthin (a carotene). There, suitable DNA vectors are described for the selection of transformants. It will be comparatively high

Transformation rates of> 1000 transformants / μg vector DNA scored. However, the benefit of the Phaffia expression system for optimizing astaxanthin production has been low. Another disadvantage of the P. rhodozyma expression system is that the fermentation must be carried out at comparatively low temperatures of 20 ° C., which makes the technical implementation more difficult.

EP 0340986 B describes the use of the enhancer protein ADRI for enhancing heterologous, recombinant protein expression, based on a prior art genetic expression system, in the baker's yeast Saccharomyces cerevisiae, a yeast from the class of the Ascomycetes. The enhancer function of ADRI is assigned a general validity, including to enhance heterologous gene expression in yeasts of the genus Sporidiobolus. However, the person skilled in the art will not receive instructions for action from this invention in order to develop a genetic expression system for strains of the genus Sporidobus and how this could be used to improve Q10 production. Neither is an ADR1 gene from Sporidiobolus known nor is it disclosed how it could be isolated. Also, no sporidiobolus active genetic regulatory elements (promoters, terminators) are disclosed. Of the described expression vectors and the transformation method it is not known whether they are suitable for Sporidio bolus. Rather, the skilled artisan expects the transfer of one for yeasts from the class of ascomycetes developed genetic expression system on a yeast from the class of Basidiomycetes is not possible.

For various filamentous fungi of the basidiomycete genus, DNA vectors suitable for the transformation and selection of transformants have been described. EP 1066393 z. B. describes a method for the transformation of Trametes versicolor and the use of this expression system for the production of the enzyme laccase for industrial applications.

Phanerochaete chrysosporium has also been described as a method of genetic transformation (Alic et al (1991) Curr Genet 19, 491-494), as well as Pleurotus ostreatus (Yanai, K., et al., (1996) Biosci Biotech. Biochem 60, 472-475). US 5362640 describes DNA vectors for the transformation of Basidiomyceten Coriolus hirsutus.

The filamentous fungi-based expression systems are usually characterized by only a low transformation rate. In addition, the transformation process is very complex. These expression systems also have the disadvantage that the fermentation on an industrial scale is often difficult because of the filamentous cell growth and high viscosity of the fermented biomass.

An instruction for an expression system of yeasts of the genus Sporidio- bolus could therefore not be derived from the present state of the art.

The object of the present invention was to provide a genetic expression system for yeasts of the genus Sporidiobolus which makes it possible to improve the production of the economically valuable metabolite coenzyme Q10 by metabolite engineering.

The object is achieved by an expression system consisting of a host organism of the genus Sporidiobolus and a DNA vector which contains a selection marker gene which is suitable for encodes a protein which after transformation of the host organism allows selection of positive transformants and is selected from the group of antibiotic resistance genes, the genes which complement an auxotrophy of the host organism and the genes which code for a protein that belong to a host in which the expression of the selection marker gene is controlled by at least one genetic regulatory element active in the host organism, characterized in that a DNA vector contains a gene to be expressed which codes for a protein in which the gene to be expressed is a gene from the Q10 biosynthesis pathway and in which the expression of the gene from the Q10 biosynthesis pathway is controlled by at least one genetic regulation element active in the host organism.

In a preferred embodiment of the invention, the selection marker gene and the gene from the Q10 biosynthesis pathway are contained together on a DNA vector, the expression of the two genes being controlled by at least one genetic regulatory element active in the host organism.

If the two genes are present on different DNA vectors, both vectors are transformed simultaneously (co-transformation).

Particularly preferred host organisms for the expression system according to the invention are strains of the species Sporidiobolus ruineniae.

Particularly preferred is the strain Sporidiobolus ruineniae

DSM 15553 (deposited with the DSMZ German Collection of Microorganisms and Cell Cultures GmbH, D-38124 Braunschweig).

In a further preferred embodiment, the invention is characterized by a DNA vector which contains a selection marker gene selected from the group of antibiotic resistance genes. In this embodiment of the invention, the expression system according to the invention is characterized in that the host organism is sensitive to an antibiotic and in its presence is no longer capable of growth and the selection marker gene is selected such that its expression after transformation of the host organism causes resistance to the antibiotic and allows growth under selective conditions.

Are suitable antibiotic resistance genes which confer resistance to an antibiotic conferment hen selected from the group Hygromy- cin, G418, geneticin ^®, glyphosate, cycloheximide, bialaphos, Ka namycin, bleomycin, oligomycin, Zeocin ™, benomyl, nystatin and phleomycin.

Preferably suitable are Antibiotikarestistenzgene that Resis- competence to an antibiotic selected from the group Hygromy- cin, G418, give Geneticin ^®, glyphosate, bialaphos or cycloheximide.

Especially preferred are antibiotic resistance genes against hygromycin, G418, Geneticin® ^, and glyphosate.

Especially preferred are antibiotic resistance genes against hygromycin.

In another embodiment of the invention, the expression system is characterized in that the host organism selected from the genus Sporidiobolus possesses a genetic defect in metabolic metabolism (auxotrophy), as a result of which one or more metabolic metabolites essential for growth are no longer synthesized and the host organism is no longer capable of growth on minimal media without the addition of this or that metabolic metabolite and the selection marker gene is selected such that it complements the auxotrophic gene defect of the host organism. Examples of selection radar genes which can complement an auxotrophic gene defect in the host organism are the pyrG gene (encoded by the enzyme orotidine-5'-phosphate decarboxylase, Goosen et al., Curr. Genet. (1987) 11: 499 - 503), the pyrF gene (codes for the enzyme orotic acid phosphoribosyltransferase, DE 199 34 408), the OCT gene (codes for the enzyme ornithine carbamoyltransferase, US Pat. No. 5,362,640) or the trpC gene (a gene , whose trifunctional gene product has enzyme activity for phosphoribosyl-anthranilic acid isomerase, glutamine amido transferase and indole-glycerol phosphate synthase, Yelton et al., Proc Natl. Acad. Sci. USA (1984) 81: 1470-1474).

Furthermore, selection marker genes are suitable which code for proteins which are capable of a coloring reaction, for. For example, the glucuronidase gene, the gene for the green fluorescent protein (GFP) (including the genetic variants derived therefrom) or the gene of a laccase.

The invention also relates to DNA vectors which are suitable for the production of the expression system according to the invention. Such a DNA vector is characterized in that it contains the selection marker gene, preferably from the group of antibiotic resistance genes.

Especially for the expression system according to the invention suitable selection marker genes are the resistance genes for the following antibiotics:

G418, or Geneticin ": The kanamycin (G418) resistance gene from the E. coli transposon Tn903 (Webster and Dickson, Gene (1983) 26: 243-252).

Hygromycin: The hygromycin B phosphotransferase gene from E. coli (Gritz and Davis, Gene (1983) 25: 179-188) or from Streptomyces hygroscopicus (Malpartida et al., Biochem. Biophys. Res. Commun. (1983) 117: 6-12). Glyphosate: The 5-enolpyruvylshikimate-3-phosphate synthase gene

(EPSP) from E. coli (Kunze et al., Curr Genet. (1989) 15: 91-98). However, any other EPSP gene is suitable, or mutated EPSP genes with increased resistance to glyphosate. Bialaphos: The Bialaphos resistance gene (Bar gene) from Streptomyces hygroscopicus (Avalos et al., Curr. Genet. (1989) 16: 369-372).

The DNA vector according to the invention for expression of the selection marker gene, selected from the group of antibiotic resistance genes, additionally contains at least one genetic regulatory element active in the host organism (promoter, terminator) which, functionally linked to the selection marker gene, expresses it ensured in the host organism.

Suitable for the expression of the selection marker gene are the promoter and terminator elements for the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH gene), for example from yeasts or filamentous fungi from the class of Basidiomycetes. The GAPDH gene can be homologous from a yeast of the genus Sporidiobolus or heterologous from a yeast or a fungus from the class of Basidiomycetes, z. From Phaffia rhodozyma, Ustilago maydis, Schizophyllum commune, Trametes verisicolor, Agaricus bisporus or Phanerochaete chrysosporium.

However, all other sporidiobolus-active promoter and terminator elements are also suitable for the preparation of the DNA vectors according to the invention.

Especially suitable are promoter and terminator elements of the GAPDH gene from Sporidiobolus ruineniae, characterized by the sequences SEQ ID NO: 1 and SEQ ID NO: 2, as well as the GAPDH gene from Phaffia rhodozyma. The promoter structures for the Phaffia rhodozyma GAPDH gene are disclosed in Verdoes et al. , Yeast (1997) 13: 1231-1242. This shows a comparison of

Transformation rates (described in the Examples) that the homologous expression signals of the Sporidiobolus GAPDH gene are clearly superior to the heterologous expression signals of the P. rhodozyma GAPDH gene.

Especially suitable are promoter and terminator elements of the GAPDH gene from Sporidiobolus ruineniae, characterized by the sequences SEQ ID NO: 1 and SEQ ID NO: 2. The invention thus also relates to the promoter and terminator elements of the Sporidiobolus GAPDH gene, disclosed for the GAPDH clone Gap5 in SEQ ID NO: 1, bp 1 - 1050 (promoter), or bp 2997 - 3500 (terminator) and for the GAPDH clone Gapl8 in SEQ ID NO: 2, bp 1-790 (promoter) and DNA sequences derived therefrom by extension, truncation or alteration, which can be active in Sporidiobolus as promoter or terminator.

The invention relates in particular to the promoter and terminator elements of the Sporidiobolus GAPDH gene, disclosed for the GAPDH clone Gap5 in SEQ ID NO: 1, bp 1-1050 (promoter) or bp 2997-3500 (terminator) and for the GAPDH clone Gapl8 in SEQ ID NO: 2, bp 1-790 (promoter).

The invention further relates to a DNA vector which contains at least one selection marker gene which codes for a protein which, after transformation of a yeast of the genus Sporidiobolus, permits a selection of positive transformants, characterized in that the selection marker gene is selected from the group of antibiotic resistance genes coding for proteins that abolish the anti-growth effect of antibiotics against which the host organism is not resistant, the genes that complement a genetic defect of the host organism (auxotrophy), and the genes that produce encode proteins which are capable of a coloring reaction, and that the selection marker gene is controlled by at least one genetic regulatory element active in the host organism.

In a preferred embodiment, the DNA vectors of the invention allow the selection of positive transformants of yeasts of the genus Sporidiobolus due to the acquired after transformation antibiotic resistance in the host organism.

Particularly preferred is the selection of transformants of the yeast Sporidiobolus ruineniae by DNA according to the invention. Enables vectors containing the antibiotic resistance genes which are considered to be preferred.

The invention furthermore comprises a DNA vector which is suitable for expressing at least one gene coding for a protein, preferably from the Q10 biosynthesis pathway, in the host organism. For the purposes of the invention, genes coding for proteins are also to be understood as meaning the cDNA genes of the proteins derived from the structural genes, as well as mutated genes. The proteins can be proteins which are heterologous to the host organism or homologous proteins for the host organism, which moreover can be present either intracellularly or extracellularly (secreted proteins).

The DNA vector according to the invention thus preferably contains at least one gene to be expressed which codes for a protein, preferably from the Q10 biosynthesis pathway.

It is obvious to a person skilled in the art that the gene to be expressed is not limited to a protein from the Q10 protein.

Biosynthetic pathway restricted. Rather, the gene to be expressed can code for any protein to be used for a technical or pharmaceutical application or else for an enzyme from any biosynthetic pathway other than the Q10 pathway. In this case, the biosynthetic pathway can already be created in the host organism and the gene to be expressed alters the production efficiency (yield) for the desired metabolite, or a new metabolic pathway is created by the gene to be expressed, as a result of which the recombinant host organism produces new metabolite. Examples of affected biosynthetic metabolic pathways are the steroid metabolism, the carotenoid metabolism, the metabolism of polyunsaturated fatty acids (also referred to as PUFA or omega fatty acids) and the polyketide metabolism (generally the metabolism of the metabolites). In a preferred embodiment, the gene to be expressed is a gene from the Q10 biosynthesis pathway. An overview of the known QlO biosynthesis genes is given by Meganathan, FEMS Microbiol Lett. (2001) 203: 131-139. Tzagoloff and Dieckmann, Microbiological Reviews (1990) 54: 211-225, describe eight complementation groups from baker's yeast called CoQ1-CoQ8, which define ubiquinone biosynthetic genes.

Examples of genes from the Q10 biosynthesis pathway are the genes for acetoacetyl-CoA thiolase, hydroxymethyl-glutaryl-CoA synthase (HMG-CoA synthase), hydroxymethyl-glutaryl-CoA reductase (HMG-CoA reductase), mevalonate kinase, phosphomevalo - natkinase, diphosphomevalonate decarboxylase, isopentenyl diphosphate isomerase, farnesyl diphosphate synthase, chorismate

Pyruvate lyase, decaprenyl diphosphate synthase (DPS, CoQ1 gene), p-hydroxybenzoic acid decaprenyl diphosphate transferase (CoQ2 gene), 3,4-dihydroxy-5-decaprenyl benzoate methylase (CoQ3 gene), a C-methyltransferase (CoQ5 gene), a flavin dependent monooxygenase (CoQ6), 5-demethoxyubichinone hydroxylase (CoQ7), a potential QlO transport protein (CoQ10, Barros et al., J. Biol. Chem. (2005) 280: 42627-42635.) and the hitherto not further characterized genes CoQ4, CoQ8 and CoQ9 (Johnson et al., J. Biol. Chem. (2005) 280: 31397-31404). However, the genes of the Q10 biosynthetic pathway according to the invention are not limited to these examples.

An example of a CoQ1 gene is the gene for decaprenyl diphosphate synthase (DPS) from the yeast Rhodotorula minuta disclosed in EP 1336657 A, SEQ ID NO: 1.

The Q10 biosynthesis genes may be homologous from Sporidioboluε or heterologous from another organism.

Preferably, these genes are homologous genes from the Q10 biosynthetic pathway of the genus Sporidiobolus. For expression in the host organism, the protein coding gene is operably linked to genetic regulatory elements such as a promoter or terminator.

The promoter can be derived from the gene to be expressed, or the promoter of a foreign gene can also be used, which is functionally linked to the coding region of the gene to be expressed.

The DNA vector according to the invention thus preferably also contains a promoter for the expression of the protein-encoding gene.

The DNA vector according to the invention preferably contains as promoter for the expression of the protein-encoding gene a promoter which ensures a high expression performance.

A promoter used particularly preferably for this purpose is the promoter of the gene for the GAPDH gene, and in this case especially the GAPDH promoter from Sporidiobolus ruineniae (SEQ ID NO: 1, bp 1 - 1050 and SEQ ID NO: 2, bp 1 - 790).

The DNA vector according to the invention preferably also contains a transcription terminator for the protein-coding gene.

The terminator of the protein-coding gene to be expressed or else the terminator of a foreign gene can be used as transcription terminator. Preferably, the transcription terminator is a GAPDH gene, in particular the GAPDH gene from Sporidiobolus ruineniae (SEQ ID NO: 1, bp 2997-3500).

In a particularly preferred embodiment, the promoter of the GAPDH gene, especially of the GAPDH gene from Sporidiopus ruineniae (SEQ ID NO: 1, bp 1 - 1050 or SEQ ID NO: 2, bp 1-790) and the like, is used Extension, truncation, or alteration-derived DNA sequences present in Sporidiobolus Promoter can be active. The isolation of the Sporidiobolus GAPDH gene is described in Example 4.

The invention thus also relates to an active in Sporidiobolus regulatory element which is characterized in that it contains the SEQ ID NO: 1 sequence section of base 1 - 1050, or contained in SEQ ID NO: 2 sequence section of base 1 - 790 and thereof by extension, truncation or alteration derived DNA sequences which may be active in Sporidi obolus as a promoter.

An example of this is the GAPDH used in the 5th example.

Promoter from Phaffia rhodozyma (gene database accession number

"PRGPDGENE. gb_pl, bp 1-717), which is 37% identical to the sequence segment of base 1 - 1050 contained in SEQ ID NO: 1, or to the sequence segment from base 1 - 790 to 35 contained in SEQ ID NO: 2 % is identical and is suitable as a heterologous promoter for regulating gene expression in S. ruineniae.

To analyze DNA sequences, the computer program "Wisconsin Package Version 10.3, Accelrys Inc." was used. used. The homology determination was carried out by comparison of the DNA sequences with the subprogram "Gap". Here, the default parameters "gap creation penalty 50" and "gap extension penalty 3" are used.

A DNA vector for the transformation of Sporidiobolus contains no genetic element that ensures its autonomous replication in the host organism. Therefore, after transformation, the DNA vector must be integrated into the genome of the host organism. The integration into the host genome can be carried out undirected by random recombination or else by homologous recombination.

The gene region of the so-called rDNA, which codes for ribosomal RNA and from which approximately 100 copies are present in the genome of the host organism, is suitable as an integration site for homologous recombination. are holding. From short, published sequences for sections of the S. ruineniae rDNS, an rDNA fragment for homologous recombination was isolated (SEQ ID NO: 3). The isolation of a sporidiobolus rDNA fragment is described in Example 2.

A DNA vector according to the invention thus preferably also contains an RNA fragment from S. ruineniae (SEQ ID NO: 3).

The preparation of the DNA vectors according to the invention is carried out by means known in the art. Various possibilities are set forth in the examples. The methods described there can be applied by the skilled person to any other vectors, resistance genes, regulatory elements and structural genes.

The DNA vectors according to the invention are suitable for the production of Sporidiobolus strains which are capable of efficient gene expression.

The invention therefore also relates to methods of producing Sporidiobolus strains capable of efficient gene expression.

This method is characterized in that a yeast of the genus Sporidiobolus is used as the host organism, which is transformed with a DNA vector which has a gene to be expressed and an antibiotic resistance gene, and transforms from the transformation batch with the DNA vector selected clones are selected by selection of antibiotics-resistant transformants, the expression of the gene to be expressed and of the antibiotic resistance gene in the host strain being respectively controlled by at least one genetic regulatory element which is active in the host strain.

However, the gene to be transformed can also be cloned into an expression vector without selection marker gene and together with a vector expressing the selection marker gene used to generate transformants (co-transformation).

The gene to be transformed is cloned in a known manner into a DNA vector according to the invention and introduced into a yeast of the genus Sporidiobolus by means of the methods mentioned.

The relevant yeast strain of the genus Sporidiobolus may be a monocaryontic or else a dikaryontisean strain.

Particularly preferred as a host for gene expression is a yeast of the species Sporidiobolus ruineniae.

Particularly preferred as a host for gene expression is the strain Sporidiobolus ruineniae DSM 15553.

The transformation of the host strain is carried out according to methods that correspond to the state of the art. These methods include transformation with the lithium acetate method, transformation by electroporation or biolistic transformation by bombardment with DNA-containing microprojectiles, or a combination of the methods mentioned. These methods are described in standard textbooks.

The selection of positive transformants is carried out, for example, by sporidiobolus cells after transformation with vector DNA on a medium to which antibiotic was added in amounts which suppresses the growth of wild-type strain and which allows the selection of antibiotic-resistant transformants. A method for the inventive transformation of Sporidiobolus is described in the 8th example.

In a preferred embodiment of the invention, the yeast Sporidiobolus ruineniae is transformed in the manner mentioned above with the gene of one or more Q10 biosynthesis enzymes. This results in an increase in the expression rate for the said gene and significantly improves the Q10 production rate. The Q10 biosynthesis genes may be derived from yeasts of the genus Sporidiobolus, or other Q10 biosynthesis genes may also be used.

The sporidiobolus expression system according to the invention is particularly suitable for expressing one or more of the described genes from the Q10 biosynthesis pathway. The application of the expression system according to the invention for improving Q10 production is described in the 12th example.

The invention thus also relates to a method for using the expression system according to the invention, characterized in that a strain of the yeast Sporidiobolus, which was produced by the process according to the invention, is cultivated in a manner known per se and the product, whether it is a recombinantly produced protein or a metabolic product synthesized with the aid of the recombinantly produced protein, including preferably Q10, isolated.

Such production methods are known, for example, from EP 1469078 A or from US 4070244.

However, the invention includes not only the use of the expression system for the genetic optimization of Q10 production. The expression system can also be used to genetically engineer or optimize any other metabolic pathway in strains of the genus Sporidiobolus. Examples include the production of steroids, carotenes, terpenes and polyunsaturated fatty acids (omega fatty acids). In addition, the expression system according to the invention is also suitable for heterologous or homologous production of proteins in recombinant form. These recombinantly produced proteins find use, for. B. as therapeutic proteins in medicine, in biotransformations in the enzyme-catalyzed organic-chemical synthesis or as technical enzymes in various industrial applications. As an example Games are called the detergent, paper, food or animal feed industries.

The following examples serve to further illustrate the invention. The standard methods used in the examples for the treatment of DNA or RNA, such as the treatment with restriction endonucleases, DNA polymerases, reverse transcriptase, etc. as well as the standard methods such as transformation of bacteria, Southern and Northern analysis, DNA sequencing, labeling of DNA Probes, screening and PCR technology were performed as recommended by the manufacturer, unless otherwise stated, or if no manufacturer's instructions were available according to the state of the art known from standard textbooks.

1st example:

Cultivation of Sporidiobolus ruineniae on minimal medium in the presence of antibiotics

The strain Sporidiobolus ruineniae Sr-1 (deposited with the DSMZ German Collection of Microorganisms and Cell Cultures GmbH, D-38124 Braunschweig under the number DSM 15553) was used.

Cultivation of Sporidiobolus ruineniae Sr-1: Culture media used were YPD medium (1% yeast extract, 2% peptone, 2% glucose), YPG medium (1% yeast extract, 2% peptone, 5% glycerol) or YNB- Minimal medium (1.34% "Yeast Nitrogen Base", 2% glucose) Cultivated in liquid media or on agar plates (YPD, YPG or YNB medium with 2% agar each), for 2 to 5 in each case Days at 28 ° C. Liquid cultures were shaken under these conditions at 140 rpm on an orbital shaker (Infors).

Antibiotic sensitivity tests were performed with the antibiotic G418 (also known under the brand name Geneticin ^®; Invitrogen), hygromycin B (Calbiochem) and glyphosate (N- (phosphonomethyl) - Glycine, Sigma, known as herbicide under the trade name RoundUp ").

Selection conditions for G418: YNB plates with a G418 concentration of 50 μg / ml, 100 μg / ml, 150 μg / ml and 200 μg / ml were prepared, Sr-1 was plated thereon and incubated at 28 ° C. Compared with control plates without G418, a significantly reduced growth was observed at a G418 concentration of 100 μg / ml and no growth of the strain Sr-1 at 200 μg / ml. For transformation experiments, YNB plates with a G418 concentration of 100 μg / ml and 150 μg / ml were used as the selective medium.

Selection conditions for hygromycin B: YNB plates having a hygromycin B concentration of 40 μg / ml, 60 μg / ml, 80 μg / ml and 100 μg / ml were prepared, Sr-1 was plated thereon and incubated at 28 ° C. Compared with control plates without hygro- mycin B, a significantly reduced growth was observed at a hygromycin B concentration of 60 μg / ml and no growth of the strain Sr-1 at 100 μg / ml. For transformation experiments, YNB plates with a hygromycin B concentration of 80 μg / ml and 100 μg / ml were used as the selective medium.

Selection conditions for glyphosate: YNB plates were prepared with a glyphosate concentration of 1 mg / ml, 1.25 mg / ml, 1.5 mg / ml 1.75 mg / ml and 2 mg / ml, Sr-1 on top plated and incubated at 28 ° C. Compared to control plates without glyphosate, a markedly reduced growth was observed at a glyphosate concentration of 1.25 mg / ml and no growth of the strain Sr-1 at 1.75 mg / ml.

Example 2 Isolation of a gene fragment of ribosomal DNA (rDNA) from Sporidiobolus ruineniae

Isolation of genomic DNA from S. ruineniae Sr-1: Sr-1 was cultured in YPD liquid medium for 2 days as described in Example 1. The Sr-1 cells were then isolated by centrifugation (10 min 3000 rpm, Heraeus Megafuge 1.0R). DNA was isolated from the Sr-1 cells using the Qiagen Genomic Tip kit for genomic DNA extraction.

Isolation of a DNA fragment of ribosomal DNA (rDNA)

S. ruineniae:

DNA sequences of short fragments of S. ruineniae rDNA are stored in gene databases under accession numbers abO21696.gb_pl and af070438.gb_pl. These DNA sequences were used to make sequence comparisons with the known rDNS region of the baker's yeast Saccharomyces cerevisiae (gene database accession number scylrl54c .gb_pll). The computer program "Wisconsin Package Version 10.3, Accelrys Inc." was used for the search and analysis of DNA sequences. used. Based on this comparison, the PCR primers SrRDIf (SEQ ID NO: 4) and SrRD2r (SEQ ID NO: 5) were selected whose DNA sequences in S. cerevisiae and S. ruineniae are identical.

The primers had the following sequences:

Primer SrRD1f: 5 '-GCTTGTCTCAAAGATTAAGC-3' (SEQ ID NO: 4)

Primer SrRD2r: 5 '-GGTCCGTGTTTCAAGACGGG-3' (SEQ ID NO: 5)

PCR reactions with the primers SrRDIf and SrRD2r with genomic DNA from S. ruineniae Sr-1 were carried out according to the prior art with a GenAmp PCR System 2400 from Applied Biosystems. The Taq Core PCR kit (Qiagen) was used with the following PCR program: After incubation of the PCR mixture for 1 'at 94 ° C, 30 reaction cycles of l ^' 94 ° C, 30 '' 5O ° C and 2, 5 '72 ° C and the reaction with an incubation for 5' at 72 ° C ended. The resulting PCR product was purified by preparative agarose gel electrophoresis. The size of the DNA fragment formed was 3 kb.

Vector pSrrDNA:

The 3 kb DNA fragment was inserted into the vector PCR script

SK (+) (Stratagene) cloned. The result was the vector pSrrDNA (Fig. 1). Sequence analysis of the cloned DNA fragment of vector pSrrDNA (SEQ ID NO: 3) confirmed that it was the expected fragment of ribosomal DNA of S. ruineniae.

3rd example

Preparation of a chromosomal gene bank from Sporidiobolus ruiniae.

S. ruineniae genomic DNA was isolated from the cells of three shake flask cultures (50 ml each YPD medium, 24 h, 140 rpm, 28 ° C.). For this purpose, the cells from the culture were first freeze-dried (lyophilizer Christ alpha 2-4) and the genomic DNA was isolated therefrom using the Qiagen "Genomic-tip" DNA isolation kit, following the instructions of the manufacturer. The yield was 100 μg of genomic DNA.

To prepare the chromosomal gene bank, 100 μg of chromosomal DNA from Sporidiobolus ruineniae Sr-1 were cut in a partial digestion with Sau 3A and fractionated by agarose gel electrophoresis. The chromosomal DNA fragments were isolated in the size range from 1 to 20 kb and, according to the manufacturer's instructions, each cloned into Lambn phage pre-cut with Barn HI (Stratagene cloning system "Lambda ZAP Express") From 1 to 20 kb of DNA fraction, 2.5 x 10 ⁶ phage / μg of vector DNA were obtained and phage were amplified by infection of E. coli strain XL-I Blue MRF '.

4th example

Cloning of the S. ruineniae GAPDH gene

A. Isolation of a GAPDH DNA probe from Sporidiobolus ruineni ae: The strain Sporidiobolus ruineniae Sr-1 (see Example 1) was used. Genomic DNA was isolated as described in the 2nd example.

From the published DNA sequences of the GAPDH gene from Phaserochaete chrysosporium (MC Harmsen et al., (1992) Curr Genet., 22, 447-454) and the GAPDH gene from Coriolus hirsutus (JP 9-47289) and Phaffia rhodozyma (JC Verdoes et al. (1997), Yeast 13, 1231-1242) primers were designed for PCR amplification of a GAPDH-specific gene fragment. The primers had the following DNA sequences:

Primer srgapl:

5 '-CTTGAGTACATGGTCTACATGTTC-3' (SEQ ID NO: 6).

Primer srgap2r:

5'-TTAACACCGCAGACGAACATGG-3 '(SEQ ID NO: 7).

PCR amplifications were carried out according to the prior art: In a PCR reaction, 200 ng chromosomal Sporidiobolus ruineniae DNA were used in a 50 μl PCR reaction containing the buffer provided by the manufacturer and additionally 1.25 U Taq polymerase, 1, 25 mM MgCl ₂ , 0.2 mM each of the four dNTPs (dATP, dCTP, cGTP, dTTP) and in each case 100 pmol of the primers srgapl and srgap2r. Further conditions for specific amplification of the desired PCR product were: 94 ° C for 5 min followed by 30 cycles of 94 ° C for 0.5 min, 50 ° C for 1 min, and 72 ° C for 1 min. A PCR product of about 0.3 kb was obtained.

The PCR product was purified by agarose gel electrophoresis, cloned into the vector PCR-Script SK (+) (Stratagene) and transformed into E. coli Top 10F '. The plasmid was isolated from the culture of transformed E. coli. DNA sequence analysis confirmed that the cloned DNA fragment was the fragment of the GAPDH gene from S. ruineniae. To prepare the DNA probe for the screening of the GAPDH gene, the GAPDH-specific PCR fragment was excised by treatment with Not I and Eco RI, isolated by agarose electrophoresis and labeled with the "AlkPhos Direct" DNA labeling kit. Amersham Biosciences), as recommended by the manufacturer.

B: Isolation of a chromosomal GAPDH gene from S. ruineniae:

The chromosomal gene bank of Sporidiobolus ruineniae Sr-I described in Example 3 was used. The screening for the chromosomal GAPDH gene was carried out according to the prior art. In a first round of screening, cells of E. coli XL-1 Blue MRF 'were first cultured on 6 petri dishes and then infected with 50,000 phages of the Genbank per petri dish. After incubation at 37 ° C overnight, the newly formed phage were transferred to nylon filters (Hybond-N ⁺ , Amersham Biosciences). The filters were then hybridized to the labeled GAPDH-specific probe (see Section A) according to the manufacturer's recommendations. The hybridization temperature was 6O ° C. The washing temperature was 6O ° C. The detection of positive clones was carried out by chemiluminescence (CDP-Star detection kit from Amersham Biosciences) and autoradiography. 18 positive clones were picked. These were purified by repeating the screening procedure. After three rounds of singling were at the

Screening five strongly hybridizing phage clones isolated, which according to a manufacturer's instructions (Stratagene) by "in vivo excision" in the pBK CMV vector (Stratagene) were recloned. Analysis of the clones by digestion with restriction endonucleases and DNA sequencing revealed that four of the five clones were GAPDH clones. Two allelic clones, designated Gap5 (SEQ ID NO: 1), and Gap18 (SEQ ID NO: 2) were sequenced.

The start ATG codon of the GAPDH gene was determined by the expert's 5'-RACE analysis (Generacer Kit from Invitrogen). The test instructions provided by the manufacturer were followed. They involved in the production of the cDNA required RNA was isolated with the "peqGOLD TriFast" reagent (PeqLab) The gene-specific primer srgaplOR, which was furthermore required for the PCR reaction, had the following sequence:

Primer srgaplOr:

5 '-TTGAACATGTAGACCATGTACTCGAG-3' (SEQ ID NO: 8).

A comparison of the DNA sequence of the 5'-RACE cDNA fragment with the sequences of the Gap5 and Gapl8 clones revealed that the start ATG codon is interrupted by an intron. The corresponding promoter regions are thus bp 1 - 1050 in clone Gap5 (SEQ ID NO: 1), or bp 1-790 in clone Gap18 (SEQ ID NO: 2). As terminator region, the DNA sequence bp 2997-3500 in clone Gap5 (SEQ ID NO: 1) was determined.

Sequence comparison of the DNA of the available coding regions (including intron sequences) of the Gap5 clone (SEQ ID NO: 1, bp 1051-2996) and the Gapl8 clone (SEQ ID NO: 2, bp 791-21226) gave a 97.8 % Identity. By contrast, the promoter regions mentioned above were only 41.8% identical. In the present invention, all homology values mentioned refer to results obtained with the computer program "Wisconsin Package Version 10.3, Accelrys Inc." were obtained. Homology was determined by comparing the DNA sequences with the subprogram "gap". The preset parameters "gap creation penalty 50" and "gap extension penalty 3" are used here.

Example 5 Expression vector pprG418Sr for the transformation of S. rueneiae

The GAPDH gene from Phaffia rhodozyma is known (gene designation in the GCG gene database: PRGPDGENE .gb_pl) and its use for the transfomation of Phaffia rhodozyma is described (Wery et al., Biotechnology Techniques (1998) 12: 399-405). The G418 resistance gene from the E. coli transposon Tn903 is contained in the Saccharomyces cerevisiae expression vector. pUG6 (described in Gueldener et al., Nucleic Acids Res. (1996) 24: 2519-2524; the DNA sequence is stored in the GCG gene database under accession number AF298793).

Promoter and terminator of the GAPDH gene were amplified by PCR

Reactions isolated from genomic DNA of strain Phaffia rhodozyma CBS 6938 (available from the strain collection Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands). Genomic DNA of P. rhodozyma was isolated as described in Example 2 for S. ruineniae.

To isolate the promoter DNA fragment, the primers prgapl and prgap2r were used. The primers prgap3 and prgap4r were used to isolate the terminator. The primers had the following DNA sequences:

Primer prgapl:

5'-CCGAAGCTTAAGTCGAGGGAACCCGAG-3 '(SEQ ID NO: 9).

Primer prgap2r:

5 '-TTCGGATCCCCGCGGCCATGGTGGTAAGAGTGTTAG-3' (SEQ ID NO: 10).

Primer prgap3:

5'-ACGGTTCTCTCCAAACCCTC-3 '(SEQ ID NO: 11).

Primer prgap4r:

5 '-GGATCCCGGAGATGATGGTGATG-3' (SEQ ID NO: 12).

PCR reactions with genomic P. rhodozyma DNA and the prgapl and prgap2r, prgap3 and prgap4r primers were carried out as described in Example 2. The following PCR program was used: After incubation of the PCR mixture for 1 'at 94 ° C 30 reaction cycles of 1' 94 ° C, 30 ^'' 5O ° C and 30 '' 72 ° C were carried out and the reaction with incubated for 5 'at 72 ° C. The resulting PCR products were analyzed by agarose gel electrophoresis. The size of the promoter DNA fragment (primer prgapl and prgap2r) was 0.7 kb. The size of the terminator DNA fragment (primer prgap3 and prgap4r) was 0.4 kb. The two DNA fragments were purified by preparative agarose gel electrophoresis.

The isolated promoter DNA fragment was digested with Hind III (cleavage was in primer prgapl) and Barn HI

(Cleavage site contained prgap2r primer) and cloned into the Hind III and Bam HI cut and dephosphorylated pUC18 vector (Amersham Biosciences). The result was the 3.4 kb vector pPRgap-pro.

The isolated terminator DNA fragment was cloned into the PCR Script Vector (Stratagene). This resulted in the 3.4 kb vector pPRgap-term. For the subsequent work, a clone was selected in which the Bam HI cleavage site was located from the prgap4r primer next to the Bam HI cleavage site of the PCR script vector.

Vector pPRgap:

Vector pPRgap-pro was cut with Sac II and Bam HI and the linearized vector was dephosphorylated.

Vector pPRgap-term was cut with Sac II and Bam HI and the resulting 0.4 kb terminator fragment isolated by preparative agarose gel electrophoresis.

The pPRgap-pro vector fragment and the terminator fragment were ligated and transformed into E. coli Top10F '(Invitrogen). The result was the 3.8 kb vector pPRgap.

Preparation of the vector pprG418:

Vector pPRgap was first cut with Sac II and the overhanging end filled in by reaction with Pfu DNA polymerase (Stratagene). Then the vector was cut with Nco I. The linearized 3.8 kb vector fragment was subsequently dephosphorylated.

The G4l8 resistance gene was excised from the pUG6 vector by digestion with Nco I and Sca I as a 0.8 kb fragment. and isolated by preparative agarose gel electrophoresis.

The linearized 3.8 kb pPRgap vector fragment was ligated with the 0.8 kb G418 resistance gene fragment and transformed into E. coli Top 10F '. This resulted in the 4.6 kb vector pprG418, in which the G418 resistance gene is functionally linked to the GapDH promoter and terminator from Phaffia rhodozyma (FIG. 2).

Preparation of expression vector pG418Sr:

Vector pprG418 with the G418 selection marker gene was cut with AfI II and Barn HI. The resulting 1.9 kb DNA fragment containing the expression cassette consisting of P. rhodocyma GapDH promoter, G418 resistance gene and P. rhodozyma GapDH terminator was isolated by agarose gel electrophoresis.

Vector pSrrDNA was cut with AfI II and Bgl II, which was thereby isolated linearized 5.9 kb vector fragment by agarose gel electrophoresis and dephosphorylated with alkaline phosphatase.

The 1.9 kb DNA fragment from the vector pprG418 was ligated with the 5.9 kb pSrrDNA vector fragment and transformed into E. coli ToplO F '. This gave rise to the 7.8 kb expression vector pG418Sr (FIG. 3). pG418Sr is a novel expression vector suitable for the transformation of Sporidioboluε ruineniae and for the selection of G418-resistant transformants.

6th example

Expression vector pprHPHsr for the transformation of S. ruineniae

Vector pPRgap:

The 3.8 kb vector fragment described in Example 3 was used, which was prepared by digestion with Sac II Pfu DNA polymerase, digestion with Nco I and dephosphorylation had been made.

Hygromycin resistance gene (HPH): The sequence of the hygromycin resistance gene from E. coli is stored in the GCG database under the file "ECAPH4" (Kaster et al., Nucleic Acids Res. (1983) 11, 6895-6911) Resistance gene was prepared from E. coli DNA by PCR with Pfu DNA polymerase (Stratagene) and the primers hphlf and hph2r, and hphlf and hph2r primers had the following DNA sequence:

Primer hphlf:

5'-GAGTCATGAAAAAGCCTGAACTCAC-3 '(SEQ ID NO: 13).

Primer hph2r:

5'-TACTCTATTCCTTTGCCCTCGG-3 '(SEQ ID NO: 14).

In the PCR reaction, a 1 kb DNA fragment was generated, which was cut with Ex. HI (contained in the primer hphlf).

Preparation of the vector pprHPH:

The 3.8 kb pPRgap vector fragment was ligated with the 1 kb DNA fragment of the hygromycin resistance gene and transformed into E. coli Top 10F '. This gave rise to the 4.8 kb vector pprHPH (FIG. 4).

Preparation of the expression vector pHPHsr:

Vector pprHPH with the hygromycin B selection marker gene was cut with AfI II and Barn HI. The resulting 2.1 kb DNA fragment containing the expression cassette consisting of P. rhodozyma GapDH promoter, hygromycin B resistance gene and P. rhodozyma GapDH terminator was isolated by agarose gel electrophoresis.

Vector pSrrDNA was cut with AfI II and Bgl II, which was thereby isolated linearized 5.9 kb vector fragment by agarose gel electrophoresis and dephosphorylated with alkaline phosphatase. The 2.1 kb DNA fragment from the vector pprG418 (containing the expression cassette with the hygromycin B resistance gene) was ligated with the 5.9 kb pSrrDNA vector fragment and transformed into E. coli ToplO F ^' (Invitrogen). This resulted in the 8 kb expression vector pHPHsr (FIG. 5). pHPHsr is a novel expression vector suitable for the transformation of Sporidiobulus ruineniae and for the selection of hygromycin B-resistant transformants.

7th example

Expression vector pG418G18R for the transformation of S. ruineniae

Preparation of vector pG418G5:

Vector pprG418 (see 5th example) was cut with AfI II and Nco I to remove the P. rhodozyma GAPDH promoter. This resulted in a 3.9 kb vector fragment which was isolated by agarose gel electrophoresis and dephosphorylated with alkaline phosphatase.

Genomic DNA of the S. ruineniae GAPDH clone Gap5 (see Example 4) was used in a PCR reaction (Taq DNA polymerase, Qiagen Taq Core Kit) together with the primers srgap5 and srgap6. srgap5 (forward) binds to the beginning of the promoter sequence segment, srgap6 (reverse) at its end. The two primers had the following sequences:

Primer srgap5: 5'-GATCTTAAGGAAGAGTCGCTCACTC-3 'SEQ ID NO: 15

Primer srgap6:

5 '-CTTCCATGGTGAGGTTGATCCGCTG-3' SEQ ID NO: 16

The conditions for the PCR reaction were: 1 '94 ° C, followed by 30 cycles of 30 "94 ° C, 30" 52 ° C, 60' 72 ° C, and finally 5 '72 ° C. The 1 kb formed in the PCR reaction DNA fragment of the GAPDH promoter was isolated and cut with AfI II and Nco I.

The 3.9 kb AfI II / Nco I cut pprG418 vector fragment and the 1 kb AfI II / Nco I-cut GAPDH promoter fragment were ligated and transformed into E. coli Top 10F '. This resulted in the 4.9 kb vector pGapPromG418, in which the Gap5 promoter was linked to the G418 resistance gene.

Vector pGapPromG418 was cut with Not I and Barn HI, removing the 0.4 kb P. rhodozyma GAPDH terminator fragment. The 4.5 kb vector fragment was isolated by agarose gel electrophoresis and dephosphorylated by phosphatase treatment.

Genomic DNA of the S. ruineniae GAPDH clone Gap5 (see Example 4) was used in a PCR reaction (Taq DNA polymerase, Qiagen Taq Core Kit) together with the primers srgapllf and srgapl2r, srgapllf contains a Not I site for linkage with the 3 'end of the G418 resistance gene and contains the sequence after the first possible stop codon of the GAPDH gene (see 4th example). srgapl2r contains a Bam HI site and is derived from the sequenced 3 'end of the Gap5 clone. The two primers had the following sequence:

Primer srgapllf:

5 '-TATGCGGCCGCTATGCGCCGTGTAAAGCGTG-3' SEQ ID NO: 17

Primer srgapl2r: 5'-TATGGATCCCAGGGCTGATCGCTCGTTGC-3 'SEQ ID NO: 18

The conditions for the PCR reaction were: 1 '94 ° C, followed by 30 cycles of 30''94 ° C, 30''52 ° C, 30''72 ° C and finally 5' 72 ° C. The 0.42 kb DNA fragment of the GAPDH promoter formed in the PCR reaction was isolated and cut with Not I and Bam HI. The 4.5 kb Not I / Bam HI cut pGapPromG418 vector fragment and the 0.42 kb Not I / Bam HI cut GAPDH terminator fragment were ligated and transformed into E. coli Top 10F '. The 4.9 kb vector pG418G5 (FIG. 6) was created, in which the promoter and terminator of the Gap5 GAPDH clone are functionally linked to the G418 resistance gene.

Vector pG418G18

Genomic DNA of the S. ruineniae GAPDH clone Gapl8 (see Example 4) was used in a PCR reaction (Taq DNA polymerase, Qiagen Taq Core Kit, see vector pG418G5) together with the primers srgap13f and srgap15r. srgap13f binds to the beginning of the promoter sequence, srgapl5r at its end. The two primers had the following sequences:

srgapl3f:

5'-TATCTTAAGTCCGGACGATTTCGTCCTC-3 '(SEQ ID NO: 19)

srgapl5r: 5'-TATCCATGGTGTGGTTGATCGAGTAG-3 '(SEQ ID NO: 20)

The 0.8 kb Gap18 promoter fragment formed in the PCR reaction was gel isolated and cut with AfI II and Nco I. Vector pG418G5 was cut with AfI II and Nco I and the 3.9 kb vector fragment gel isolated and dephosphorylated. The

0.8 kb of Gap18 promoter fragment was cloned into the 3.9 kb vector fragment, resulting in the 4.7 kb vector pG418G18 (Figure 7).

Preparation of vector pG418G18R:

From the vector pG418G18, the G418 expression cassette was excised by digestion with Xma I and Bsp EI as a 2.1 kb DNA fragment and isolated by agarose gel electrophoresis.

Vector pSrrDNA was cut with Xma I, the 5.8 kb vector fragment was isolated by agarose gel electrophoresis and dephosphorylated. The two DNA fragments were ligated and transformed into E. coli Top 10F '. The result was the 7.9 kb vector pG418G18R (FIG. 8). pG418G18R is a novel expression vector suitable for the transformation of yeasts of the genus Sporidiobolus.

8th example

Transformation of Sporidiobolus ruineniae

Cells from S. ruineniae strain Sr-1 were prepared in a pre-culture in YPD medium (28 ° C., 140 rpm, 2 days' growth) as described in Example 1. The preculture was used to inoculate a daily culture of 200 ml YPD medium to a cell density OD _600nm of 0.2. The daily culture was then incubated at 28 ° C. and 140 rpm until a cell density OD _{600 nm} of _{1.0-1.5 was} reached.

The cells were then harvested by centrifugation (10 min 3000 rpm, Heraeus Megafuge 1.0R). The cell pellet was suspended in 40 ml of DTT buffer (100 mM lithium acetate, 10 mM Tris-HCl, pH 7.5, 10 mM dithiothreitol, 0.6 M mannitol) and incubated for 15 min at 28 ° C and 140 rpm. Thereafter, the cells were isolated by centrifugation and the cell pellet washed twice with 200 ml of ice-cold 0.9 M mannitol and the cell pellet finally taken up in 1 ml of 0.9 M mannitol. The cells thus prepared were used for transformation by electroporation.

For an electroporation approach, 100 μl cells were mixed with 1-10 μg DNA (maximum 10 μl volume) in an electroporation cuvette and cooled on ice. The DNA was previously linearized by digestion with Nhe I. The DNA to be transformed was the vectors pG418Sr (5th example, G418 selection), pHPHsr (6th example, hygromycin B selection) and pG418G18R (7th example, G418 selection). In a control approach, cells without added DNA were used. For electroporation, a Gene Pulser from BioRad was used. Conditions of electroporation were: voltage 7.5 kV / cm; Capacity 25 μF and a resistance of 200 ohms. The discharge time was about 4 msec.

After electroporation, the transformation mixture was admixed with 1 ml of YNB medium and incubated overnight at 28 ° C. with gentle shaking. The cells were then isolated by centrifugation (Eppendorf microcentrifuge 2 min 13,000 rpm), taken up in 1 ml of YNB medium and plated out in aliquots of 0.25 ml each on 4 selective agar plates. Selective agar plates each contained YNB medium with the appropriate antibiotic. In G418 selection, the agar plates contained G418 in a concentration of 100-150 μg / ml (depending on the respective antibiotic batch). In the

Hygromycin selection contained the agar plates Hygromycin B in a concentration of 60 - 100 ug / ml (depending on the respective antibiotic lot).

The transformants plated on selective plates were incubated at 28 ° C. Colonies of antibiotic-resistant transformants were observed after an incubation period of 5 to 10 days.

Obtained transformants were first inoculated onto selective plates and incubated at 28 ° C to confirm the expression of the antibiotic-resistant phenotype. Of the fastest growing transfoments, selective smears were then made on selective plates.

Typical transformation rates from transformation experiments with the expression constructs pG418Sr, pHPHsr and pG418G18R are given in Table 1. As shown in Tab. 1, significantly higher transformation rates were achieved with the homologous Sporidiobolus GAPDH promoter (construct pG418G18R) than with the heterologous P. rhodozyma promoter (constructs pG418Sr, pHPHsr). To control the background, transformation approaches without DNA were carried out in an equivalent manner. In doing so, observed only in the case of G418 selection background clones.

Due to the special combination of the expression vectors according to the invention, consisting of promoter, terminator, antibiotic resistance gene and rDMS, it was possible to transform a yeast strain of the genus Sporidiobolus in an unexpected manner. This transformation system forms the basis for the genetic optimization according to the invention Q10 production in Sporidiobolus. pG418sr, PG418G18R and pHPHsr are thus novel expression vectors suitable for the transformation of S. ruineniae and, in combination with a Q10

Biosynthesis gene can be used to improve Q10 production.

9th example

Genetic analysis of the transformants

Transformants of Sporidiobolus ruineniae, as obtained in Example 8, were analyzed by PCR and Southern blot analysis for the integration of plasmids pG418Sr, pHPHsr, and pG418G18R, respectively. Genomic DNA was isolated from various transformants with the plasmids pG418Sr, pG418G18R, pHPHsr and as control of the strain Sr-1 after culturing in YPD medium (see Example 1) as described in Example 2.

Plasmids pG418Sr and pG418G18R: Of the G418-resistant strains, 1 μg each of the genomic DNA and 50 ng of the plasmid pG418Sr with Nco I and Not I, then separated by agarose gel electrophoresis, blotted onto nylon filters (Hybond +, Amersham Biosciences) and hybridized with a DNA probe specific for the G418 resistance gene.

The DNA probe was prepared by cutting the plasmid pG418Sr with Nco I and Not I and isolating the resultant 0.8 kb DNA fragment (containing the G418 resistance gene) by preparative gel electrophoresis. The 0.8 kb G418-specific DNA fragment was labeled according to the manufacturer's instructions with the AlkPhos DNA labeling kit from Amersham Biosciences. The hybridization temperature for DNA spiked on nylon filters (Hybond +, Amersham Biosciences) with the labeled DNA probe was 60 ° C. Hybridized DNA probe detection was carried out using the Amersham Biosciences "CDP Star Detection Kit", which complied with the Southernblot conditions described in the technical literature and by the manufacturer, and was evaluated by autoradiography In this case, the 0.8 kb G418-specific DNA fragment could only be detected in the transformants, but not in the Sr-I wild-type strain.

Plasmid pHPHsr: Genomic DNA of hygromycin resistant transformants (200 ng each) and S. ruineniae Sr-I (200 ng, negative control) and pHPHsr plasmid DNA (50 ng, positive control) were used in PCR reactions (Taq Core Kit, Qiagen) with the primers hphlf (SEQ ID NO: 13) and hph2r (SEQ ID NO: 14). The conditions of the PCR reaction were: 1 '94 ° C followed by 30 cycles of 30' '94 ° C, 30' '55 ° C, 1'

72 ° C and finally 5 '72 ° C. Aliquots of the PCR reactions were analyzed by agarose gel electrophoresis. The hygromycin resistance gene was observed as a 1 kb PCR fragment (see Example 6) only in transformants and in the pHPHsr positive control, but not in the non-transformed S. ruineniae Sr-1 negative control. These results confirm that, in the transformation of the strain Sporidiobolus ruineniae Sr-I, the G418 or hygromycin resistance gene from the expression vectors pG418Sr and PG418G18R, or pHPHsr, was integrated into the genome and used for the productive expression of the G418 gene. or hygromycin resistance gene, which allowed transformants to grow on G418 or hygromycin B antibiotic-containing media. Surprisingly, it was also found for the first time that the expression signals of the GAPDH gene from the basidiomycete yeast Phaffia rhodozyma are also functional in Sporidiobolus ruineniae, even though the transformation efficiency with the heterologous expression signals was markedly lower (see Example 8).

10th example

RNA analysis of Sporidiobolus transformants

Sporidiobolus ruineniae Sr-I was transformed with the expression vectors pG418Sr and pG418G18R as described in Example 8. Selected clones of transformation were cultured in YPG medium as described in Example 1. RNA from the cells of the culture was isolated with the "peqGOLD TriFast" reagent (PeqLab) according to the manufacturer's instructions RNA of the heterologously expressed G418 resistance gene was synthesized by reverse transcription combined with a PCR reaction (so-called RT-PCR). was detected as cDNA and the "SuperScript ™ II Reverse Transcriptase" RT-PCR cloning kit from Invitrogen was used. RNA was first treated with DNase I to remove any remaining DNA residues. 500 ng of the RNA thus obtained were then used for the RT-PCR and followed the manufacturer's protocol for the RT-PCR cloning kit. The cDNA obtained in this way was used in PCR reactions (Taq Core Kit, Qiagen) with the primers Greslf and Gres2r to detect the G418 resistance gene. The primers Greslf and Gres2r originated from the 5 'and 3' ends, respectively, of the 0.8 kb G418 resistance gene (see 5th example) and had the following sequences: Primer Greslf:

Greslf: 5 '-GGTAAGGAAAAGACTCACGT-3' (SEQ ID NO: 21).

Primer Gres2r:

Gres2r: 5'-TTAGAAAAACTCATCGAGCATC-3 '(SEQ ID NO: 22).

RT-PCR reactions were carried out with RNA samples isolated from the wild-type strain S. ruineniae Sr-I and from G418-resistant clones of S. ruineniae transformation with the expression vectors pG418Sr and pG418G18R, respectively. The cDNA products from the RT-PCR were analyzed by agarose gel electrophoresis. In RNA of the G418-resistant transformants, the expected 0.8 kb cDNA fragment could be detected, but not in RNA from nontransformed S. ruineniae Sr-I cells.

This result shows that S. ruineniae can be transformed with the expression vectors pG418Sr, pG418G18R and pHPHsr according to the invention, resulting in the functional expression of a heterologous gene.

11. Example

Phenotypic analysis of Sporidiobolus transformants

G418-resistant transformants of S. ruineniae strain Sr-I were purified on G418-selective YNB plates. Purified strains were used for cultivation in shake flasks. Legal media were YNB medium (non-selective) and YNB medium with G418 (selective). Growth conditions were 28 ° C on an orbital shaker (Infors, 140 rpm). Cell growth was measured by photometric determination of the cell density OD at 600 nm. The control strain used was untransformed S. ruineniae Sr-I. The result is shown in Tab. 2 (cell growth after six days of cultivation). While everyone

Strains in nonselective medium had good cell growth (column YNB without G418 in Tab. 2), no growth could be observed in the wild-type strain in selective medium, as expected. the. On the other hand, transforra- nants could also grow on selective medium after an initial lag phase of about two days. This demonstrates that the G418 resistance gene is operatively expressed in transformed S. ruineniae strains, resulting in inactivation of the G418 antibiotic and growth of the transformants.

This experiment shows that S. ruineniae can be transformed and, under the control of the homologous S. ruineniae GAPDH promoter or the heterologous P. rhodozyma GAPDH promoter, a heterologous gene is expressed in an active form, giving the transformants an antibiotic-resistant phenotype lent.

12. Example

Improvement of QlO Production in S. ruineniae by Metabolism Engineering

A: Isolation of the decaprenyl diphosphate synthase gene (DPS gene) from Rhodotorula minuta.

The DNA sequence DPS gene from Rhodotorula minuta is disclosed in EP 1336657 A, SEQ ID NO: 1. The strain Rhodotorula minuta DSM 3016 (available from the DSMZ German Collection of Microorganisms and Cell Cultures GmbH, D-38124 Braunschweig) was cultured at 28 ° C. in YPD medium (see Example 1) and genomic DNA was isolated from the resulting cells (see 2nd example). The DPS gene was isolated from genomic R. minuta DNA, according to the prior art, by PCR (Qiagen Taq Core Kit) with the primers rmlf and rm2r as a 1.6 kb DNA fragment. The primers rmlf and rm2r had the following DNA sequences:

Primer rmlf:

5'-TATATCATGATGCACCGACAAGCTGC-3 '(SEQ ID NO: 23)

Primer rm2r: 5'-TATGCGGCCGCTACTTTGTTCGGTTGAGCAC-3 '(SEQ ID NO: 24)

B: Preparation of vector pDPSrmGap

The PCR generated DNA fragment of the DPS gene was purified by preparative agarose gel electrophoresis and cut with Ex HI and Not I (the cleavage sites were contained in the primers rmlf and rm2r) and in the vector cut with Nco I and Not I pG418G5 (see 7th example). The result was the 5.8 kb vector pDPSrmGap (FIG. 9), in which the R. minuta DPS gene had been cloned behind the S. ruineniae GAPDH promoter. By DNA sequence analysis, it was verified that the DPS gene was correctly isolated from the genomic R. minuta DNA.

C: Production of the vector pDPSrm The expression cassette was isolated from the vector pDPSrmGap by digestion with AfI II and Bam HI as a 3.1 kb DNA fragment and cloned into the vector pG418G18R cleaved with AfI II and Bgl II (see Example 7). The result was the 11 kb expression vector pDPSrm (FIG. 10).

D: Transformation of S. ruineniae

The strain Sr220-159 disclosed in DE 10317877, 5th example, was used. The transformation with the vector pDPSrm linearized by digestion with Bsr GI was carried out as described in the 8th example. 100 G418-resistant transformants were isolated, purified and analyzed for Q10 production by cultivation in a shake flask, as described in DE 10317877, 5th example. From the master comparison, the recom- binary strain DPSrm-65 was selected for the fermentation analysis.

E: Fermentation analysis The fermentation analysis of the recombinant strain DPSrm-65 was carried out as disclosed in DE 10317877, 7th example. For the strain DPSrm-65, the biomass at the end of the fermentation was 80.3 g / l and the Q10 yield was 4.5 mg / g dry biomass. This corresponds to a marked increase compared to the prior art disclosed in DE 10317877, 5th example, for S. ruineniae.

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0-1 Form PCT / RO / 134 (SAFE) Information on a deposited microorganism and / or other stored biological material

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1-3-1 Name of the depository DSMZ DSMZ -German collection of microorganisms and cell cultures GmbH

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Brunswick, Geπnany

1-3-3 Date of deposit 07. April 2003 (07-04-2003)

1-3-4 entry number DSMZ 15553

1-5 States of destination for which special information is given to all the countries of destination

TO BE COMPLETED BY THE REGISTRATION OFFICE

0-4 This form has been received by the international application (yes or no)

0-4-1 authorized agent

Delimon, Krista

TO BE COMPLETED BY THE INTERNATIONAL OFFICE -5 This form was received by the International Bureau on the following date: -5-1 Authorized Agent

Claims

Claims:

1. Expression system consisting of a host organism of the genus Sporidiobolus and a DNA vector which contains a selection marker gene which codes for a protein which, after transformation of the host organism, permits a selection of positive transformants and is selected from the group of antibiotic resistance genes, the genes which complement an auxotrophy of the host organism and the genes coding for a protein which are capable of a coloring reaction, wherein the expression of the selection marker gene is controlled by at least one genetic regulatory element active in the host organism, characterized in that DNA vector containing a gene to be expressed which codes for a protein, wherein the gene to be expressed is a gene from the Q10 biosynthesis pathway and wherein the expression of the gene from the Q10 biosynthesis pathway by at least one in the host organism active genetic regulatory element kon - is trolled.

2. Expression system according to claim 1, characterized in that the selection marker gene and the gene from the Q10 biosynthetic pathway are contained together on a DNA vector, wherein the expression of the two genes by at least one active in the host organism geneti - Controlling regulatory element is controlled.

3. Expression system according to claim 1 or 2, character- ized in that the host organism is a strain of the species Sporidiobolus ruineniae.

4. Expression system according to claim 1, 2 or 3, character- ized in that the DNA vector contains a selection marker gene selected from the group of antibiotic resistance genes.

5. Expression system according to one of claims 2 to 4, data carried in that the antibiotic resistance genes confer resistance to an antibiotic selected from the group hygromycin, G418, loheximid Geneticin ^®, glyphosate, Cyc-, bialaphos, kanamycin, bleomycin, oligomycin, Zeocin ™, benomyl, nystatin and phleomycin.

6. Expression System to claim 5, characterized net gekennzeich- accordance that the antibiotic resistance genes resistance conferment hen to an antibiotic selected from the group hy- gromycin, G418, geneticin ^® and glyphosate.

7. Expression system according to claim 1 to 6, characterized in that the vector as promoter or Terminatore- ele- ments the promoter or terminator elements of GAPDH

From Sporidiobolus ruineniae, or the GAPDH gene from Phaffia rhodozyma, Ustilago maydis, Schizophyllum commune, Trametes versicolor, Agaricus bisporus or Phanero chaete chrysosporium.

8. Expression system according to claim 7, characterized in that the active in Sporidiobolus promoter elements are selected from the DNA sequences SEQ ID NO: 1, bp 1 to bp 1050 or SEQ ID NO: 2, bp 1 to bp 790th

9. Expression system according to claim 1 to 8, characterized in that the gene to be expressed from the QlO biosynthetic pathway is selected from the group acetoacetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase , Diphosphomevalonate decarboxylase, isopentenyl diphosphate isomerase, farnesyl diphosphate synthase, chorismate pyruvate lyase and the genes CoQ1 to CoQ10.

10. A process for the production of Sporidiobolus strains which are capable of efficiently gene expression and production of proteins, characterized in that a yeast of the genus Sporidiobolus is used as the host organism. which is transformed with a DNA vector which has a gene to be expressed and an antibiotic resistance gene, and from the transformation batch the clones transformed with the DNA vector are selected by selection of antibiotics of resistant transformants In each case, the expression of the gene to be expressed and of the antibiotic resistance gene in the host strain is controlled by a genetic regulatory element that is active in the host strain.

11. Process for the production of proteins, characterized in that an expression system according to one of claims 1 to 9 is used for protein production.

12. The method according to claim 11, characterized in that one or more genes selected from the group acetoacetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase, isopentenyl diphosphatidase, farnesyl diphosphate synthase, chorismate

Pyruvatlyase and CoQl to CoQ10 are expressed.

13. promoter and terminator elements of the sporidiobolus GAPDH gene, characterized in that they have a sequence selected from the group SEQ ID NO: 1, bp 1-1050 (promoter), SEQ ID NO: 2, bp 1-790 ( Promoter) and SEQ ID NO: 1 bp 2997-3500 (terminator), as well as DNA sequences derived therefrom by prolongation, truncation or alteration, which may be active in Sporidiobolus as promoter or terminator.

14. DNA vector which contains at least one selection marker gene which codes for a protein which, after transformation of a yeast of the genus Sporidiobolus, allows a selection of positive transformants, characterized in that the selection marker gene is selected from the group of Antibiotic resistance genes encoding proteins that inhibit the growth inhibition of antibiotics abrogate the host organism, the genes that complement a genetic defect of the host organism (auxotrophy) and the genes that code for proteins that are capable of a color-producing reaction, and that the selection marker gene at least one active in the host organism genetic regulatory element is controlled.