DK175647B1 - New DNA contg. formate dehydrogenase gene and its control regions - providing high level expression of foreign proteins under stringent control - Google Patents

Abstract

New DNA molecule (I) contains the control regions and the structural gene for a protein having formate dehydrogenase (FMDH) activity. Also new are recombinant vector contg. (I) and microorganisms contg. such vectors. (I) may encode wild-type or modified FMDH protein and can include (1) foreign genes under stringent control of the FMDH regulatory sequences and/or (2) a secretory signal. The specification includes the complete sequence of the FMDH gene and its 5' and 3' control regions (2276 bases) and of the protein encoded by its 1020 base open reading frame. Specified foreign genes are those of hepatitis B virus S1-S2-S or S antigens; alpha-amylase or glucoamylase from Schwanniomyces castelli (Sc.c.) or invertase of Saccharomyces cerevisiae (S.c.). Secretion signals are e.g., Hansenula polymorpha membrane translocation signals; Sc.c. alpha-amylase or glucoamylase signals, or S.c. alpha-factor or invertase signals. Pref. microorganisms are yeasts of the genera Candia, Hansenula or Tichia; these may contain (I) as extra-chromosomal DNA or integrated into the genome.

Description

in DK 175647 B1

The present invention relates to a DNA fragment comprising a promoter region of a gene encoding a protein derived from a methylotrophic yeast, which has dehydrogenase (FMHD) activity, recombinant vectors containing this fragment. microorganisms containing these vectors and a process for producing a substance using the microorganisms of the present invention.

Over the past decade, several yeast strains have been isolated which are capable of utilizing methanol as the sole source of carbon and energy. Until recently, the 10 studies were limited to the enzymatic level and mainly concerned two species, namely Hansenula polymorpha and Candida boidinii.

The enzymatic studies revealed that in methylotrophic yeast strains, methanol is oxidized via formaldehyde and formate to CO 2 by methanol 15 oxidase (MOX), formaldehyde dehydrogenase (FMD) and formate dehydrogenase (FMDH), respectively. H 2 O 2, which is generated during the first oxidation step, is degraded by catalase. Cl compound is assimilated by a transketalase reaction between xylulose-5- (P) and formaldehyde, the latter of which originates from the dissimilation pathway. The reaction is catalyzed by dihydroxyacetone synthase (DHAS).

Growth of methylotrophic yeast on methanol is followed by changes in the total protein composition. There are 3 large and approx. 5 minor proteins that are newly synthesized. The growth of methanol is also followed by the emergence of enormous peroxisomes. These organelles carry some of the key enzymes involved in methanol metabolism, namely MOX, DHAS and catalase (1). The other two methanol enzymes, FMD and FMDH, are cytoplasmic proteins. In methanol-grown cells, the enzymes FMDH, MOX and DHAS make up to 40% of the total cell protein. The methanol utilization reaction pathway is very divided and the integration of these reactions is very complex.

30

The enzymes for dissimilation of methanol are regulated by the glucose catabolite repression / repression mechanism (2). Methanol has an additional inducing effect by increasing the expression level by a factor of 2-3. In H. polymorpha, the assimilatory DHAS enzyme follows this general regulatory scheme, but during growth on

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limiting amounts of glucose play derepression, an additional post-transcript.

however, a role in regulation. IN

Recently, 3 genes encoding peroxisomal enzymes were cloned from Η. H

5 polymorpha and Pichia pastoris, and the analysis of nucleotide sequences of MOX genes from

H. polymorpha (3) and P. pastoris (4) and the DAS gene encoding DHAS from Η. IN

polymorpha (5), revealed that a repeatable signal sequence is not required for H

the transport of MOX and DHAS into the peroxisome. IN

10 The promoters of certain methanol genes are very effective and their mode of regulation is I

advantageous for industrial use. The expression of foreign proteins can I

promoted and placed under strict control. The large amounts of proteins (MOX, I

DHAS), produced in this way by methylotrophic yeast, is stored in peroxisol

clips. Understanding this mechanism will help to solve some problems

15 with the stability of foreign proteins in yeast. IN

There is a need in the industrial biotechnology field for microbiological regulation

ionization systems whereby large amounts of a particularly desired protein can be prepared H

under strict control. Although promoter / terminator systems already exist, H

20 which can be used in genetic engineering systems to control the amount of proteins, H

to be manufactured, there is still a great need for additional regulatory H

systems become available, having been found to be advantageous in biological H

systems to provide multiple systems so that the most efficient system can

is selected. The current systems are far from effective, especially when string I

25 regulation and high mitotic stability are required. IN

It is therefore an object of the present invention to provide a more I

efficient and very easily controllable regulatory system. IN

The advantage of the present invention comes from the provision of a DNA-I

fragment comprising a promoter region of a gene encoding a protein from

a methylotrophic yeast strain and having the format I

dehydrogenase (FMDH) activity, which gene is identical to or corresponds to an H

FMDH gene obtainable from the genome of Hansenula polymorpha and in which I

35 DNA fragment The FMDH gene is found in a 3.5 kb BamHI-HindIII fragment. IN

DK 175647 B1 3

To begin more comprehensive studies of basic research and biotechnological aspects of methanol utilization, the gene encoding the cytoplasmic methanol key enzyme FMDA was cloned. This 1020 bp gene sequence and its 5 regulatory regions have been cloned. FMDA is regulated at the transcription level by the glucose catabolite repressions / derepressions / methanol induction mechanism. The DNA fragment of the present invention is extremely useful in the biotechnology industry because of the above-described feature that the expression of foreign proteins can be promoted and placed under strict control.

) DNA fragments of the present invention can be modified by recombinant DNA technology techniques well known in the art, resulting in a modified promoter still retaining its promoter activity.

15

A preferred embodiment of the DNA molecule of the invention is shown in FIG. 5th

Examples of the use of the FMDA regulatory sequences of the present invention are combinations of the DNA sequences with foreign genes encoding hepatitis B virus S1-S2-S antigen and hepatitis B virus S antigen α-amylase from S. castellii and glucoamylase from S. castellii or invertase from Saccharomyces cerevisiae.

The DNA fragments of the invention can be further combined into DNA sequences encoding secretory signals such as Hansenula polymorpha membrane translocation signals, preferably those of peroxisomal proteins, methanol oxidase and dihydroxyacetone synthase, Schwanniomyces castellii α-amylase and glucoamy α-factor and invertase signals.

Preparation of the DNA fragments encoding control regions and the structural gene for protein with FMDH activity can be obtained from natural DNA and / or cDNA and / or chemically synthesized DNA.

Recombinant vectors can be prepared which contain the DNA sequences of the invention as such, which DNA sequences encode the regulatory regions

DK 175647 B1 I

and / or the structural genes for FMDH protein, and they can be combined to further I

DNA sequences as described above. Recombinant Vectors to Transfer I

DNA sequences for an expression system are commonly used in the art and I

can be chosen appropriately. For example, the λ-Chano 4A phage may carry the I

5 DNA molecules described. IN

The microorganisms suitable for the expression of the desired genes can be I

is selected from known microorganisms in the field adapted for recombinant I

DNA technologies. However, microorganisms which are capable of

10 tolerate high concentrations of foreign proteins. IN

Most preferred are microorganisms of the genus Candida, Hansenula or Pichia. IN

Said microorganisms are capable of producing the desired substances either

15 by integrating the DNA molecules of the invention into the microorganism I

chromosome or by maintaining the DNA molecules on an extrachromosomal DNA-I

molecule via episomal vectors. IN

The proteins encoded by foreign genes associated with DNA molecules

20 of the present invention, which are made from the transformed I

microorganisms, can be obtained by growing the microorganisms in a manner which is H

known in the art, and by isolating the proteins according to standard knowledge in the art. IN

The invention is now illustrated in a more detailed manner by the following description I

25 and figures. The figures show: H

Figure 1: Analysis of protein crude extracts and in vitro translation products at I

SDS-polyacrylamidgelelektoforese. IN

5 DK 175647 B1 lane 1. Lane 5, translation of hybrid-selected mRNA. Lane 6, immunoprecipitation of translation products from Lane 5.

Figure 2: DNA fragment restriction map comprising the FMDH gene.

5

The arrow shows the direction of transcription.

j

Figure 3: S1 mapping; Lanes M1, 1, 2, 3, 4, M2, a, b, c, d, separation on alkaline agarose gel.

Lanes 5, M3, separation of 6% polyacrylamide gel / 8M urea. Lanes M1-M3-MW, markers. Lanes 1, 2-total protection (1) of the 4.1 kb Eco RI / Hind III fragment (2) comprising the gene. Lanes 3, 4 - protection of the 3 'end tag 1.4 kb Barn HI / Hind III fragment; 3-protected band; 4-1.4 kb intact band. Lane 5 - protection of 1 kb Barn HI / Pst I fragment with a single mark on the Barn Hi site.

Lanes a, b, c, d - protection of 3 'end-labeled DNA fragment containing part of the gene by mRNA preparation isolated from: induced culture, respectively, depressed (1% glycerol) culture, 20 stationary phase culture with 3 % glucose and mid-log phase culture with 3% glucose.

Figure 4: Sequencing strategy - schematic representation.

Twenty-five DNA fragments containing the gene were subjected to Bal31 degradation and the resulting fragments were subcloned into M13 and / or. pUC-type vectors. The fragments were sequenced by the Sanger and, in case of doubt, the Maxam-Gilbert methods.

Figure 5a: Nucleotide sequence of the FMDH gene and its 5 ', 3' control regions.

Figure 5b: Nucleotide sequence of the FMDH gene and its 5 ', 3' control regions.

Figure 5c: Nucleotide sequence of the FMDH gene and its 5 ', 3' control regions.

35

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I I

In Figure 6: Plasmid containing the fusion of bacterial β-lactamase gene with I

FMDH-promoter. IN

In Figure 7: Plasmid containing the hepatitis S gene; HAIR - H. polymorpha- I

5 autonomously replicating sequence; URA3 - S. cerevisiae gene; FMDH- I promoter (-9 type promoter).

In Figure 8: Western blot stained by peroxidase / protein A method. Polyclonal I

Antibodies (not clarified) were used in this experiment:

I I

In Lane a: LR9 growth in methanol I

In Lane I: transformant without S-gene I

In 15 Pathways k,!, M,: transformants with S-gene grown on

glucose (repression) I

In lanes b, c, d, e, different transformants with S gene I

f, g: grown on methanol I

20 I

Lanes n, o: 500 and 450 ng, respectively, purified

HSBAg I

Figure 9: Plasmid expressing α-amylase gene; symbols are the same I

In FIG. 25 as in FIG. 7. I

In Figure 10: Growth of Transformants on Medium Containing Methanol I

I (induction). Enzyme activity (U / ml) was measured in the medium and i

cells (intracellular enzyme level). The latter value I

I 30 was expressed as 1 ml of the medium. IN

In Figure 11: The formation of a ring after the plate has been applied 50 µΙ off

In the medium from transformants (top row) and from control I

In non-transformed strain LR9 (bottom row). IN

B 35 I

7 DK 175647 B1

Strains, media, vectors

A thermophilic homothallic H. polymorpha strain (ATCC 34438) was used. Yeast 5 was grown at 37 ° C on minimal YNB medium as described {3, 5). Induction of the methanol utilization system was achieved by growth in minimal medium containing 1% methanol; growth of 3% glucose minimal medium resulted in system repression.

E. coii L90; c600recA, hsdM, araB was used for transformation; E. coli JM103, thi, strA, supE, endA, sbcB, hsdR, F'traD36, proAB, lad, ZM15 and E. coli KH802 gal, met, supE were used to host phage M13 and λ-15 vector Charon, respectively. 4A. Plasmid DNA and RF M 13 were isolated by scaled-up alkaline minilysate methods (6) followed by CsCl ultracentrifugation.

λ vector Charon 4A and Charon 4 recombinant cions were isolated by scaled plate lysate methods (6).

20 H. polymorpha total DNA of size greater than 50 kb was isolated from spheroplasts as previously described (5).

Charon 4 H. polymorpha DNA library was constructed by ligating partially EcoRI-25 cut H. po / ymo / p / ia DNA with Chardn 4 arms as previously described (5).

H. polymorpha polyA mRNA and analysis of the mRNA by an in vitro cell-free rabbit reticulocyte system has been described previously (5).

30 mRNA labeling: mRNA was partially fragmented by mild alkaline treatment (7) and labeled at the 5 'end with λ-32Ρ-ΑΤΡ (Amersham).

The differential plaque filter hybridization was essentially performed as described in (12). Recombinant phages were plated to ca. 3000 pfu per plate.

35 plaques from each plate were exposed to a set of 5-6 replica nitrocellulose filters

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(BA85, Schleicher and Schull). The filters were hybridized to appropriate 32P mRNA-I

or 32P DNA probes in 5 x SSPE, 50% formamide containing an additional 150 ng / ml I

tRNA, 10 ng / ml poly A, 5 x Denhardt's solution, 5 pg / ml H. polymorpha I rRNA

isolated as described in (5, 6). IN

S1 mapping experiments were performed essentially as described by

Favarolo et al. (8). S1 nuclease from NEN at a concentration of 1000 units / ml I

Was used. IN

The hybrid selection technique was performed as described by Buneman et al. (9). Card I

told, DNA from recombinant subclones was covalently bound to a DPTE derivative of

Sephacryl S-500. Total mRNA was then hybridized with DNA / S-500 matrix. IN

mRNA molecules that were not complementary to the immobilized DNA became I

washed out under very severe conditions (5, 9). Hybridized mRNA was eluted with I

15 H 2 O at 100 ° C. Hybrid-selected mRNA was then translated into a cell-free I

system, and the translational products were analyzed by immunoprecipitation as I

previously described. IN

Sequence Analysis: Various overlapping fragments derived from exonuclease I

The Bal31 degradation of DNA fragments comprising the FMDH gene was cloned into

M13 phages mp9, mp8 and into plasmids pUC12, pUC13. The subcloned fragments I

were sequenced by the methods of Sanger et al. (10) and Maxam-Gilbert (11). IN

Formate dehydrogenase was purified to homogeneity from methanol grown Η. IN

25 polymorph cells as described elsewhere. Antibodies to FMDH, denatured

mold, was prepared in rabbits according to standard procedures. IN

Identification of mRNA molecules encoding FMDH

30 In vitro translation products of total mRNA isolated from cells grown on 3% glucose (repression) or 1% methanol (induction) were analyzed on SDS-PAGE

gels. Figure 1 shows a comparison of in vitro translational products of I mRNA

induced (lane 1) and uninduced (lane 2) cells, as well as immunoprecipitates I

from the first preparation with specific antibodies directed against FMDH (lane 4). IN

In addition, the electrophoretic patterns of crude protein extracts from 1% methanol, I

9 DK 175647 B1 0.5% glycerol / 0.1% glucose (derepression) and 3% glucose cultures compared with the electrophoretic mobility of purified FMDH (lanes 7, 8, 9 and 10, respectively).

5 The results obtained clearly identified the location of the FMDH protein on SDS-PAGE and suggest that FMDH protein and its mRNA are dominant molecules in cells grown on methanol (induction). The location of the other two dominant proteins, MOX and DHAS, is also indicated. FIG. Figure 1 also shows that considerable expression is obtained under depressed conditions (lane 8) and that 3% glucose inhibits the enzymes of the methanol utilization system. The foregoing conclusions enabled the present inventors to isolate, through sucrose gradient centrifugation, an mRNA fraction rich in mRNA encoding FMDH (lane 3) for use in the screening method.

Screening for FMDH gene

The H. polymorpha DNA bank of the Charon 4 phage was screened by differential plaque hybridization (Materials and Methods) with radioactive 32P-labeled mRNA from induced and uninduced cells and with 32P mRNA from a fraction rich in FMDH-20 mRNA (Fig. 1, lane 3). Further, replica filters were hybridized with 32P DNA probes from clones encoding MOX and DAS genes (3, 5). The latter was performed to identify and eliminate clones encoding the other two highly inducible genes. Desired phages were selected and their DNA further characterized.

25

Characterization of recombinant clones

The initial identification of a clone was obtained by the hybrid selection technique, restriction mapping and determination of the size of the mRNA encoded by a given clone.

Hybrid selection DNA from Charon 4 recombinant clone JM was covalently linked to DPTE S-500 matrix, mRNA complementary to the JM clone was selected and its

I DK 175647 B1 I

I I

v / faith translation products were analyzed. FIG. 1 shows that the hybrid selected I

mRNA by in vitro translation yields a large peptide product with the same I

electrophoretic mobility such as FMDH pep time (lane 5). When lane 5 peptides became I

precipitated with specific antibodies (lane 6) became a large band of size I

5 with the FMDH band and other weak bands visible. In a control experiment with non- I

induced mRNA, non-detectable FMDH type mRNA was selected by this I

technique. The presence of other weak bands visible in lanes 5 and 6 is I

probably artifacts of the hybrid selection technique used. IN

10 These data strongly suggest that clone JM contains the FMDH gene. IN

Restriction cards and the size and direction of the transcript

The restriction map for clone JM and its subclones is shown in FIG. 2. DNA fragments I

15 comprising the gene was identified by hybridizing Southern blots with 32P-I

labeled induced mRNA. IN

An 8.5 kb EcoRI H. polymorpha DNA fragment from clone JM contains a gene. And I

In further analysis, the gene and its putative regulatory regions enabled I

In 20, subcloned into HindIII / EcoRI 4.1 kb fragments into pBR325. IN

In Si mapping I

In a non-radioactive HindIII / EcoRI 4.1 kb fragment from plasmid p3M1 was isolated and I

25 associated with induced and uninduced mRNA. The size of DNA protected by I

In its related mRNA against the action of nuclease Si was analyzed by agarose I

In electrophoresis followed by Southern blotting and hybridization with appropriate 32 P-I

In DNA to visualize the fragment. FIG. 3, lane 1 shows that induced mRNA protects I

It is a 1.2 kb fragment. This suggests that the gene encodes a protein of approx. IN

I 30 35000-37000 dalton. This value was found for the FMDH protein. Because FMDH is you

In the only highly inducible protein in this molecular weight range, this supports I

result in the identification of the gene. IN

The 3 'end of the gene, transcription direction and the amount of FMDH transcript DK 175647 B1 11

Two fragments containing the gene, 1.0 kb BamHl / Pstl and 1.4 kb Hind-III / BamHI, were isolated and a 3 'end tag was introduced at the BamHI site. Only the label on the right (Fig. 3, lanes 3-4) 1.4 kb HindIII / BamHI fragment was protected by being linked to mRNA, suggesting that the transcription direction goes from left to right (arrow in Fig. 2). The size of this binding (lane 4) suggests that the 3 'end of the gene is found 850 bp to the right of the BamHI site. This type of experiment was also used to roughly establish the amount of FMDH mRNA molecule in the 10 total polyA + mRNA isolated from cells grown under different conditions. A known amount of 32P-3 'end-tagged DNA containing part of the gene was hybridized to varying amounts of mRNA. In excess of DNA, the radioactivity present in a band protected from SI by a given amount of mRNA is a measure of the amount of FMDH mRNA in the preparation. These data suggest that FMDH mRNA contributes 15 and ca. 7% ± 1% and 3% to 4% of the total polyA + mRNA in preparations from induced and uninduced growth conditions. FIG. 3, lanes a, b, c and d show the comparison between the intensity of the DNA band resulting from S1 experiments where 3 µg of DNA was hybridized with 10 µg of total polyA + mRNA.

It is also clearly shown that in mid-log phases of 3% glucose (repression) cultures, only 20 negligible amounts of FMDH transcript are visible, while the same culture at the stationary phase already shows considerable amounts of transcript. This is a good example of the phenomenon of depression - in the stationary phase glucose is depleted.

The 5 'end of the gene 25

A 1.0 db BAmHI / PstI fragment with a single 5 'end label at BamHI gave, by S1 mapping, multiple bands ranging from 255 to 265 bp (Fig. 3, lane 5). The comparison of this value with sequence data indicated that transcription started approx. at the -12 position from the first ATG. The headband shows the start at "A" 30 surrounded by pyrimidine grooves.

nucleotide

The nucleotide sequence of the FMDH gene and the surrounding region was determined by the methods of Sanger (10) and Maxam-Gilbert (11). The fragments that should

DK 175647 B1 I

sequenced, was generated by removing DNA containing the gene with Bal31. IN

FIG. 4 shows that all the regions of the gene were sequenced several times in both directions. H

In case of doubt, data from the M13 method were corrected with data from Maxam-I

Gilbert methods. The nucleotide sequence is shown in FIG. 5. The gene contains an open I

5 reading frame (ORF) of 1020 nucleotides and encodes a protein of 340 Da. IN

The molecular weight of the protein, as calculated from these data, is 35700 Da, which is in H

good agreement with the values obtained by SDS-PAGE for purified H

protein. The gene was positively identified as being the FMDH gene by co-I

compared the N-end of the gene derived from the DNA sequence with data obtained at NH-end I

10 analysis of the purified protein.

5'-3 'end regions H

In the 5 'control regulatory region of the eukaryotes, a common sequence is H

15 -3A (9) XXlAUG4GX6py has been reported to be necessary for efficient transcription.

prepared and translated genes (12, 13). In the FMDH gene, the rule is only partially followed where

the sequence -3AUC + 1AUG + 4AX + 6A is present. The first ATG is followed by I

stop codons in all reading frames. The sequence CTATAAATA that in eukaryotes is I

involved in transcription initiation, found at the -40 position. Other features that

20 is thought to play a role in transcriptional control in the yeast S. cerevisiae, such as H

CAACAA or CACACA (12) are not present in FMDH. IN

In most yeast strains that have been studied to date, the gene contains 3'-I

end region characteristic sequences which, according to some authors, play a role in I

25 the correct transcription termination and acts as polyadenylation signals (14, 1

15). Zared and Sherman (16) and Bennetzen and Hall (17) assumed that a sequence I

T-rig ... TAG ..-. TAG (or TATGT) ... AT ... TTT or T ... TAAATAA ... A (or. I

G) ... T ... A - .. AT plays these roles. Similarities to these common sequences can be found

rarely in the FMDH gene. Some repetitive sequences were found when some H

Thirty potential signals were sought. The sequences TTGGA and TAGG are repeated twice. IN

A A ATAT AA, similar to an animal polyadenylation signal, is found 30 bp downstream I

for the end of the ORF. IN

EXAMPLE 1

In order to be able to investigate the functional regions of the FMDH 5 'upstream region, a series of deletions from this region were isolated. To obtain the promoter without the structural gene, a pUC-type plasmid containing the 1.4 kb Barn HI fragment was first subjected to Bal31 exonuclease treatment after the plasmid had been linearized at a proper point. Initially, attention was focused on the promoter fragment that had the deletion at position -5 from the first ATG; the fragment is called "-5 promoter". Also the "-9-" deleted promoter was used in certain experiments.

The "-5 promoter" was fused to the open reading frame of the bacterial β-lactamase (Bla) gene. The gene was used in the laboratory as a very suitable model to study the expression of foreign protein under the control of yeast promoters.

The signal sequence of the β-lactamase was not present in the obtained construct, enabling the measurement of enzyme activity in yeast protein extracts. The fused DNA fragment was cloned into the plasmid containing H. polymorpha autonomously replicating sequence (HARS1) (Fig. 6) and the S. cerev / s / ae Ura3 gene, which acts as a marker for H. polymorpha transformation. . The amount of β-lactamase produced in H. poiymorpha transformants was measured by enzymatic tests and immune tests. Table 1 shows the synthesis of β-lactamase under control of FMDH promoter in cells grown in different media (different carbon sources).

Table 1 shows that the isolated FMDH promoter is properly and strictly controlled by the repressor / derepression / induction mechanism. An estimate of the amount of synthesized protein shows that the system of the invention is characterized by highly efficient transcription and translation of the foreign protein. In the control experiment, β-lactamase was expressed in S. cerevisiae under the control of a strong S.

30 cerev / s / ae PDC (pyruvate decarboxylase yeast promoter on a 2-pm plasmid (50 copies per cell). The values obtained were lower by a factor of 5-6 than the values for H. polymorpha.

I DK 175647 B1 I

IN

Table 1: Production of β-lactamase I

In clone enzymatic test immunoassay I

5 (U / mg protein) (% of total cell protein) I

I GLU GLIC Met-OH GLU GLIC Met-OH I

I 10 I

I Ir 45 30 4000 15000 3-4 6-8 I

I L 5 70 10000 28000 6-8 10-12 I

In 15 GLU - growth in 3% glucose (repression) In GLIC - growth in 1% glycerol (derepression)

Met-OH - growth of 1% methanol (induction) I

In all cases, late-logarithmic phase cells were sampled. The plasmid that I

20 contains the merger, has 50-60 copies per cell. IN

EXAMPLE 2 I

Expression of genes encoding hepatitis B surface antigens (HSBAg) under I

In control of FMDH promoter I

Construction of the plasmid expressing hepatitis proteins I

In A hepatitis B 1.2 kb DNA fragment encodes a long S2-S1-S protein (pre-S), I

In 30 as after treatment (removal of the S2-S1 moiety), the S protein is converted. Virus I

The envelope consists of both proteins. IN

I For the present expression experiments, the 1.2 kb fragment I has been used

In as well as a shorter portion of this DNA, which encodes only S protein. The latter is you

I also capable of forming antigenic pseudo-virus particles. IN

DK 175647 B1 I

Both hepatitis S genes have been inserted into the universal vector. As shown in FIG. 1 I

and Scheme 1, the vector contains an autonomously replicating sequence (HAIR), UraS4 I

the gene from S. cerevisiae as a selective marker and H. polymorpha promoter I

5 followed by a short linker. After the S gene, a DNA fragment I is located

I derived from the H. polymorpha-MOX gene, which exhibits transcription terminator I

function. FIG. Figure 7 shows the construct containing the S gene. IN

2. Transformation of H. polymorpha and screening for clones expressing HSBAg I

H. polymorpha Ura3 mutant LR9 was transformed with the I described above

plasmids. The yeast transformants were then immediately screened for HSBAg-I

expression using polyclonal antibodies. As an immune screening, you are

used western blotting (peroxidase protein A or, to improve sensitive I)

Presently, 25J protein A). The screening method was significantly inhibited by I

the strong cross-reactivity of the sera with H. polymorpha crude extract proteins. The ten

however, succeeded in showing the expressed antigen. IN

FIG. 8 shows the western blotting protein extracts from cells transformed with I

20 hepatitis genes grown on methanol and it further shows an antigenic band with I

Expected molecular weight of the S protein. The control extracts from transformants I

grown on glucose (repression of FMDH promoter) does not have this band. The re- I

results shown in FIG. 8, are derived from transformants containing FMDH -9- I

promoter, ie promoter derived by removing the DNA fragment inside I

25 holding the promoter function to position -9 from the first ATG.

Also, by testing S1 nuclease mapping, mRNA produced in the transformants was analyzed. The results indicate that the transformants produce many S-gene mRNA molecules and that the transcription is strictly controlled by the repression / derepression / induction mechanism.

The above results were confirmed by a positive R1A test of protein extracts derived from transformed cells. The test used monoclonal antibodies directed against native S protein.

------ I DK 175647 B1

EXAMPLE 3 I

Expression and secretion of α-amylase from Schwanniomyces castellii in Η. IN

polymorpha under the control of FMDH promoter I

To investigate the possibility of expression in H. polymorpha, I

selected an α-amylase gene from the yeast S. castellii. The gene encodes the 56 kb protein, I

which in S. castellii is completely secreted to the medium; this secretion process is followed

of glycolysis of the protein. IN

An EcoRI fragment comprising the structural gene and its terminator was inserted into I

the expression plasmid (Fig. 9). IN

H. polymorpha was transformed with this plasmid and the transformants became I

15 tested for expression and secretion of amylase using an I

starch degradation test (starch iodine plate annealing) or an enzyme kinetics test kit (α-amylase Merkotest A). The results clearly show that α-amylase is produced under the control of FMDH promoter. Furthermore, the protein is secreted into the medium. FIG. 10 shows that in the mid-log phase, approx. 90% of the protein 20 to the medium. The starch iodine plate test confirmed these results (Fig. 11).

These data also show that it is possible to achieve a high level of expression under depressed conditions. In particular, this property of the system is very valuable and important for biotechnological purposes, ie. that the synthesis of foreign proteins can start without the addition of methanol as an inducer by simply depleting glucose in the medium and / or by adding glycerol. A system which can be processed in such a simple manner, and at the same time provides a very efficient expression, giving quantities of proteins suitable for use in the biotechnology industry, has not been previously provided.

In separate studies, it has been shown that other H. polymorpha promoters such as MOX and DAS do not respond as strongly to derepression signals. With DAS promoter, expression under derepressed conditions is further diminished by post-transcriptional control.

DK 175647 B1 I

REFERENCES I

11. Douma, A.L., Veenhuis, M., King, W.r Evers, M., and Harder, W., Arch, of I

Microbiol., (1985), 145, 237. I

2. Roggenkamp, R., Janowicz, Z., Stanikowski, B. and Hollenberg, C.P., (1984) I

Moth. Gen. Genet. 194, 489 * 493. IN

3. Ledeboer, A.M., Maat, J., Visser, C., Bos, J.W., Verrips, C.T., Janowicz, Z.; IN

10 Eckart, M., Roggenkamp, R. and Hollenberg, C.P. (1985) Nucleic Acid Res., I

3063. I

4. Ellis, S. B., et al. Isolation of alcohol oxidase and two other methanol I

regulatory genes from the yeast Pichia pastoris. Molecular and Cellular I

Biology (1985) 5: 1111-21. IN

5. Janowicz, Z.A., Eckart, M.R., Drewke, C., Roggenkamp, R.O., Hollenberg, I.

C.P., Maat, J., Ledeboer, A.M., Visser, C. and Verrips, C.T., Nucl. acid Res.

(1985) 13, 3043.

6. Williams, B.G. and Blattner, F.R. (1979) J. Virol. 29, 555.

7. Goldbach, R.W., Borst, P., Bollen-de-Boer, J.E. and van Bruggen, E.F.J.

(1978) Biochem. and Biophys. Acta 521, 169-186.

8. Favarolo, J., Treisman, R. and Kamen, R. (1980) Methods in Enzymology 65, 718.

9. BOnemann, H., Westhoff, P. and Hermann, R.G. (1982) Nucl. Acid. Res. 10, 30 7163-7178.

10. Singer, F., Coulson, A.R., Barreli, B.G., Smith, A.J.H. and Roe, B.A. (1980) J.

Moth. Biol. 143, 161-178.

11. Maxam, A.M. and Gilbert, Methods Enzymol, (1980) 65, 499.

In DK 175647 B1

12. van Dijken, S.P. Veenhuis, M. and Harder, W. (1982) Ann. NEW. Acad. Sci. IN

386, 200-216. IN

13. Kozak, M. (1981) Nucl. Acid Res. 9, 5233-5252. IN

14. Sahm, H. (1977) Adv. Biochem. Meadow. 6, 77-103. IN

15. Veenhuis, M., van Dijken, J.P. and Harder, W. (1983) Adv. Microbiol. Physiol. IN

10 23, 2-76. IN

16. Zaret, K.S. and Sherman, F. (1982) Cell 28, 563-573. IN

17. Bennetsen, J.H. and Hall, B.D. (1982) J. Biol. Chem. 257, 3018-3025. IN

Claims (34)

A DNA fragment, characterized in that it comprises a promoter region of a gene encoding a protein derived from a methylotrophic yeast strain and having format dehydrogenase (FMDH) activity, which is identical to or corresponding to an FMDH gene. gene obtainable from the Hansenula polymorpha genome and in which DNA fragment the FMDH gene is found in a 3.5 kb BamHI-HindHI fragment. DNA fragment according to claim 1, characterized in that the gene encodes a wild type FMDH protein. DNA fragment according to claim 1 or 2, characterized in that it has been modified using 15 recombinant DNA technologies while preserving its promoter function. DNA fragment according to claim 1, characterized in that it comprises the 5 'region of the nucleotide sequence shown in Figure 5. DNA fragment according to any one of claims 1 to 4, characterized in that it further comprises at least one DNA sequence encoding a foreign gene whose transcription is controlled by the promoter region. 25 DNA fragment according to claim 5, characterized in that the foreign gene is selected from the genes encoding: (a) Hepatitis B virus S1-S2-S antigen, (b) Hepatitis B virus S antigen, ( c) α-amylase from Schwanniomyces casteliii, (d) glucoamylase from Schwanniomyces casteliii or (e) invertase from Saccharomyces cerevisiae. DNA fragment according to any of claims 1 to 6, characterized in that it comprises a DNA sequence encoding a secretory signal. IN DNA fragment according to claim 7, H 5 characterized in that the secretory signal is selected from: H Hansenula polyamorpha membrane translocation signals, preferably those of H peroxisomal proteins, methanol oxidase and dihydroxyacetone synthase H Saccharomyces cerevisiae α-factor and invertase signals. IN DNA fragment according to any one of claims 1 to 8, H characterized in that it has been obtained from natural DIMA and / or cDNA I and / or chemically synthesized DNA, wherein the promoter region is obtained from natural H DNA and / or chemically synthesized DNA. IN A recombinant vector, H characterized in that it contains a DNA fragment according to any one of claims 1 to 9. H Microorganism, H characterized in that it comprises a vector according to claim 10. H Microorganism according to claim 11, characterized in that it is a methylotrophic yeast strain. H Microorganism according to claim 12, characterized in that the yeast is selected from the genera Candida, Hansenula or Pischia. H Microorganism according to any one of claims 11 to 13, H characterized in that it has received the vector of claim 10 by H transformation. IN Microorganism according to any one of claims 11 to 14, characterized in that the vector is integrated into the genome of the microorganism or is retained as an extrachromosomal DNA molecule. Microorganism according to any of claims 11 to 15, characterized in that it tolerates high concentrations of foreign protein. Process for the preparation of a useful substance, characterized in that a microorganism according to any one of claims 11 to 16 is grown and the foreign substance is isolated and purified in a manner known in the art. A method of producing a DNA fragment, characterized in that the DNA fragment comprises a promoter region of a gene encoding a protein derived from a methylotrophic yeast strain and having format dehydrogenase (FMDH) activity, which gene is identical. with or corresponding to an FMDH gene obtainable from the Hansenula polymorpha genome and in which DNA fragment The FMDH gene is found in a 3.5 kb BamHI-HindHI fragment, which involves isolating the DNA fragment on a in a manner known per se. 20 The method of claim 18, characterized in that the gene encodes a wild type FMDH protein. 20 I A method according to any one of claims 18 and 19, 25, characterized in that the DNA fragment has been modified using recombinant DNA technologies while retaining its promoter function. The method of claim 18, characterized in that the DNA fragment comprises the 5 'region of the 30 nucleotide sequence shown in Figure 5. Method according to any one of claims 18 to 21, characterized in that the DNA fragment further comprises at least one DNA sequence encoding a foreign gene whose transcription is controlled by the promoter region. DK 175647 B1 I A method according to claim 22, characterized in that the foreign gene is selected from the genes encoding I (a) Hepatitis B virus S1-S2-S antigen, I (b) Hepatitis B virus S antigen, I ( c) α-amylase from Schwanniomyces castellii, I (d) glucoamylase from Schwanniomyces castellii or I (e) invertase from Saccharomyces cerevisiae. IN Method according to any of claims 18 to 23, characterized in that the DNA fragment comprises a DNA sequence encoding a secretory signal. H Method according to claim 24, 15, characterized in that the secretory signal is selected from: Saccharomyces cerevisiae α-factor and invertase signals. IN Method according to any one of claims 18 to 25, H characterized in that the DNA fragment is obtained from natural DNA and / or I cDNA and / or chemically synthesized DNA and the promoter region is obtained from H natural DNA and / or chemically synthesized DNA. H Process for producing a recombinant vector, characterized in that the DNA molecule prepared according to any one of claims 18 to 26 is inserted into a cloning vector. IN Process for producing a microorganism, characterized in that a vector according to claim 27 is introduced into the microorganism. IN Process according to claim 28, characterized in that the microorganism is a methylotrophic yeast strain. In DK 175647 B1 The method according to claim 29, characterized in that the yeast strain is selected from the genera Candida, Hansenula or Pischia. Method according to any of claims 28 to 30, characterized in that the vector produced according to claim 27 is introduced by transformation. Method according to any of claims 28 to 31, 10, characterized in that the vector is integrated into the microorganism genome or retained as extrachromosomal DNA molecule. Process according to any of claims 28 to 32, characterized in that the microorganism tolerates high concentrations of 15 foreign protein. A process for producing a useful substance, characterized in that a microorganism prepared according to any one of claims 18 to 33 is cultured and the useful substance is recovered and purified in a manner known in the art. in the art.