Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
  • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts

Definitions

• the present invention relates to the field of biotechnology, in particular to an improvement brought to the production of a polypeptide of commercial or therapeutic interest in yeast and, in particular, in Saccharomyces cerevisiae. It relates, firstly, to new nucleic acid fragments isolated from the genomic DNA of Saccharomyces cerevisiae and exhibiting transcriptional promoter activity and, secondly, to expression cassettes, expression vectors and host cells. containing as well as their use for the production of polypeptides of interest.
• the yeast Saccharomyces cerevisiae is considered one of the preferred hosts for the production of recombinant proteins for many reasons.
• this organism is non-pathogenic and it is commonly used in the food industry.
• it can be grown on a large scale and at high density in a relatively inexpensive environment and can be easily adapted to an industrial environment.
• it has been particularly studied so that multiple data concerning its genetics and physiology are available.
• it is capable of carrying out certain typically eukaryotic modifications (glycosylation, disulfide bridges, etc.).
• PGK genes (3-phosphoglycerate kinase; Hitzeman et al., 1983, Science, 219, 620-625), TDH coding for GAPDH (glyceraldehyde phosphate dehydrogenase; Holland and Holland, 1979, J. Biol Chem., 254, 9839-9845), TEF1 (Elongation factor 1; Cottrelle et al., 1985, J. Biol. Chem., 260, 3090-3096), MF al (precursor of the sexual pheromone a; Inokuchi et al., 1987, Mol. Cell.
• a promoter region is located in the 5 ′ region of the genes and comprises all of the elements allowing the transcription of a DNA fragment placed under their dependence, in particular:
• a so-called minimal promoter region comprising the TATA box and the transcription initiation site, which determines the position of the initiation site as well as the basal level of transcription.
• the length of the minimum promoter region is relatively variable. Indeed, the exact location of the TATA box varies from one gene to another and can be from -40 to -120 nucleotides upstream of the initiation site (Chen and Struhl, 1985, EMBO J .; 4, 3273-3280)
• sequences located upstream of the TATA box (immediately upstream of up to several hundred nucleotides) which make it possible to ensure an efficient level of transcription either constitutively (relatively constant level of transcription throughout the cell cycle , whatever the culture conditions) is in a controllable manner (activation of transcription in the presence of an activator and / or repression in the presence of a repressor).
• modulators can be of several types: activator, inhibitor, enhancer, inducible, repressible and respond to various cellular factors or culture conditions.
• the subject of the present invention is an isolated nucleic acid fragment comprising all or part of a nucleotide sequence homologous to the sequence shown in the sequence identifier NO: 1, 2 or 3 or homologous to its complement, said fragment exhibiting transcriptional promoter activity.
• nucleic acid fragment is meant a polymer of nucleotides which may be of DNA or RNA type. These terms are defined in all basic molecular biology works.
• a nucleic acid fragment according to the invention is a double stranded DNA fragment.
• nucleic acid fragment In general, all or part of one of the nucleotide sequences specified in SEQ ID NO: 1, 2 and 3, its complement or one of its homologs can be used in the context of the present invention.
• the term “part” designates a fragment comprising a portion of at least 17 continuous nucleotides identical to a portion of equivalent length of one of the nucleotide sequences indicated in the sequence identifiers or of its complement. But, of course, a The nucleic acid fragment according to the invention is not limited to the sequences described and can extend beyond this.
• homologous means a sequence capable of hybridizing under stringent conditions with all or part of the sequence reported in SEQ ID NO: 1, 2 or 3. It more particularly refers to any nucleic acid retaining the promoter function and having one or more sequence modification (s) with respect to one of these sequences. These modifications can be obtained by mutation, deletion and / or addition of one or more nucleotide (s) relative to the native sequence. They can be introduced in particular to improve the promoter activity, to suppress a region inhibiting transcription, to make a constitutive promoter regulable or vice versa, to introduce a restriction site facilitating the subsequent cloning steps, to eliminate the sequences not essential to the activity. transcriptional ...
• a nucleic acid fragment according to the invention is identical to all or part of one of the nucleotide sequences shown in the sequence identifier NO: 1, 2 or 3 or of its complement.
• nucleic acid fragment having a sequence as shown in:
• sequence identifier NO: 1 starting at the nucleotide at position 197 and ending at the nucleotide at position 1016, or (iii) the sequence identifier NO: 3, starting at the nucleotide at position 5 and ending at nucleotide at position 523.
• a nucleic acid fragment according to the invention can be constituted by the assembly of elements of various origins to form a so-called hybrid promoter functional in the host cell considered.
• a hybrid promoter can comprise:
• nucleic acid fragment according to the invention comprising a minimum promoter region; said minimal promoter region being placed downstream of one or more modulating sequence (s) heterologous to said minimal promoter region, or (ii) a nucleic acid fragment according to the invention, comprising at least one modulating sequence; said modulator sequence being placed upstream of a minimal promoter region heterologous to said modulator sequence.
• Regulatory modulator sequences will preferably be chosen which make it possible to vary the transcription as a function of the culture conditions or of the growth phase.
• such sequences are derived from or derive from regulable genes and are known to those skilled in the art.
• these sequences can comprise modifications (mutation, deletion and / or substitution of one or more nucleotides) compared to the native sequence, as long as they do not alter their modulatory function drastically.
• nucleic acid fragment according to the invention can be used as a bi-directional promoter capable of exercising its function independently of its orientation with respect to the gene to be transcribed (in a sense orientation of 5 ' towards 3 'as indicated in the SEQ IDs or reversed).
• a nucleic acid fragment according to the invention can be obtained by any technique used in the art, for example by cloning, hybridization using an appropriate probe, by PCR (Polymerase Chain Reaction) using suitable primers or by chemical synthesis.
• a nucleic acid fragment according to the invention is intended to allow the expression of a gene of interest in a host cell and, for this purpose, is linked in an operational manner to it in an expression cassette. This is why the present invention also extends to an expression cassette comprising a nucleic acid fragment according to the invention and a gene of interest placed under its control.
• an expression cassette according to the invention can contain several genes of interest, either in the context of a multicistronic cassette (shown schematically by the arrangement "promoter-gene 1-gene 2 ") in which the different genes are placed downstream of a nucleic acid fragment according to the invention and are separated from each other by suitable sequences, such as the elements IRES (for Internai Ribosome Entry Site in English) allowing the reinitiation of translation or in the context of a bidirectional cassette ("gene 1 -promotor-gene 2”) in which a nucleic acid fragment according to the invention is inserted between two genes of interest to simultaneously govern their expression.
• a multicistronic cassette shown schematically by the arrangement "promoter-gene 1-gene 2 "
• suitable sequences such as the elements IRES (for Internai Ribosome Entry Site in English) allowing the reinitiation of translation or in the context of a bidirectional cassette (“gene 1 -promotor-gene 2”) in which a nucleic acid fragment according to
• a gene of interest can be derived from a eukaryotic, prokaryotic organism or from a virus. It can be isolated by any conventional molecular biology technique or can be synthesized chemically. Furthermore, it can code for a protein of interest (i) intracellular, (ii) membrane or anchored to the cell membrane or (iii) secreted into the culture medium. It can therefore include additional elements such as, for example, a sequence coding for a secretion signal.
• the signal sequence BGL2 (EP 0 423 302), the pre or pre-pro sequences MF al (Kurjan and Herskowitz, 1982, Cell, 30, 933-943) and also the pro sequence of the defensin A (EP 0 607 080).
• Endogenous secretion signals of the gene in question can also be used.
• the choice of secretion signals possible in the context of the present invention is within the reach of ordinary skill in the art.
• a gene of interest can code for a polypeptide of interest corresponding to all or part of a protein as found in nature (native or truncated protein). It may also be a chimeric protein, for example originating from the fusion of polypeptides of various origins or a mutant exhibiting improved and / or modified biological properties. Such a mutant can be obtained by conventional molecular biology techniques.
• cytokines and in particular interleukins IL-2, 4, 5, 6, 12 ...), interferons ⁇ , ⁇ and ⁇ , colony stimulating factors (GM-CSF, C-CSF, M-CSF); - growth factors (growth hormone, erythropoietin, insulin, etc.) or cellular or nuclear receptors; - the anticoagulants, preferably hirudin and, in particular the hirudin variants described in European application EP 273 800 and, most preferably the variant HV2 Lys47; - enzymes (trypsin, ribonucleases, P450 cytochromes, lipases, amylases, etc.); - structural proteins (albumin, etc.); - enzyme inhibitors (cc-1 antitrypsin, antithrombin III, inhibitors of viral proteases ...); - polypeptides capable of inhibiting the initiation or progression of tumors, or cancer
• an expression cassette according to the invention can, in addition, comprise additional elements necessary for the expression of the gene of interest (intronic sequence, transcription terminator sequence, etc.) or even for its maintenance in the host cell considered (origin of replication such as ARS or 2 ⁇ , gene coding for a phenotypic selection marker such as URA3 or LEU2, gene coding for a product conferring resistance to an antibiotic for example to hygromycin, cycloheximide, neomycin, phleomycin .).
• additional elements necessary for the expression of the gene of interest intraonic sequence, transcription terminator sequence, etc.
• a phenotypic selection marker such as URA3 or LEU2
• gene coding for a product conferring resistance to an antibiotic for example to hygromycin, cycloheximide, neomycin, phleomycin are known to those skilled in the art.
• the invention also relates to an expression vector comprising one or more expression cassette (s) according to the invention. It can be a multicopy or centromeric plasmid vector, a cosmid or a YAC type vector. Finally, it can be integrative or self-replicating.
• the present invention also relates to a host cell comprising an expression cassette or a vector according to the invention. It can be generated by any method allowing the introduction of foreign DNA into a cell (transformation, transfection, microinjection, electroporation, liposomes, etc.). It is indicated that any host cell, eukaryotic or prokaryotic, can be used in the context of the present invention insofar as it has the appropriate factors to enable a nucleic acid fragment according to the invention to exercise its promoter function . It is within the reach of the skilled person to verify whether a particular cell can be used as a host by measuring promoter activity as indicated above.
• a host cell according to the invention can be derived from an animal cell (CHO, Vero, BHK, etc.) or from a bacterium such as Escherichia coli, but it is preferable to use a lower eukaryote and, in particular, a yeast.
• yeast of the genus Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, Hansemtla, Phaffia or Yarrowia is chosen from the species Schizosaccharomyces pombe, Pichia pastons, Kluy ⁇ 'eromyces lactis, Hansenula po / ymorpha, Yarrowia hpolylica and, preferably, Saccharomyces cerevisiae. It is particularly preferred to use a yeast deficient in protease (s) such as TGY73 4 or that described in European application EP 390 676. A large number of these strains are available commercially in organizations such as the AFRC (Agriculture and Food Research Council, Norfolk, UK) and ATCC (Rockville, MA, USA).
• the present invention also relates to a process for producing a polypeptide of interest comprising the culture of a host cell according to the invention under appropriate culture conditions allowing the production of said polypeptide of interest and its recovery in cell culture It is preferable to use a defined culture medium comprising glucose as carbon source
• this method is preferably applicable to the production of a protein of therapeutic interest and, in particular hirudin, in a yeast Saccharomyces cerevisiae.
• the protein can be recovered directly from the culture medium or after lysis of the cells according to the conventional methodology. It can be purified by applying standard techniques known to a person skilled in the art, for example ion exchange chromatography, differential precipitation, immunopurification or else filtration on gel at high or low pressure.
• Figure 1 is a schematic representation of the vector pTG9852 for the selection of DNA fragments exhibiting transcriptional promoter activity. It includes the URA3-d gene, a multiple cloning site (from M13tgl31; Kieny et al., 1983, Gene, 26, 91-99), the ble gene conferring resistance to phleomycin, the transcription terminator of the gene PGK (PGKt), a fragment of pBR322 carrying an origin of bacterial replication and the Amp gene conferring resistance to ampicillin and the origin of replication 2 ⁇ (indicated 2 m).
• URA3-d gene includes the URA3-d gene, a multiple cloning site (from M13tgl31; Kieny et al., 1983, Gene, 26, 91-99), the ble gene conferring resistance to phleomycin, the transcription terminator of the gene PGK (PGKt), a fragment of pBR322 carrying an origin of bacterial replication and the Amp gene confer
• Figure 2 is a schematic representation of the vector pTG 10231 (multicopy vector) comprising the URA 3-d gene, the promoter of the CYCl gene (pCYCl), the coding part of the GUS gene, the PGK terminator, a fragment of pBR322 and the origins phage replication F.ori and yeast 2 ⁇ .
• Figure 3 is a schematic representation of the vector pTG8795 (single-copy vector) similar to pTG10231, except that the marker gene consists of the complete URA3 gene and that the origin ARSH4-CEN6 replaces most of the 2 ⁇ fragment included in this vector.
• Figure 4 is a diagram schematizing the promoter activities of the inserts D64, R13, J1, and their subfragments (J1.2, R13.2 and R13.3) compared to the promoter of the KEX2 gene. The bars represent the level of activity of the GUS protein in the Saccharomyces cerevisiae TGY74.3 strain tested in a multicopy system.
• Figure 5 is a diagram schematizing the promoter activities of the inserts D64, R13, J1, and their subfragments (J1.2, R13.2 and R13.3) compared to the promoters of the genes KEX2, PGK, MF al (pMF1 ) and CYCl. The bars represent the level of activity of the GUS protein in the Saccharomyces cerevisiae TGY74.3 strain tested in a single-copy system.
• Figure 6 is a diagram showing the influence of the culture medium on the promoter activities of the inserts D64, J1.2, R13 and, as a control, of the TEF1 promoter. The values indicated represent an average of two samples taken during the growth phase.
• FIG. 7 is a schematic representation of the R13 subfragments generated by deletion of the 5 ′ region and of the GUS activity measured for each of them in a single-copy expression system and at the start of growth.
• the techniques described below are carried out according to the general techniques of genetic engineering and molecular cloning detailed in Maniatis et al. (1989, Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) or as recommended by the manufacturer when using a commercial kit.
• the stages of cloning into bacteria are carried out in the strain Escherichia coli (E. coli) 5K (Hubacek and Glover, 1970, J. Mol. Biol., 50, 11-1127).
• Amplification techniques by PCR are known to those skilled in the art (see for example PCR Protocols, A Guide to Methods and Applications, 1990, ed Innis, Gelfand, Sninsky and White, Académie Press Inc).
• the technique used consists of filling the protruding 5 ′ ends with the large fragment of DNA polymerase I from E. coli (Klenow).
• the non-transformed yeasts are generally cultivated at 28 ° C. in a non-selective YPG medium (Yeast Extract 1%, Bactopeptone 1% and Glucose 2%) while the transformed cells are maintained in selective conditions according to the nature of the selection gene contained in the construction.
• a non-selective YPG medium Yeast Extract 1%, Bactopeptone 1% and Glucose 26%
• the culture is carried out in YEG medium (Yeast Extract 0.5%, Glucose 2%) buffered at pH7 by addition of 0.1 M MOPS and in the presence of phleomycin at a minimum concentration of 50 ⁇ g / ml.
• YEG medium Yeast Extract 0.5%, Glucose 2%) buffered at pH7 by addition of 0.1 M MOPS and in the presence of phleomycin at a minimum concentration of 50 ⁇ g / ml.
• URA3 or URA 3-d gene complementing urotac auxotrophy
• the culture medium is composed of YNBG + cases (Yeast nitrogen base 0.675%, Glucose 1% and casamino acids 0.5 %).
• EXAMPLE 1 Cloning of yeast DNA fragments exhibiting promoter activity.
• a DNA library enriched in DNA fragments originating from chromosome IX is constituted from the yeast Saccharomyces cerevisiae TGY73.4 (MA T ⁇ , ura3, his3, pra1, prb1, prc1, cps1) treated by the so-called agarose technique solid (Rose et al; 1990, supra, Preparation of chromosome-size yeast DNA molecules in so ⁇ d agarose). Briefly, the yeasts treated with zymolyase are included in agarose which is then solidified.
• the chromosomes are separated by electrophoresis in pulsed fields (CHEF-DRII, Biorad) on 1% agarose gel (Biorad chromosomal grade agarose) in 0.5x TBE buffer (45 mM Tris-HCl, 45 mM Borate and EDTA 2 mM).
• the electrophoresis takes place for a total duration of 24 hours (h) by applying the following parameters: 40 sec pulses for 16 h then 90 sec for 8 h with a voltage of 200 volts. These conditions are optimal for the separation of small chromosomes (I, VI, III and IX).
• the band corresponding to chromosome IX (the fourth from the bottom) is cut from the gel and the DNA extracted from the agarose (Gene Clean kit, Bio 101 Inc). It was necessary to carry out several electrophoreses of this type in order to be able to have approximately 1 ⁇ g of chromosomal DNA.
• the preparation enriched in chromosome IX is partially digested with the enzyme Sau 3A (10 digests of 100 ng of DNA per 0.2 unit of enzyme for 1 h at 37 ° C. in a reaction volume of 50 ⁇ l).
• the enzymatic reaction is stopped by adding 1 ⁇ l of 0.5 M EDTA at pH8.
• After alcoholic precipitation the fragments of a size between 0.5 and 1.5 kb are isolated on 1% LMP agarose gel (Low Melting Point, BRL) and eluted by the Gene Clean method.
• the isolated fragments are cloned into the plasmid pTG9852 ( Figure 1) linearized with BamHI and treated with alkaline calf phosphatase (Boehringer).
• the latter derives from plasmids pTG6888 and pUT332 (Gatignol et al., 1987, Mol. Gen. Genêt., 207, 342-348).
• the first corresponds to the vector pTG3828 (Achstetter et al., 1992, Gene, 110, 25-31) modified by deletion of the Xbal site contained in the 2 ⁇ origin (by partial Xbal digestion and Klenow treatment).
• the coding part of the ble gene is purified from pUT332 in the form of a BamHI-EcoRI fragment which is subjected to the action of Klenow before being introduced into the BglI I site (made blunt by treatment with Klenow ) of the vector pTG6888.
• the insertion of DNA fragments into pTG9852 at the unique BamHI site placed upstream of the coding sequences of the ble gene, will make it possible to select those which have transcriptional promoter activity (by selection of yeasts transformed with phleomycin).
• the amplification In the absence of an insert (parental vector pTG9852), the amplification generates a band of 269 bp. On the other hand, after insertion of a yeast fragment, the size of the amplified band is increased by the size of the insert. The results indicate an insertion frequency of 90% and an average size of the inserts close to 700 bp.
• a library is created by extracting the plasmid content of all of the clones generated.
• C Selection of potential functional promoters in the yeast Saccharomyces cerevisiae.
• the bank obtained in the previous step is transformed into the yeast strain
• the electroporated cells are spread in parallel on two different media in order to evaluate, on the one hand, if they are transformed (spreading on YNBG medium + case for the selection of the Ura + phenotype) and, on the other hand, if the insert has transcriptional promoter activity (spreading on YEG medium supplemented with 250 ⁇ g / ml of phleomycin). It is indicated that the untransformed strain TGY73.4 or transformed by the vector pTG9852 (carrying the ble gene devoid of promoter) is incapable of growing beyond a phleomycin concentration of 20 ⁇ g / ml.
• the vector pTG9851 comprising a functional expression cassette for the ble gene (under the control of the TEF1 promoter) can resist an antibiotic concentration of 2 mg / ml. This is obtained by introduction of the BamHI fragment isolated from the vector pUT332 in the BglII site of pTG6888
• the promoter used is weak (pTG9895 comprising the ble gene under the control of the promoter of the KEX2 gene, see example 2)
• the cells grow up to 50 ⁇ g / ml.
• the retained concentration of 250 ⁇ g / ml is intermediate in order to be able to select promoter fragments of variable force.
• 9300 transformants (Ura + ) are generated, of which approximately 1% exhibit resistance to a phleomycin concentration of 250 ⁇ g / ml (106 clones) PCR analysis with the previous primers indicates an insert frequency of 80%, their size varying from 0.5 to 1.6 kb with an average of 0.7-0.8 kb.
• Replicas of these 106 candidates were carried out on selective medium with increasing phleomycin concentration and 20 clones resist 2 mg / ml
• the plasmid content of the 20 transformants selected is electroduct in E. coli 5K (Nacken et al., 1994, Nucleic, Acids Res., 22, 1509-1510) before being reintroduced into the yeast TGY73.4.
• Four clones still have a band amplifiable by PCR and a growth capacity in the presence of phleomycin. Three of them have been characterized. These are the clones transformed by the plasmids pTG8732, pTG8733 and pTG8734 carrying the inserts D64, R13 and J1 respectively.
• ORF putative reading frames
• EXAMPLE 2 Vector for expression of the GUS gene under the control of the preceding inserts.
• the activity of transcriptional promoter is evaluated with respect to the GUS gene, the expression product of which is easily measurable. Indeed, its enzymatic activity can be detected by colorimetry, fluorometric assay on cellular extracts (Jefferson et al., 1987, Molecular Biology Reporter, 5, 387-405) or by histochemical test on Nylon filters (Hirt, 1991, Current Genetics , 20, 437-439).
• the inserts are isolated by PCR from the vectors pTG8732 (carrying D64), pTG8733 (R13) and pTG8734 (J1) and using primers provided with a ClaI restriction site in 5 ′ of the sense primer and Dirty in 5 'of the antisense. These are indicated in the sequence identifiers 6 to 9 (oTG6302 and oTG6293 for D64; oTG6292 and oTG6293 for R13 and oTG6298 and oTG6293 for J 1).
• R13 Characterization of sound-fragments of selected registrants.
• the insert R13 exceeding 1 kb, it may be useful to have sub-fragments of reduced size therefore more easily manipulated and nevertheless retaining a transcriptional promoter activity.
• the 5 'and 3' regions of R13 are isolated from pTG8733 using the primers oTG6292 and oTG6294 (SEQ ID NO: 10) and oTG6301 (SEQ ID NO: 11) and oTG6293 respectively.
• the 416 bp fragment covering the 5 ′ part is designated R13.2 while that corresponding to the 3 ′ half with a length of 599 bp is designated R13.3.
• Other subfragments were created by progressive deletion of the 5 'region (see example 5 below).
• the insert is deleted at 3 ′ in order to eliminate the putative 25 aa ORF and, in particular, its initiating ATG capable of interfering with the translation of the protein of interest.
• a 547 bp fragment (J1.2) lacking putative coding sequences is isolated by amplification from pTG8374 and the primers oTG6298 and oTG6299 (SEQ ID NO: 12).
• the basic vector designated pTG10231 ( Figure 2, Degryse et al., Yeast, in press) is derived from pTG3828 (Achstetter et al., 1992, supra). It includes three origins of replication, yeast (2 ⁇ ), bacterial (ori) and finally phage (f.ori allowing the production of single-stranded DNA) as well as two selection markers
• URA3-d and Amp genes As an indication, the URA3-d gene corresponds to the gene
• URA3 deleted from its promoter thereby ensuring a large number of copies of plasmid in the cell. Finally, it carries an expression cassette in which the sequences coding for the GUS protein (Jefferson et al., 1986, Proc. Natl. Acad.
• the vector pTG10231 is digested with ClaI and SalI in order to eliminate the fragment carrying the CYCl promoter.
• the amplified fragments (corresponding to the inserts and their respective sub-fragments) are cloned into the linearized vector.
• Clones having plasmids with a correct restriction profile are selected, respectively named pTG8784 (carrying D64), pTG8781 (R13), pTG8785 (J1), pTG8782 (R13.2), pTG8783 (R13.3) and pTG8786 (J1.2 ).
• a negative control is generated by digestion of pTG 10231 with Clal and SalI, Klenow treatment and religation. This control lacking a promoter is designated pTG8793.
• promoter sequences of the present invention are also useful to compare with other promoters, either deemed strong like those of the MF al and PGK genes or weaker like the promoter of the KEX2 gene (Fuller et al., 1989, Proc. Natl Acad. Sci. USA, 86, 1434-1438).
• the corresponding sequences are obtained by PCR: for the promoter of the KEX2 gene: amplification of a 524 bp fragment from pTG9895 and the primers oTG6304 (SEQ ID NO: 13) and oTG6293.
• pTG9895 is obtained by insertion into the BamHI site of pTG9852 (Example 1) of a PCR fragment carrying the promoter of the KEX2 gene.
• the latter is amplified from the matrix pTG4812 (described in EP 396 436) and oligonucleotides oTG5739 and oTG5740 (SEQ ID NO: 14 and 15), for the promoter of the MF al gene: amplification of a fragment from 974 bp to from a genomic DNA preparation of the yeast strain FL 100 (ATCC 28383) and the primers oTG6929 and oTG6930 (SEQ ID NO: 16 and 17), and for the promoter of the PGK gene: amplification of a fragment of 779 bp from a genomic DNA preparation of the yeast strain FL 100 and the primers oTG7002 and oTG6928 (SEQ ID NO: 18 and 19).
• the amplified fragments are then inserted between the ClaI and SalI sites of the vector pTG10231 in place of the CYCl promoter, to give pTG8780 (KEX2), pTG8789 (MFa 1) and pTG8791 (PGK), respectively.
• pTG8780 KEX2
• MFa 1 pTG8789
• PGK pTG8791
• the vector pTG8795 ( Figure 3) is the equivalent of the vector pTG10231 except that it comprises an autonomous replication unit ARSH6-CEN4 (Sikorski and Hieter, 1989, Genetics, 122, 19-27) in place of the origin 2 ⁇ and the UPA3 gene in place of UM3-d.
• ARSH6-CEN4 autonomous replication unit
• the promoter fragments are introduced into pTG8795 by homologous recombination in replacement of the CYCl promoter.
• the E. coli strain BJ5183 endA, sbcBC, galK, met, thi-1, bioT, hsdR, strR
• one of the plasmids obtained in the previous step (donor plasmids pTG8781 to pTG8786) digested with Scal and, on the other hand, the base vector pTG8795 (recipient plasmid) linearized by NotI.
• a first analysis of the restriction profile is made on the clones generated in order to select those with the expected profile (designated pTG9704 (D64), pTG9701 (R13), pTG9705 (J1), pTG9702 (R13.2), pTG9703 (R13.3 ) and pTG9706 (J1.2)). Their plasmid content is then transferred to the 5K strain in order to obtain higher quantities of plasmid DNA.
• Example 2C and D are used to transform the strain TGY73.4 or W303a (MATa, ura3, leu2, his3, trp1, ade2; Crivellone et al., 1988, J. Biol. Chem., 263, 14323 -14333).
• the expression of the GUS gene can be evaluated directly on the colonies resulting from the transformation by a semi-quantitative technique described by Hirt (1991, supra), the protocol of which has been modified as indicated below. A portion of colony is taken with a toothpick and deposited on a Nylon N membrane (Amersham).
• the colonies are thawed on 3M Whatman paper soaked in Na 2 HPO 4 buffer 50 mM pH7 containing reagent 5-bromo-4-chloro-3-indolylglucuronide (X-gluc) at 50 ⁇ g / ml (dilution of a stock solution to 5 mg / ml in DMSO, dimethylsulfoxide). The reaction takes place in the dark at 37 ° C. The colonies producing the GUS protein appear in blue, the intensity and the speed of appearance of the coloration being all the stronger the higher the level of expression. In order to have more quantitative measurements, the transformed yeasts are cultivated in a liquid medium (YNBG + case) at 28 ° C.
• YNBG + case liquid medium
• a culture sample (10 ml) is taken during growth, the cells are recovered by centrifugation and taken up in 500 ⁇ l of GUS extraction buffer supplemented with 1 mM Pefabloc (Jefferson et al., 1987, supra), before being ground for 15 min (Retsch mill). The ground material is then centrifuged for 10 min at 10,000 rpm at 4 ° C. The protein concentration is measured on the supernatant (Biorad kit) and the enzymatic activity of the GUS protein determined by fluorimetry using the methyl umbelliferyl ⁇ -glucuronide substrate. There is generally a difference in the protein extraction yields between the strains TGY73.4 and W303 ⁇ . However, the GUS activities reduced to the amount of protein are comparable in the two strains.
• Figure 4 shows the activity levels of the GUS protein produced in the TGY73.4 strain transformed by the multicopy vectors of Example 2C.
• the samples are taken in the stationary growth phase and GUS activity is given in nmoles of methyl umbelliferone (MU) produced per min and mg of protein.
• the insert R13 has a promoter activity clearly superior to that measured with all the fragments tested as well as the promoter KEX2 (factor 58).
• the D64 insert can also be considered as a strong promoter in view of the levels of GUS protein produced under its control (28 times greater than those obtained with the KEX2 promoter).
• the promoter capacities of the complete insert J 1 are of the same order of magnitude as KEX2 (to within a factor of 2).
• the deletion of the 25 aa ORF located at its 3 ′ end proves to be advantageous since the promoter activity of the J 1.2 subfragment is clearly improved.
• the R13.3 sub-fragment retains a significant promoter activity although it is less than the complete insert from which it derives, while the R13.2 sub-fragment constitutes a very weak promoter.
• no GUS activity is measured with the negative controls (strain TGY73.4 not transformed or transformed by the plasmid pTG8793).
• Figure 5 summarizes the data concerning the single-copy vectors of example 2D (sampling at an OD600nm of approximately 1). There is a roughly comparable profile.
• the GUS activity levels obtained under the control of the complete R13 insert greatly exceed those produced from the clones tested as well as from the PGK and MF al promoters pondered to be strong.
• the promoter activity of the inserts D64 and J 1.2 is of the same order of magnitude as that of the reference promoters PGK and MF al.
• the transcriptional capacity of R13.3 although notable, is lower than that determined with the complete insert.
• the low activity of the CYCl promoter is explained by the large amount of glucose in the culture medium because the samples are taken in the exponential phase.
• the characterization of the GUS protein produced by the various transformed yeasts was carried out by 7.5% SDS PAGE gel (mini-protean II dual slab cell system, Biorad). Is highlighted in yeasts containing pTG8784, pTG8781 and pTG8786, after staining with Comassie blue, a band of expected molecular weight (68 kDa) corresponding to the expression product of the GUS gene, which represents approximately 5% of the total proteins of the extract.
• the regulatory capacity of these promoter fragments can be studied as a function of growth and culture conditions (measurement of the activity at different culture times, addition of specific nutrients in the culture medium, such as glucose, thiamine, etc. .).
• the level of GUS activity produced by TGY73.4 strains transformed by the monocopy vectors was determined as a function of the growth of yeasts in minimum medium.
• the insert R13 is active at the very start of the exponential phase and its activity decreases as the OD 600 nm increases.
• the J.12 subfragment exhibits similar behavior. With regard to D64, its promoter activity is also less in the stationary phase but the profile observed is slightly different in the sense that the maximum activity is located in the middle of the exponential phase (bell-shaped profile). In contrast, the promoter activity of the R13.3 subfragment is increased in the stationary phase (as observed with the CYCl promoter).
• the single-copy vectors pTG9704 (D64), pTG9706 (J1.2) and pTG9701 (R13) were cultivated in 3 different media, in parallel with the plasmid pTG9707 equivalent to the previous ones except that it is the TEF1 promoter (Cottrelle et al. ., 1985, supra) which directs the expression of the GUS gene.
• the study is carried out on defined medium YNBG + glucose-based case, rich medium YPG and defined oxidative medium YNBGly + case whose carbon source is glycerol. Two samples are taken in the exponential growth phase (OD 600 ⁇ 1). GUS activities are measured in fluorimetry on the acellular protein extracts of each sample ( Figure 6).
• the protein extracts of the clones corresponding to the three promoter inserts of the invention all have a GUS activity reduced by 50% on YNBGly + case medium, in comparison with the glucose medium YNBG + case
• the GUS activity controlled by pTEFl is identical on YNBG + case and YNBGly + case media, confirming that this promoter is effective on the two carbon sources contained in these culture media.
• the GUS activity is less on YPG than on YNBG + cases.
• the optimal activity of the promoter fragments D64, J1 .2 and R13 seems to be obtained in a defined medium with glucose as a carbon source. However, as indicated previously (example 3), the activity decreases regularly during the growth as the glucose disappears from the culture medium, (reduction of the level of GUS production by a factor 2 in the stationary phase) . It is indicated that on a defined glycerol medium (YNBGly + case), such activity variation is not observed during cell growth (constant GUS level).
• the RI 39 fragment is amplified by PCR from pTG9701 (Example 2D) using oTG 7659 and 7234 (SEQ ID No 20 and 21) and makes it possible to generate pTG9754 after cloning in the form of a ClaI-SalI fragment in the monocopy expression vector of the GUS gene.
• R1329 corresponds to R13 deleted by 80 bp in 5 '
• the insert R13.5 is also amplified by PCR from the pTG9701 matrix using the oligonucleotides oTG7422 (SEQ ID No: 22) and oTG7234, and gives rise to the vector GUS pTG9719 expression.
• R13.5 corresponds to a 190 bp deletion in 5 'of the insert R13.
• the sub-fragment R13.6 which is obtained using the primers oTG7423 (SEQ ID NO: 23) and oTG7234 results from a deletion of 300 bp in 5 ′ of R13. Its cloning into the GUS monocopy expression plasmid generates pTG9720.
• the fragment R13.3 already described, comprises a deletion of 450 bp in 5 ′ of R13.
• the yeasts TGY73.4 are cultured in defined medium YNBG + cases and collected at OD 600 ⁇ 1.
• Figure 7 represents the GUS activities corresponding to the means of fluorimetric measurements at OD 600 ⁇ 1 (three clones tested by contruction).
• the original RI 3 insert has a GUS activity of 225 U / mg in the exponential growth phase.
• the deletion of approximately 100 bp in 5 ′ generating R13.9 is accompanied by a loss of activity of approximately 40% (140 U / mg).
• the promoter activity significantly increases after an additional deletion of approximately 100 bp (R13.5) (GUS activity of 290 U / mg exceeding that obtained with R13 and R13.9), which suggests that the deleted region includes a potential element of downregulation of gene expression.
• additional 5 'deletions (R13.6 and R13.3) considerably reduce the promoter activity (95% loss compared to RI 3.5).
• the deleted zone contains the consensus sequence motifs for binding the activator / repressor product encoded by the RAP1 gene from S. cerevisiae.
• the R13.5 fragment exhibits maximum promoter activity which can be used for the expression of the gene of interest.

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Engineering & Computer Science (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Biomedical Technology (AREA)
• Organic Chemistry (AREA)
• Biotechnology (AREA)
• General Engineering & Computer Science (AREA)
• Chemical & Material Sciences (AREA)
• Mycology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Microbiology (AREA)
• Physics & Mathematics (AREA)
• Plant Pathology (AREA)
• Molecular Biology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Biophysics (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=9477880

Family Applications (1)

Country Status (6)

Families Citing this family (6)

• 1995
  • 1995-04-07 FR FR9504171A patent/FR2732691B1/fr not_active Expired - Fee Related
• 1996
  • 1996-04-05 WO PCT/FR1996/000523 patent/WO1996031611A1/fr not_active Application Discontinuation
  • 1996-04-05 US US08/930,617 patent/US5952195A/en not_active Expired - Fee Related
  • 1996-04-05 CA CA002218405A patent/CA2218405A1/fr not_active Abandoned
  • 1996-04-05 EP EP96912075A patent/EP0819175A1/de not_active Withdrawn
  • 1996-04-05 JP JP8530050A patent/JPH11503318A/ja not_active Ceased

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events