DE19963690A1 - Vectors and methods for producing recombinant proteins in fungi - Google Patents

Abstract

The invention relates to integration vectors for the targeted and stable integration of heterologous DNA into the genome of a host organism, especially a fungus, and to the use thereof. The invention also relates to a method for producing one or more recombinant proteins using the inventive vectors. The aim of the invention is to provide integration vectors which enable foreign DNA to be integrated into the genome of a host organism in a stable manner and with a high copy number and if necessary, to enable different genes to be expressed for a simultaneous coproduction of different proteins. Another aim of the invention is provide stable production strains and a method for producing heterologous proteins. To this end, an integration vector comprising the following is provided: a) at least one rDNA sequence from yeast; b) at least one non-deficient selection marker gene; and c) at least one expression cassette, said expression cassette comprising a promoter element that is functional in the host organism, an area of a heterologous or genuine gene that code for a desired protein and a terminator element that is functional in the host organism. The invention also comprises a host organism containing at least one inventive vector. A preferred organism is the methylotrophic yeast <i>Hansenula polymorpha </i>.

Description

The present invention relates to integration vectors for targeted and stable integra tion of heterologous DNA in the genome of a host organism, especially a fungus, and their use. The invention further relates to a method for producing a or several recombinant proteins using the Vek according to the invention goals.

Mushrooms and especially yeasts are attractive systems for the recombi production nanter proteins. A prominent representative is the optional methylotrophic yeast Han senula polymorpha, which is used for the production of pharmaceutical substances and indu strategic enzymes is used. The generation of recombinant strains of H, polymorpha is routinely done with vectors integrated into the yeast genome become. Due to the genomic localization of the heterologous DNA, the recombinant strains of H. polymorpha are characterized by high mitotic stability. However, each transformation approach results in different recombinant ones Strains that differ in the place of integration and number of copies of the integrated heterologous DNA differ, the number of copies can vary from 1 to 80. Because the place of integration is accidental, a non-homologous recombination in is not closer to the integration characterized gene loci accepted.

The targeted integration of heterologous DNA into the H. polymorpha genome has turned out to be very difficult. More recently, first opportunities have emerged described for targeted homologous integration into defined gene locations. These few Examples are limited to a disruption integration approach in which the recom binant DNA targeted in the MOX (methanol oxidase) locus (Agaphonov et al., 1995) or in telomer-associated HARS sequences from H. polymorpha (Sohn et al., 1999) was integrated.

A targeted homologous integration into a gene locus would be advantageous Offers requirements for expressibility. This usually applies to such areas a chromosome containing a highly expressed gene, such as B. the above introduced MOX gene. For a targeted integration of foreign sequences was also a highly expressed genomic region that contains the genes for the ribosomal RNAs (the so-called rDNA).  

This approach was developed for two types of yeast, the brewer's yeast Saccharomyces cerevisiae (Lopes et al., 1989; Lopes et al., 1991) and the milk yeast Kluyveromyces lactis (Bergkamp et al., 1992) tested. In these cases, the integration was carried out by homologous recombination using rDNA fragments of the corresponding host. Generated in this way recombinant strains contained multiple copies of the heterologous DNA integrated in the rDNA locus of the transformants. As an important, indispensable factor for the int Gration of the vector in high copy number was the presence of a complement tion gene with a defective promoter (i.e. Leu2-d or Ura3-d) with a high called selective effect. The combination of a defective promoter and one rDNA segment proved to be indispensable.

The generation of recombinant strains with different foreign genes in a desired number of copies was previously only possible by successive transforms steps possible. To do this, the plasmids used must have different Selek tion marker genes. Examples of this are strains for the coexpression of the genes for the HbsAg-L and S antigen (Janowicz et al., 1991), for the development of a Hepatitis B vaccine or the coexpression of two enzyme genes for development of a biocatalyst (Gellissen et al., 1996). So there is a need for vectors which the simultaneous integration of different heterologous DNA sequences into the genome allow a host organism.

The object of the invention is therefore to provide further integration vectors that it allow the foreign DNA in high copy number in the genome of a host organism stable integration and, if necessary, different genes for a simultaneous co-product tion of different proteins to express. Another object of the invention is stable production strains and a process for the production of heterologous proteins Semi note.

This problem is solved by an integration vector, which

• a) at least one rDNA sequence from yeast,
• b) at least one non-deficient selection marker gene and
• c) at least one expression cassette

comprises, the expression cassette a functional in the host organism Pro motor element, a region of a heterologous coding for a desired protein Genes and includes a terminator element functional in the host organism.

Surprisingly, it has been found that a stable multi-copy integration an integration vector with at least one rDNA sequence from yeast, without to a deficient selection marker gene can be achieved, and that different Integration vectors with identical, non-deficient marker genes integrated at the same time can be. When several vectors are integrated at the same time, the stoichio remains Mitometrically stable. The establishment of a "universal" yeast vector, basie Based on the newly identified elements, the Erfin can be widely used dung.

Under a "rDNA sequence from yeast" a sequence is ver Stand out from a section of the genome of a yeast that contains the genes for the riboso paint contains RNAs, is derived. Possible embodiments of the invention rDNA sequences come from yeasts of the Hansenula, Pichia, Saccharomyces, Kluyveromyces, Candida, Schwanniomyces, Yarrowia, Arxula or Trichosporon, and especially from the methylotrophic yeast Hansenula polymorpha and Pichia pastoris.

A "non-deficient selection marker gene" as used herein means a gene whose protein product has certain selectable properties for the host organism gives it before the transformation and integration of the vector according to the invention or the vectors of the invention did not possess (for example resistance to Antibiotic, complementation of an auxotrophy), the activity of which by a tens copy of the selection marker gene encoded product for normal growth of the host organism is sufficient. Examples of non-deficient selection marker genes that suitable for carrying out the invention include the URA3, HIS3, ADE2, LYS2, AR07 and TRP1 genes from S. cerevisiae and the LEU2 gene from H. polymorpha. Alternatively, a dominant selection marker gene such as e.g. B. the bacts rielle kanamycin resistance gene can be used.

"Expression cassette", as used here, refers to a DNA fragment which in min an mRNA is transcribed, which subsequently translates into a gene product  becomes. According to the invention, the expression cassette comprises a radio in the host organism capable promoter element, a region coding for a desired protein of a heterologous gene and a terminator that works in the host organism element. The expression cassette can also have a sequence for a signal peptide and / or contain a polylinker sequence. The aforementioned components of an Ex The pressure cassette can come from one gene or from different genes.

A "promoter element functional in the host organism", as used here, be draws a DNA fragment through which the initiation point and the initiation frequency the transcription (RNA synthesis) of one under the control of the promoter element gene in the host organism. To carry out the invention in Promoter elements under consideration include the FMD, MOX, TPS1, PMA1 and DAS promoters from Hansenula polymorpha, the MOX, AOX1 and GAP1 promoters from Pichle pastoris and the ADH1, PDC1, GAP1 and CUP1 promoters from S. cerevisiae.

A "heterologous gene" as used herein means a gene that is different from another Organism comes from the host organism in which it is to be expressed. At games for expressible heterologous genes include the genes for insulin, Phytase, HGH, hirudin, lactoferrin or G-CSF.

A "functional terminator element in the host organism", as used here, be records a DNA fragment that contains signal structures for RNA polymerase that lead to the termination of the transcription. Examples of terminator elements that can be used are the H. polymorpha MOX or PHO1 terminator.

Particularly suitable host organisms for carrying out the present invention Fungi, for example filamentous fungi such as Aspergillus, Neurospora, Mucor, Trichoderma, Acremonium, Sordaria and Penicillium or yeasts like Saccha romyces, Hansenula, Pichia, Kluyveromyces, Schwanniomyces, Yarrowia, Arxula, Trichosporon and Candida. Preferably, the host organism in which the express sion of heterologous genes for the production of one or more proteins using the Integration vectors according to the invention takes place, a yeast, in particular a me thylotrophic yeast. In the preferred embodiment of the invention, the inventions Integration vectors according to the invention for the production of proteins in a yeast Genus Hansenula, preferably Hansenula polymorpha, used.  

Integration vectors according to the invention have proven to be particularly advantageous in which at least one rDNA sequence from a region of the rDNA of a methylo trophic yeast, which contains a 400 bp fragment of the 3 'region of the 25S rDNA Gene and a 600 bp fragment of the 5 'region of the 18S rDNA gene and the intermediate sequences including the 5S rDNA sequence. Go one contains from a similar rDNA structure as in Saccharomyces cerevisiae this approx. 3 Kb long area is a TOP sequence, one or more HOT1 sequences as well as NTS1 and NTS2 sequences. Vectors according to the invention that such rDNA sequence included, are integrated in a high copy number and stably. Prefers these rDNA sequences originate from Hansenula polymorpha. Alternatively, you can rDNA sequences from other methylotrophic yeasts such as B. Pichia pastoris, Pichia methanolica or Candida boidinü can be used.

In a preferred embodiment of the invention, the integration vectors according to the invention comprise at least one rDNA sequence which is selected from the following sequences:

• a) a sequence according to SEQ ID NO 1;
• b) a fragment of the sequence according to a), which is necessary for the stable integration of Se sequences of the vector in high copy number into the genome of a host organ nism, in particular a yeast is suitable;
• c) a sequence which is at least 70% complete with a sequence according to a) or b), preferably at least 80%, particularly preferably at least 90% is identical;
• d) a sequence which coexists with the sequence of a) or b) hybridized;
• e) one by substitution, addition, inversion and / or deletion of one or Multiple base derivative of a sequence according to one of the Groups a) to c), which are responsible for the stable integration of sequences of the Vek tors in high copy number in the genome of a host organism, in particular which is suitable for a yeast, and  f) a sequence complementary to a sequence according to one of the groups a) to e).

The 2.4 Kb long rDNA sequence which can be isolated as a BamHI / EcoRI fragment according to SEQ ID NO 1 comprises 400 bp of the 3 'region of the 25S rDNA gene and TOP, HOT1, NTS1 and NTS2 sequences flanking the 55 rDNA gene. To carry out the invention fragments of this sequence can also be used, which are responsible for the stable integra tion of sequences of the vector in high copy number in the genome of a host organ mus, in particular a yeast, for example a fragment that does not Contains sequences of the 25S rDNA gene, or a fragment that the between the 3 'end of the 25S gene and the 5' end of the 5S gene located TOP, HOT1, NTS and 5S sequences. Frag also comes for the implementation of the invention elements in which one or more of the HOT and / or NTS sequences are missing, as well as fragments in which the TOP and / or the 5S rDNA sequence is missing. There however, it is believed that the TOP and HOT sequences are important components of the rDNA sequence contained in the integration vectors according to the invention, fragments of SEQ ID NO 1 are preferred which have at least one TOP sequence and contain at least one HOT sequence.

The term "stable integration" as used herein means an integration at the restriction pattern and number of copies after at least 100 generations of permanent culture remain essentially unchanged in non-selective medium.

For the purposes of the invention, the expression “at least 70%, preferably at least 80%, particularly preferably at least 90% identical "on identity on DNA Level that according to known methods, e.g. B. the computer-aided sequence comparisons (Basic local alignment search tool, S.F. Altschul et al., J. Mol. Biol. 215 (1990), 403-410) can be determined.

The term "identity" known to those skilled in the art denotes the degree of relatedness shaft between two or more DNA molecules, which is determined by the match between the sequences is determined. The percentage of "identity" results from the Percentage of identical areas in two or more sequences taking into account of gaps or other sequence peculiarities.  

The identity of related DNA molecules can be determined using known methods be determined. As a rule, special computer programs are used algorithms that meet their requirements. Preferred procedures to determine identity first generate the greatest match between the sequences examined. Computer programs for determining identity between two sequences, but are not limited to that GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12 (12): 387 (1984); Genetics Computer Group University of Wisconsin, Madison, (WI)); BLASTP, BLASTN and FASTA (Altschul, S. et al., J. Molec Biol 215: 403/410 (1990)). The BLAST X program can be obtained from the National Center for Biotechnology Information (NCBI) and from other sources (BLAST Handbuch, Altschul S., et al., NCB NLM NIH Bethesda MD 20894; Altschul, S., et al., J. Mol. 215: 403/410 (1990)). The well-known Smith Waterman algorithm can also be used to determine identity be used.

Preferred parameters for sequence comparison include the following:
Algorithm: Needleman and Wunsch, J. Mol. Biol 48: 443-453 (1970)
Comparison matrix: match = +10,
Mismatch = 0
Gap penalty: 15
Gap length value:

(Gap Length Penalty): 1.

The GAP program is also suitable for use with the above parameters net. The above parameters are the default parameters for Nucleic acid sequence comparisons.

Further exemplary algorithms, gap opening penalties, Gap extension penalties, comparison matrices included those mentioned in the Program Guide, Wisconsin Package, Version 9, September 1997 can be used. The selection will depend on the comparison to be made depend and continue whether the comparison between sequence pairs, where GAP or Best Fit are preferred, or between a sequence and an extensive one Sequence database, with FASTA or BLAST being preferred.  

The feature "sequence which is the opposite strand of a sequence according to a) or d) hybri disiert "indicates a sequence that is under stringent conditions with the counterpart strand of a sequence hybridized with the features specified under a) or b). For example, the hybridizations at 68 ° C in 2 × SSC or after the proto coll of the dioxygenin labeling kit from Boehringer (Mannheim) become.

In one embodiment of the invention, the integrations according to the invention comprise vectors an additional selection marker gene for E. coli, for example an ampicillin Resistance gene, a tetracycline resistance gene or a kanamycin resistance gene. As Se less marker marker gene for E. coli also comes the URA3 gene from S. cerevisiae in Be which is a selection of transformants of the uracil auxotrophic E. col / strain MC1066 allowed.

The vectors according to the invention preferably have a length of 7 to 12 Kb. Such vectors offer good handling in E. coli and problem-free trans formation of yeast.

A host organism that is stable to at least one integration vector according to the invention Integrated in the genome is also provided by the invention. Preferential wise the host organism is a yeast, especially a methylotrophic yeast. In a preferred embodiment of the invention, the host organism is a yeast of the Genus Hansenula, preferably Hansenula polymorpha.

The invention further provides a host organism that can be achieved by stable integration of one or more integration vectors according to the invention into the genome of a Yeast is available. The yeast preferably belongs to the Hansenula genus; especially the yeast Hansenula polymorpha is preferably used. In a special version Rungsform the invention is the host organism through stable integration of several Vectors comprising different expression cassettes are available.

The invention further provides a method of making one or more Proteins in a eukaryotic microorganism. According to the invention cultivated for a host organism according to the invention in a manner known per se and the protein produced is obtained.  

The use of the integration vectors and host organisms according to the invention for Production of one or more proteins is also encompassed by the invention sums up.

The following figures and examples illustrate the invention.

Fig. 1 shows the organization of an rDNA unit of S. cerevisiae (A) and H. polymorpha (B) and the deduced position of the cloned rDNA sequence (SEQ ID NO 1) in a unit rDNA of H. polymorpha. The gray arrows indicate the direction of transcription of the rmA genes. The dashed boxes indicate the primary amplificate and the integration sequence used in the integration vectors, the representation not being to scale. NTS = non-transcribed spacer; 5S = 5S rDNA; HOT = hot spot of recombination; TOP = topoisomerase binding site. (A) The rDNA unit from S. cerevisiae according to Lopes et al. (1996). (B) Position of the primary amplicon. The primers homologous to S. cerevisiae bind to the highly conserved 25S rDNA (primer A) and to the 5 'end of the 18S rDNA (primer B) within the H. polymorpha rDNA unit. The black arrows indicate the primary PCR amplificate. (C) Position of the 2.4 Kb integration sequence (SEQ ID NO 1) in the rDNA unit of H. polymorpha. This sequence contains the 5S gene, the NTS1 sequence and partial regions of the NTS2 sequence and 25S sequence.

Fig. 2 represents the 2.4 Kb long rDNA sequence of an integration element (SEQ ID NO 1) of the present invention rDNA integration vector. The region of the 5S rDNA is shaded gray.

Fig. 3 illustrates the rDNA integration vector pM2.

Fig. 4 represents the rDNA integration vector pM2Δ.

Fig. 5 illustrates pM2ZT (lacZ, TPS1 promoter), a rDNA integration vector for Expres sion is in H. polymorpha of lacZ.

Fig. 6 shows pM2GT (GFP3, TPS1 promoter), an rDNA integration vector for the expression of GFP in H. polymorpha.

Fig. 7 shows the result of a Southern blot analysis for the detection of the integration of pB14 in the rDNA of H. polymorpha.

Figure 8 shows a model for the integration of an rDNA integration vector (pB14) into the DNA. The figure shows the assignment of the fragments from the Southem blot analysis in FIG. 7. The assignment of the fragment lengths from the Southern blot analysis results from the restriction site for Bg / II and the known lengths of the fusion fragments from heterologous DNA and rDNA.

Fig. 9 shows the result of a Southern blot analysis for a selection of recombinant strains after transformation of RB11 with three different vectors.

Fig. 10 shows the result of a Southern Blof analysis to determine the copy number of the cointegrated vectors in the recombinant strain RB3T-19.

FIG. 11 shows the result of a Southern B / of analysis to demonstrate the mitotic stability of the recombinant strain RB3T-19. The numbers indicate the day of sampling.

Figure 12 shows a Southern blot analysis of four recombinant strains generated by simultaneous transformation with five vectors. Three of these strains contain sequences from at least one rDNA integration vector.

Figure 13 shows the TPS9 promoter-controlled expression of lacZ in recombinant strains of H. polymorpha. The colonies are numbered from top left to bottom right.

Fig. 14 shows the identification of recombinant insulin-secreting strains of H. polymorpha in an immunological test filter. The numbers on the filters correspond to the numbers of the recombinant strains. Filter A shows strains RB4T 1-66. The positive control is at position 67. Filter B shows the strains RB4T 67-132. The positive control is at position 133.

Examples material and methods Abbreviations

ampicillin
amp ^R

Ampicillin resistance
ARS Autonomously Replicating Sequence
BSA Bovine Serum Albumin
Bp base pairs
DNA deoxyribonucleic acid
DTT dithiothreitol
EDTA ethylenediaminetetraacetic acid
ELISA Enzyme Linked Immuno Assay
FMD gene for formate dehydrogenase
HARS H. polymorpha Autonomously Replicating Sequences
HOT hot spot of recombination
Kb kilobase pairs
LB Luria Broth medium
MOX gene for the methanol oxidase from Hansenula polymorpha
NTS non-transcribed spacer
ori origin of replication of a plasmid
ODX Optical density at _x

nm wavelength
PCR polymerase chain reaction
pH potentia hydrogenii
rDNA ribosomal DNA
rmA ribosomal RNA
rpm revolutions per minute (rounds per minute)
SDS sodium dodecyl sulfate
TOP topoisomerase binding site
TPS1 gene for trehalose-6-phosphate synthase
Tris tris (hydroxymethyl) methylamine
ÜK overpower culture
X-Gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside
YNB Yeast Nitrogen Base (selection medium without uracil)
YPD full medium

2. Chemicals and enzymes

Unless specifically mentioned, chemicals were obtained from the following manufacturers Gen: Acros Organics (New Jersey); Amersham International plc. (Buckinghamshire, England); Baker (Deventer, The Netherlands); Biorad (Richmond, USA); Biozyme (Hess. Oldendorf); Caesar & Loretz GmbH (Hilden); Difco Laboratories (Detroit, USA); Fluka AG (Basel, Switzerland); Gibco (Paisly, Scotland); Janssen (Beerse, Belgium); Merck (intestine city); Oxoid Ltd. (Basingstone, England); Pharmacia (Freiburg); Riedel de Haen (Seelze); Roth (Karlsruhe); Serva (Heidelberg); Sigma (St.Louis, USA); Restriction enzymes were produced by the manufacturers Boehringer (Mannheim), Gibco BRL (Paisly, Scotland) and New England Biolabs (Schwalmbach). It was in the respective supplied buffers worked. The enzyme zymolyase was developed by Seikaguko Corp. based. DNA preparations were made using various DNA isolation kits Quiagen (Hilden) carried out according to the manufacturer's instructions.

3. Tribes 3.1 E. coli

DHSαF 'F'endA1hsdR17r _k

m _k

+ supE44thi-9recA1gyra relA (lacZYA-argF) U169 (ϕ80Δ (lacZ) M15) (Gibco BRL, Gaithersburg MD, USA)
MC 1066: Δ (lacI POZYA) A74, galU, galK, strA ^-

, usdR ^-

, trpC 9830, leu B6, pyr F74 :: Tn5 (Km ^r

) (Casadaban et al., 1983).

3.2 H. polymorpha

RB11 odc1 orotidine-5-phosphate decarboxylase deficient (uracil auxotrophic) H. polymorpha strain (Weydemann et al., 1995).

4. Media 4.1 Media for the cultivation of H. polymorpha

Complete medium: 2% glucose or glycerin, 1% yeast extract, 2% Bacto-Peptone.
Selection medium: 0.17% yeast nitrogen base, 0.5% ammonium sulfate, 2% glucose or glycerol, 38.4 mg / l arginine, 57.6 mg / l isoleucine, 48 mg / l phenylalanine, 57.6 mg / l Valine, 6 mg / ml threonine, 50 mg / l inositol, 40 mg / l tryptophan, 15 mg / l tyrosine, 60 mg / l leucine, 4 mg / l histidine. Uracil is missing in the selection medium.

4.2 Media for the cultivation of E. coli

Luria Broth medium (LB): 10 g / l casein hydrolyzate (Peptone No.140, Gibco), 5 g / l yeast extract, 5 g / l NaCl. Ampicillin (Roth) was used as an antibiotic in a concentration of 500 mg / l, added after autoclaving.

M9 medium: 2% glucose, 30 g / l Na ₂ HPO ₄ , 15 g / l KH ₂ PO ₄ , 5 g / l NH ₄ Cl, 2.5 g / l NaCl, 0.015 g / l CaCl ₂ , 2 , 28 g / l, MgSO ₄ , 0.05% vitamin B1 (not autoclaved) 38.4 mg / l arginine, 57.6 mg / l isoleucine, 48 mg / l phenylalanine, 57.6 mg / l valine, 6 mg / ml threonine, 50 mg / l inositol, 40 mg / l tryptophan, 15 mg / l tyrosine, 60 mg / l leucine, 4 mg / l histidine.

Solid culture media were prepared by adding 1.6% agar to the liquid media poses.

5. PCR (Polymerase Chain Reaction)

In a personal cycler (Biometra, Göttingen) PCR reactions with the PC Long expand kit or the PCR high fidelity kit from Boehringer (Mannheim) in 50 µl batches carried out. The products were purified using the PCR puri fication kit from Qiagen (Hilden) according to the manufacturer's instructions.

6. Transformation 6.1 Transformation of E.coli 6.1.1 Electroporation

400 ml LB medium were diluted 1: 100 from a ÜK and inoculated at 37 ° C up to cultivated an ODsoo of 0.5-1. After 30 min. The culture was incubated on ice at 4 ° C and 3000 rpm for 15 min. centrifuged. The cells were washed 2 × with ice cold water (1 vol./l/4 vol.) And washed 1 × with 1/50 vol. 10% glycerol. Then was the cell pellet was taken up in 1.5 ml of 10% glycerol and 40 ul aliquots at -70 ° C maintains. For the transformation an aliquot was thawed on ice and with 0.01-1 µg Plas mid-DNA with a Gene Pulser (Bio Rad) at 1.6 KV, 25 µF and 200 Ohm in one 1 mm cuvette transformed. The cells were then recovered for 30-45 min. at Incubated 37 ° C in 1 ml LB medium and then plated on LB-amp plates. The plates were incubated at 37 ° C.

6.1.2 RbCl ₂ method

RbCl2 cells were transformed according to the Hanahan (1985) protocol.  

For each transformation 20-500 ng DNA with 200 µl thawed on ice became more competent Cells mixed. After 20 min. Incubation on ice, 45 sec. Heat shock (42 ° C) and he new 2 min. The cells were incubated on ice for recovery with 1 ml LB medium added and incubated for 30-45 min at 37 ° C. The cells were then opened LB-amp plates plated, which are then incubated overnight at 37 ° C.

6.2 Transformation of H. polymorpha by electroporation

100 ml of YPD were inoculated with 5 ml of a tightly grown ÜK. The culture was shaken at 37 ° C. to an ODsoo of 0.8-1.2 (approx. 3 hours). The cells were harvested by centrifugation at 3000 rpm and in 20 ml KP; Buffer (50 mM / pH 7.5) resuspended. After adding 0.5 ml of DTT and shaking for 1 minute at 37 ° C., the cells were centrifuged at 2500 rpm and washed 2 × with STM buffer (270 mM sucrose, 10 mM TrisCl, 1 mM MgCl ₂ , pH 7.5). The cells were then taken up in 0.25 ml of STM buffer and 60 μl aliquots were stored at -70 ° C. For transformation with rDNA integration vectors, the plasmid DNA was first linearized with Xhol or Sacl. 0.1-1 µg of the linearized plasmid DNA were mixed with fresh, competent cells thawed on ice, these batches were then placed in a cuvette in 2 mm. The transformation was carried out in a gene pulser (Bio-Rad, Munich) at 2.0 kV, 25 µF and 200 ohms. The cells were then incubated for recovery in 1 ml of YPD for 1 hour at 37 ° C. and then plated out on selection medium. After 2-4 days of incubation at 37 ° C, macroscopic colonies became visible.

7. Generation of H. polymorpha strains

To integrate vectors into the yeast H. polymorpha, those from the Transforma tion-derived uracil-prototrophic clones three times in succession on selection medium plates, once on full medium plates, and then again on selection Transfer medium plates (depending on the 24 hour growth at 37 ° C).

8. Continuous cultivation of H. polymorpha

For stability studies, 50 ml liquid cultures (YPD, 2% glucose) were used over 27 days shaken at 37 ° C, freshly vaccinated every day. The H. polymorpha cultures at the time of vaccination and sampling, an ODsoo between 3 and 4, so the cells were before entering the stationary phase. There were Prepare 2 ml glycerol cultures (27%) every other day and store at -70 ° C.  

9. Preparation of nucleic acids 9.1 Fragment isolation from low-melting gels

The DNA preparation from low-melting agarose gels was carried out using the Phenoll Chloroform extraction. Gel blocks with DNA fragments cut out under UV light 130 μl of water were added and the mixture was heated at 70 ° C. until the agarose melted. After adding 150 μl phenol and shaking 50 times, the sample was centrifuged (2 min. At 13000 rpm) and the lower phenol phase discarded. Now 150 ul Phe nol / chloroform (1: 1 mixture) added to the aqueous phase, mixed and again centrifuged. Again the lower phase was discarded and the aqueous phase washed twice with chloroform / isoamyl alcohol (1:24) (150 µl each). Subsequently a 1/10 volume of NaAc (3 M, pH 8) and 1 ml of 96% ethanol was added and the DNA through 15 min. Incubation at -70 ° C. After centrifuging the DNA, washing once with 70% ethanol and then drying the DNA in 15-30 µl of water added.

9.2. Fragment isolation from PCR batches

The fragment isolation from PCR batches and from low-melting gels is carried out with the help PCR purification kit (Quiagen, Hilden) according to the manufacturer's instructions.

9.3 Plasmid preparation E. coli (mini-prep)

E. coli plasmid DNA was prepared according to the method described by Maniatis et al (1982) described method of alkaline lysis. 1.5 ml of a ÜK were at 13000 rpm centrifuged (2 min.). The cell pellet was in 100 ul P1 (50 mM Tris / HCl, 10 mM EDTA) resuspended. The lysis is carried out by adding 100 μl P2 (200 mM NaOH, 1% SDS). Proteins and other cell components were then removed by adding 100 µl P3 (2.55 M KOAc, pH 4.8) precipitated. After centrifugation for 10 min. at 13000 rpm the DNA from the supernatant was doubled in volume with 96% ethanol falls. After washing and drying, the pellet was taken up in 20 ul water.

The degree of purity of this DNA was sufficient for restriction analyzes.

9.4. Plasmid preparation from E. coli (midi prep)

This was the case for DNA preparations of higher purity and larger quantities Plasmid midi kit from Quiagen used according to the manufacturer's instructions.

9.5. Concentration determinations of nucleic acids

Low molecular weight DNA (oligonucleotides) was concentrated by determination of absorption at 260 nm. The concentration of high molecular weight DNA  was compared to a calibrated quantity standard (KB-Leiter, Pharmacia) Estimated agarose gel.

9.6 Preparation of chromosomal DNA from H. polymorpha

Cells from a 2 ml ÜK were harvested at 13,000 rpm and placed in 0.5 ml STEHp Buffer (0.5 M sorbitol; 0.1 M Tris / HCl; pH 8; 0.1 M EDTA) resuspended. The cell wall was by adding 10 ul zymolyase (2.5 mg / ml) and 1 ul of beta-mercaptoethanol and Incubation for 1 hour at 37 ° C removed. It was checked under the microscope whether the Cells are present as protoplasts. The protoplasts were then carefully centrifuged giert (5 min at 3000 rpm), in TEHp buffer (50 mM Tris / HCl, pH 8, 20 mM EDTA) resus oscillated and mixed with 25 ul SDS (20%). The lysis was carried out by incubation for 20 min. at 65 ° C. By adding 200 µl KOAc and incubation on ice (30 min.) Pro teine and cell debris felled. After centrifugation at 13,000 rpm for 10 min. was the Filter the supernatant through cotton wool and the DNA dissolved in the filtrate with 0.7 volumes of isopropa nol like. After washing with 70% ethanol, the dried DNA pellet was washed in 20-30 µl of water added.

9.7 Preparation of chromosomal DNA to obtain large fragments

The standard protocol described in 9.6 was modified in order to obtain genomic DNA of better quality. H. polymorpha cells were harvested from 50 ml ÜK at 3000 rpm. The cells were resuspended in 30 ml STEHp buffer, centrifuged and then taken up in 1.5 ml STEHp. 30 µl zymolyase was added to dissolve the cell wall. The protoplasts were mixed with 2 ml of 5 M KOAc, the lysate for 30 min. centrifuged at 13,000 rpm (4 ° C). Now the supernatant was mixed with 2 ml of 5 M NH ₄ OAc and the dissolved DNA was precipitated with 10 ml of cold 96% ethanol. After 5 min. Incubation on ice was the DNA at 3000 rpm for 5 min. centrifuged, washed with 50% ethanol and taken up in 2 ml TE (10 mM Tris / HCl, 0.1 mM EDTA, pH 7.5). This entire procedure was repeated several times in decreasing volumes. The DNA was finally taken up in a volume of 200 ul TE.

10. Southern blot analysis

5 µg of chromosomal DNA (H. polymorpha) or 0.5 µg of plasmid DNA were used overnight treated with 1 U / µg of the corresponding restriction endonuclease. The separation the DNA was carried out in 0.6-0.8% agarose gels. Due to long separation times (2-12 hours at 200 mA), relatively large (5-12 Kb) DNA fragments could also be distinguished become. After photographing the gel, the DNA transfer to Ny  lonmembran (Hybound M, Amersham) in a vacuum blot apparatus (LKB 2016 Vacu gene, Pharmacia). This was first done for 15 min. with depurination solution (0.25 M HCl) transferred. This additional step also enables the transfer very large fragments. With denaturing solution (1.5 M NaCl, 0.5 N NaOH) and new Tralization buffer (3 M NaAc, pH 5, 5), the gel was each 20 min. long layered. From was closed for 20 min. with 20 × SSC transfer buffer (3 M NaCl, 0.3 M NaCitrate, pH 7) overlaid. By irradiating the membrane with UV light for 2 minutes (254 nm) and subsequent incubation at 80 ° C. for 30 minutes, the DNA was transferred to the Fixed nylon membrane.

The preparation of the probe, the hybridization and the detection of the hybrid were carried out with the help of an enzymatic reaction according to the protocol of the dioxygenin Labeling kit from Boehringer (Mannheim). In some cases, the Southern Blots hybridized and developed with a second probe. For this, the first probe removed from the membrane by a series of washes (1 hour in dimethyl formamide at 60 ° C, then washing in solution 1 (0.2 M NaOH, 0.1% SDS) and solution 2 (0.3 M NaCl, 0.03 M citrate) for 10 min each).

11. X-Gal Overlay Assay - Detection of β-galactosidase

The p-galactosidase activity was detected according to Suckow and Hollenberg (1998). The strains to be tested were cultivated for 4-6 hours in selection medium at 37 ° C. A 4 ul drop of each culture was placed on a selection plate and incubated overnight at 37 ° C. The plate was washed with fresh overlay agar (0.5% agarose, 0.5 M Na ₂ HPO ₄ / NaH ₂ PO ₄ (pH 7); 0.2% SDS; 2% DMF (Dimethylforma mid) 2 mg / ml X- gal (o-nitrophenyl-β-D-galactopyranoside) was overlaid at 70 ° C. After a few minutes, the clones with lacZ expression showed a blue color.

12. Phytase detection

The H. polymorpha cells were harvested from 3 ml of ÜK and taken up in 200 μl of YNB medium and 1 ml of 5% glycerol. After 1-2 days of growth, the OD ₆₀₀ was first determined. The cells were then centrifuged off and 25 ul of the supernatant were used again. 25 μl of 5 M NaAc and 50 μl of 4-nitrophenyl phosphate were added to this aliquot. The batch was 30 min. incubated at 37 ° C. The enzymatic conversion of the substrate was stopped by adding 100 μl of 15% trichloroacetic acid. After adding 100 µl of 1 M NaOH, positive culture supernatant samples appeared colored intensely yellow. The yellowing was quantified by OD ₄₀₅ measurement in a photometer.

13. Immunological detection of insulin

The cells were cultured for 1-2 days in 2 ml of selection medium (2% glycerol) at 37 ° C fourth. 4 µl aliquots of these cultures were dropped onto a selection plate and this incubated overnight at 37 ° C. A nitrocellulose membrane (Amersham Int., England) was placed on the plate, then incubated again overnight 37 ° C. The membrane was carefully peeled off and all adhered to the membrane Cells were rinsed carefully with water. After drying the membrane the secreted gene product was immunolo on the filters with insulin antibodies be proven. The proof was carried out according to standard methods with the help of the NOVEX anti-mouse kit. Insulin-secreting clones were able to use this method be clearly demonstrated.

14. Detection of GFP expression

For this study, an especially for the translation in the yeast S. cerevisiae opti mated mutant of wt-GFP, the yeast enhanced green fluorescent protein3 (yEGFP). In S. cerevisiae this protein shows a stronger fluorescence than wt-GFP (Cormack et al., 1997). The gene for yEGFP is located on the plasmid pM2GT control of the TPS1 promoter from H. polymorpha. It could be shown that yEGFP in H. polymorpha leads to fluorescence comparable to S. cerevisiae. In This study qualitatively analyzed GFP expression. For this, the Strains in duplicate for 12 hours at 37 ° C in YNB medium (2 ml cultures) moved and examined for a green glow under the fluorescence microscope.

example 1 Obtaining and characterizing an rDNA fragment for construction an rDNA integration vector

The prerequisite for the integration of a vector into the H. polymorpha rDNA is the presence of a sequence which is homologous to this region. For this purpose, a 2.4 Kb rDNA fragment from the rDNA region of H. polymorpha was determined. In the following this 2.4 Kb long sequence (SEQ ID NO 1) (Figure 2) is referred to as the integration sequence. For the cloning of this fragment from the H. polymorpha ribosomal DNA, a 3 Kb fragment from the H. polymorpha chromosomal DNA was first amplified with the aid of a PCR reaction. The primers were determined according to sequences of the 18S and 26S rDNA from S. cerevisiae with flanking BamHI recognition sequences. The amplificate was later shortened to 2.4 Kb by EcoRI / BamHI restriction (the fragment contains an internal EcoRI recognition sequence). The 2.4 Kb fragment was cloned into the vector pM1 and completely sequenced. The base vector pM2 ( FIG. 3) was formed, from which all the plasmids described in this application have arisen.

By comparison with known sequences from the rDNA unit of S. cerevisiae, the 5S rmA gene could be detected within the cloned sequence. The 5S DNA from H. polymorpha has a 96% homology to the corresponding gene from S. cerevisiae. The 5S gene is located at position 958-1079 of the integration sequence, counted from the flanking EcoRI interface. With this finding and knowledge of the isolation of the integration sequence, the position of the cloned rDNA fragment in an rDNA repeat of H. polymorpha could be determined and a similar organization of the rDNA sequences as in S. cerevisiae assumed. Portions of the ITS2 and the 18S sequence are lost when shortening. The position of the cloned fragment in the rDNA unit of H. polymorpha is shown in Fig. 1 (C). Its complete sequence is documented in FIG. 2 below. The gray shaded sequence shows the location of the 121 bp long 5S rDNA.

Example 2 Preparation of the basic rDNA integration vectors pM2 and pM2A

The 2.4 Kb EcoRI / BamHI rDNA fragment described in the previous example was introduced into an E. coli I H. polymorpha shuttle vector pM1 which contains the following elements: HARS1 and MOX terminator from H. polymorpha; ori and amp ^R from E. coli. A polycloning site for the reception of heterologous promoter / gene fusions is positioned between HARS1 and MOX terminator. Some examples of the resulting integration / expression vectors are given in Examples 3 and 4. The integrated rDNA contains a BrsGI, SacI, SnaI and an Xhol recognition sequence for the linearization of the resulting vector M2. The vector is shown in Fig. 2.

To generate the basic vector pM2Δ, the vector pM2 was digested with DraI / AatII and the resulting 8.7 Kb fragment was religated with T4 polymerase after removal of the 3 ′ overhang of the AatII cohesive end. The result is a shortened shuttle vector without amp ^R. The propagation of this vector and its derivatives was carried out in the E.coli strain MC1066, which is uracil auxotrophic, and thus could be selected for uracil prototrophy via the Ura3 gene. The basic vector M2Δ is shown in FIG. 4.

Example 3 Integration / expression vector for the expression of IacZ

The vector pM2ZT shown in Fig. 5 was generated by integrating an expression cassette for the bacterial lacZ gene in the polycloning site of pM2. The TPS1 promoter from H. polymorpha (pM2ZT) was used as the promoter element; in Example 4, an expression cassette for GFP was generated by integrating a suitable fragment into pM2. The H. polymorpha TPS1 promoter was used as the promoter element.

Example 4 Integration / expression vector for the expression of GPF

The vector pM2GT shown in FIG. 6 was generated by integrating an expression cassette for GFP into the polycloning site of pM2. The H. polymorpha TPS1 promoter was used as the promoter element.

Example 5 Integration / expression vectors for the expression of an insulin and a phytase Gene under the control of the TPS1 and FMD promoters

The following example describes further expression vectors. They are derivatives of pM2 and pM2Δ and, as shown for Examples 3 and 4, were generated by inserting a suitable fragment into the polycloning site of the two basic vectors. They contain expression cassettes for the synthesis of insulin and phytase. In addition to TPS9, the FMD promoter was used as a control element for the heterologous gene expression. The following vectors were generated:
pB11 derivative of pM2 with heterologous expression cassette for insulin (FMD promoter before an insulin gene), H. polymorpha rDNA integration sequence, HARS1, URA3 (S. cerevisiae), MOX terminator, ori, amp ^R.
pB14 Like pB11, but with a synthetic phytase gene instead of the insulin gene.
pB14X M2Δ derivative with heterologous expression cassette. Like pB14, but without amp ^R

Example 6 Generation of recombinant H. polymorpha strains

The transformation of RB11 with rDNA integration vectors proceeded according to a gene real principle that applies to all the transformations shown here. By the restriction with an enzyme for which only a singular interface within the integration sequence is present (e.g. Sacl or Xhol), the integration sequence was in two terminal parts shared sequences that are homologous with corresponding sequences on the rDNA unit can recombine. For non-linearized plasmids, no integration into the rDNA are detected. The rDNA integration vectors contained as Selek tion marker gene the intact URA3 gene from S. cerevisiae. The use of such intact genes, as shown in the examples below, leads to mitotic sta bile strains in which the recombinant integrated DNA is present in 40-50 copies can.

The transformation was carried out by electroporation (see Material and Metho the). The integration into the genome takes place through growth under selection pressure 30 generations (YNB plates or media that do not contain uracil). At the forth conventional method for generating recombinant H. polymorpha strains with circu Lär plasmids are at least 80 generations of growth on selective media necessary (Gatzke et al. 1995). This means that when using rDNA integration vectors a simpler and faster method of ge compared to the established protocols Generation of recombinant H. polymorpha strains for extermination.  

Example 7 Transformation with a vector - generation of recombinant strains with pB14 and their characterization

The rDNA in H. polymorpha consists of approx. 40 consecutive units that are constructed identically (Example 1). The length of a unit is approximately 8 Kb. This Length can be determined by hybridization experiments with rDNA probes, if in Restrictions Enzymes are used that cut only once in an rDNA repeat. One of these enzymes is Bglll. The integration is in one or more of these rDNA-Ein possible.

The uracil auxotrophic strain RB11 was transformed with pB14 according to the method described above. The linearization was done by Sacl. As a result, the rDNA fragment of the vector was distributed over approximately two equally long edge sequences. A representative example of the generated recombinant strains is documented below. The structural detection of the heterologous DNA integrated in the rDNA is based on the following preliminary considerations: If an enzyme is used which cuts in the vector but not in the rDNA (BamHl), and this is used together with Bglll (cuts in the rDNA unit but not in the vector), it would have to be possible to identify fragment sizes which indicate the transition from an rDNA repeating unit into a vector copy. In the event that several copies of the vector are integrated at different positions, very long bridging fragments would also have to be detected, since BamHI only cuts in the vector and BgIII only in the rDNA. Fig. 7 shows this analysis.

The DNA cut with the specified restriction enzymes hybridizes with the Probe resulting from the labeling reaction of the integration sequence (see material and methods). In the lane with the BamHI-treated DNA is a star kes signal at 7.4 Kb. This corresponds to the vector fragment. In this track a weakly recognizable signal is still visible at over 21 Kb. This fragment is interpreted as a bridging fragment. On track with BglII-treated DNA a strong signal can be seen at 8 Kb, which corresponds to the length of an rDNA unit. In Both the rDNA and the vector signals are on the double digest track recognizable. At 8.8 Kb and 6.7 Kb weak signals can be seen, the transition represent fragments of rDNA in plasmid DNA. In the first lane, the DNA  Standard length is the kilobase ladder, in the second lane EcoRI / HindIII-digested λ DNA applied.

This leads to the following finding: The heterologous DNA is present in at least two clusters. The number of plasmid copies per cluster must be significantly more than two. At least one intact rDNA unit lies between the plasmid clusters. Fig. 8 shows the deduced from the analysis of structure.

Example 8 Simultaneous integration of three vectors into the rDNA region of the genome H. polymorpha and characterization of the resulting strains

The possibility should be explored of multiple vectors with an identical basis integrating design into the rDNA in different clusters at the same time. Thereby he gives the option of recombinant strains for co-production with little effort of two or more proteins. RB11 was therefore with the three vectors ren pM2, pB11 and pB14 (see Examples 2 and 5) and the 40 resulting a recombinant strains were examined for the presence of integrated vectors. The vectors pB11 and pB14 were by SacI restriction, pM2 by Xhol restriction linearized within the integration sequence and in a transformation approach in equimolar amounts used. The three vectors have the same 2.4 Kb long integration sequence for homologous recombination in the rDNA region of the Ge noms of H, polymorpha and the URA3 gene from S. cerevisiae (see Example 1).

The chromosomal DNA of the recombinant strains resulting from the mixture was cut with AatII (cuts only once in each of the three vectors), the fragments were separated in an agarose gel and subjected to Southern blot analysis. The digoxygenin-labeled integration sequence was used as the probe. By restricting the heterologous DNA with a "single cutter" it was ensured that fragments would arise for the vectors which could be clearly assigned and which would hybridize with the probe. Figure 9 shows a selection of the strains analyzed.

The bold digits identify the trunks, the two or three different ones Carry vectors integrated at the same time. For the RB3T-13, RB3T-17 and RB3T- 37 are recognizable at the level of pM2 and pB14, they both carry vectors genomically integrated. RB3T-19 shows a signal for all three vectors. Through the restriction  tion with AatII, a signal at 6.5 Kb appears in the control RB11, which signals the rDNA Represents unity and also appears in the recombinant strains. Strain RB3T-34 shows a strong band for pB14 and a weaker band for pM2. At this recombinant strain had a different combination of cointegration. The DNA length standard (KBL) is plotted in the last lane.

The vector pM2 could be found in 20 strains (No. 1-13, 17, 19, 22, 33-40), pB14 in seven strains men (No. 4, 6, 11, 19, 21, 22,34) and pB11 in seven strains (No. 4, 6, 11, 19, 21, 22, 34) be detected. Four recombinant strains were identified in which two different vectors were integrated (No. 17, 13 and 37 with pM2 and pB11; No. 34 with pM2 and pB14). One strain (No. 19) contains all three vectors. The right Combined strains were designated according to the following nomenclature: RB for H. polymorpha strain RB11; 3T for transforming RB11 with 3 different ones Vectors.

Example 9 Copy number determination of the integrated vectors

For all generated recombinant strains of the Transfor described in Example 8 copy number between 40 and 50 have been demonstrated. You ent therefore corresponds approximately to the total number of rDNA repetition units in the rDNA H. polymorpha. This total is shared when different vectors are integrated number on the number of copies of the individual vectors. As an example of the number of copies The strain RB3T-19 was determined with three different integrated vectors chosen. A MOX terminator probe was used for the hybridization. The Se The MOX terminator sequence occurs only once in the genome of H, polymorpha and can be found on all three vectors. A probe was thus generated which can be used with both Vector fragments, as well as with the genuine "single copy" MOX terminator sequence of the host hybridizes. The chromosomal DNA of RB3T-19 was cut with Aatll and analyzed. The cut DNA was broken down into defined dilution levels carried. The strength of the hybridization signal of the heterologous sequence in the dilutions can be compared with that of the genuine gene. All three signals for the in tegrated vectors increase with decreasing intensity in the respective dilutions detect. The signal for the genuine MOX terminator fragment is only weak detect. The signals for pB11 and pM2 in the 1/20 dilution correspond approximately the MOX terminator signal. A comparison is shown for pB14 in the 1/5 dilution  bar strong signal. This results in the number of 20 copies mentioned in the text for the vectors pM2 and pB11, and five copies for pB14. For the co-integrated vector Ren pB11 and pM2 a copy number of 20 could be determined. For pB14 the result is a copy number of 5. This results in a total number of copies for all three coins free vectors of 45 reached.

Example 10 Mitotic stability of recombinant H. polymorpha strains

The mitotic stability of recombinant strains, which had been generated by transforming H, polymorpha with three rDNA integration vectors, was examined in a playful manner on the strains RB3T-13, RB3T-17, RB3T-19 and RB3T-37. For this purpose, the strains were cultivated for 27 days in a 50 ml liquid culture in non-selective (YPD) medium. Samples were taken and collected at regular intervals. The genomic DNA of the collected samples was prepared and subjected to Southern blot analysis after restriction with AatII. The AatII / Bg / II MOX terminator fragment from the vector pM2 was used as the probe. Mitotic stability was demonstrated for all four recombined strains in Southern blot analyzes. Fig. 11 shows the result of Southern blot analysis shows an example for the strain RB3T-19. The unchanged intensity of all signals for the vector fragments over the entire period shows the mitotic stability of the analyzed strain.

Example 11 Transform RB11 with five different vectors

The aim of this experiment was to increase the number of vectors used simultaneously in a transformation and thus to determine an upper limit for cointegration. RB11 was co-transformed with the vectors pB11 (10.7 Kb), pB14 (9.7 Kb), pB14X (8.7 Kb), pM2 (8.2 Kb) and pM2X (6.7 Kb). The recombinant strains were checked in Southem blot analyzes. 80 recombinant strains were analyzed. The genomic DNA of the recombinant strains was cut with NcoI, for which there is also only one restriction site in all vectors. The digoxygenin-labeled MOX terminator sequence was used as the probe. In 66 cases only one vector species was integrated, in seven cases two, in six cases three and in one case four of the total of five vectors. A strain that carried all five vectors genomically integrated could not be found among the 80 recombinant strains analyzed. Figure 12 shows Southern blot analysis for four selected strains, three of which contain sequences from at least one integration vector. The chromosomal DNA was cut with Xhol. The analysis of the cut DNA was carried out as in FIG. 11. Strain RB5T-31 contains four different vector species (pM2, pM2X, pB14X and pB11). The signal for pBl4 at 9.7 Kb is particularly strong. This speaks for a much higher number of copies than the other vectors.

Examples 12-15 Expression of different reporter genes in recombinant H. polymorpha strains, generated by integrating at least one rDNA vector

After the previous examples showed that cointegration several different vectors into the H. polymorpha rDNA to mitotically stable recombinant strains, the expressibility of four different reporter genes were detected. As reporter genes, GFP, lacZ, a gene for an insulin variant and a phytase gene chosen because of the detection the gene products are easy to carry out. The reporter genes are on the vectors pM2GT (GFP) pM2TZ (lacZ), pB11 (insulin) and pB14 (phytase) included. The four Vectors were each generated by Sacl restriction within the linea integration sequence rized and placed in equimolar amounts in a co-transformation approach. 132 right Resulting recombinant strains were based on the presence of the gene products analyzed.

Example 12 Expression of lacZ under the control of the TPSI promoter

In the vector pM2ZT used here, the lacZ gene lies behind the TPS1 promoter from H. polymorpha. This promoter is activated by higher cultivation temperatures. When growing at 40 ° C on YNB plates with glucose as the only carbon source, the promoter is sufficiently activated. The cells were cultivated for 6 hours at 40 ° C. in selection medium and then 5 μl of the cultures were dropped onto selection plates. After 24 hours of growth, the colonies were examined for β-galactosidase activity using an X-Gal overlay assay (see material and methods). After an hour of incubation, the result was documented ( Fig. 13). A blackening of the colony corresponds to a strong β-galactosidase activity. Gray colonies show moderate or weak activity. 61 of the 132 recombinant strains showed p-galactosidase activity. This result was confirmed in a repeat experiment.

Example 13 Expression of the insulin gene under the control of the FMD promoter

The vector pB14 used carries an expression cassette with the gene for an insulin variant under the control of the FMD promoter. For this purpose, the cells were incubated in a preculture (YNB, 5% glycerol) for 6 hours at 37 ° C. and 5 μl of each was dropped onto YNB plates (2% glycerol). In this case, glycerol was used as the carbon source, since the insulin gene is under the control of the FMD promoter. This promoter is repressed by glucose and derepressed by glycerin. An immunological filter test was used to identify the insulin-producing strains (see material and methods). After the filter had been developed, stains appeared at the positions of insulin-positive colonies (see FIG. 14). The color of the filter is proportional to the amount of insulin secreted. Secretion of insulin was shown in 17 of the 132 recombinant strains. On filter A in Figure 14, four strains show a moderately strong signal. Strain No. 51 and Strain No. 56 show a weak color. Strain No. 126 shows a signal comparable to the positive control. Strains No. 80, 94 and 114 show weak colorations.

Example 14 FMD promoter-controlled expression of a phytase gene

The vector pB11 carries the gene for the con-phytase under the control of the FMD-Pro motors. This phytase is a mutein (Hoffman La Roche, Basel). To determine the phytase activity, the strains were grown in 3 ml overnight cultures (YPD, 37 ° C) and the phytase was then Activity determined photometrically (see material and methods). 20 strains showed Phytase activity.

Example 15 TPS1 promoter controlled expression of the GFP gene

The vector pM2GT carries the yEGFP gene under the control of the TPS1 promoter  H. polymorpha. The reporter protein in the recombinant cell is in sufficient Amounts accumulated, so after excitation with light of the wavelength of 395 nm fluorescence with the emission maximum at 510 nm (Morise et al., 1974); the Cells glow intensely green (see material and methods). The tribes were made for Incubated for 24 hours in YNB medium at 37 ° C. Subsequently, under the fluo Rescence microscope strains identified with GFP signal. The experiment was total repeated three times and such strains determined as positive that in all three Analyzes showed a signal. GFP was detected in 36 of the 132 strains.

Example 16 Coexpression of different reporter genes

By cotransformation with four different vectors, those in the examples 12 to 15 documented 132 recombinant strains were generated. All the tribes were up the expression of the reporter genes described above was examined. 28 showed none Expression. In the remaining H. polymorpha strains, a random ver division of the vectors. 52 strains have an expression of lacZ, which lies behind the very strong TPS1 promoter in H. polymorpha. In 36 In some cases the GFP gene was expressed. The GFP gene is located on the vector used also behind the TPSI promoter. Expression of the Phytase gene, expression of the insulin gene was observed in 17. 24 tribes show coexpression of two reporter genes. In these cases, the occurrence observed all combinations of the four reporter genes. There were two recombinant Strains discovered that show co-expression of three reporter genes: In strain RB4T-75 could be a combination of GFP, lacZ and insulin, in strain RB4T-81 lacZ, Phytase and insulin can be detected.

Table 1 shows an overview of the expression of four reporter genes in recombinant H. polymorpha strains. 132 recombinant strains were analyzed. The tribes, which show a coexpression of several reporter genes are highlighted in gray, strains with the coexpression of three genes is highlighted by framing (+ = strong expression O = medium expression - = weak expression).  

Table 1

Example 17 Stable phytase production

A recombinant strain, which was transformed by transformation with the vector pB14 (FMD-Pro motor-controlled phytase production), contained approximately 40 copies of the recombinant DNA. Mitotic stability has been growing over 100 generations checked. Cultural aliquots at the start of stability studies and after 100 genera ions were used to inoculate 3 ml medium, the 2% glycerin as a carbon source contained. The secreted phytase was quantified after 30 hours of cultivation. The In both cases, the concentration was determined to be approximately 60 mg / l.

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SEQUENCE LOG

Claims (20)

1. Integration vector for the expression of at least one protein in a host organism, in particular in a fungus, which

• a) at least one rDNA sequence from yeast,
• b) at least one non-deficient selection marker gene and
• c) at least one expression cassette

The expression cassette comprises a promoter element which is functional in the host organism, a region of a heterologous gene which codes for a desired protein and a terminator element which is functional in the host organism. 2. Integration vector according to claim 1, characterized in that the host organ is a methylotrophic yeast. 3. Integration vector according to claim 2, characterized in that the methylotro phe yeast belongs to the genus Hansenula. 4. Integration vector according to claim 3, characterized in that the methylotro phe yeast is Hansenula polymorpha. 5. Integration vector according to one of claims 1-4, characterized in that at least one rDNA sequence from a region of the rDNA of a methylotrophic Yeast is derived from a 400 bp fragment of the 3 'region of the 25S rDNA gene and a 600 bp fragment of the 5 'region of the 18S rDNA gene and the intervening sequences including the 5S rDNA sequence includes. 6. Integration vector according to claim 5, characterized in that the methylotro phe yeast is Hansenula polymorpha.   7. Integration vector according to claim 5, characterized in that the methylotro phe yeast is Pichia pastoris. 8. Integration vector according to one of claims 5 to 7, characterized in that at least one rDNA sequence is selected from the following sequences:

• a) a sequence according to SEQ ID NO 1;
• b) a fragment of the sequence according to a), which is suitable for the stable integration of sequences of the vector in high copy number into the genome of a host organism, in particular a yeast;
• c) a sequence which is at least 70%, preferably at least 80%, particularly preferably at least 90% identical to a sequence according to a) or b);
• d) a sequence which hybridizes with the opposite strand of a sequence according to a) or b);
• e) a derivative of a sequence according to one of groups a) to c) obtained by substitution, addition, inversion and / or deletion of one or more bases, which is used for the stable integration of sequences of the vector in high copy number into the genome of a host organism, a yeast is particularly suitable, and
• f) a sequence complementary to a sequence according to one of groups a) to e).

9. Integration vector according to one of claims 1 to 8, characterized in that it comprises an additional selection marker gene for E. coli. 10. Integration vector according to one of claims 1 to 9 with a length between 7 and 12 Kb. 11. Host organism which has at least one integration vector according to one of the claims che 1 to 10 stably integrated in the genome.   12. Host organism according to claim 11, characterized in that it is a yeast. 13. Host organism according to claim 12, characterized in that the yeast a is methylotrophic yeast. 14. Host organism according to claim 13, characterized in that the methylotro phe yeast belongs to the genus Hansenula. 15. Host organism according to claim 14, characterized in that the methylotro phe yeast is Hansenula polymorpha. 16. Host organism, which through stable integration of one or more integra tion vectors according to one of claims 1 to 10 in the genome of a yeast is. 17. Host organism according to claim 16, characterized in that it by Integra tion of several vectors, the mutually different expression cassettes around grasp, is available. 18. Process for the production of one or more proteins, characterized thereby records that a host organism according to one of claims 11 to 17 in itself cultivated in a known manner and the protein produced is obtained. 19. Use of an integration vector according to one of claims 1 to 10 provision of one or more proteins in a host organism, preferably wise in a yeast. 20. Use of a host organism according to one of claims 11 to 17 provision of one or more proteins.