Background of the Invention The use of microorganisms as host cells for the expression of commercially interesting polypeptides makes it desirable to be able to control the expression of the polypeptide of interest. It is often desirable to be able to turn on the expression by use of e. g. inducible promoters. Inducible promoters can be induced by adding an inducer to the growth medium or some promoters can be controlled by temperature wherein the binding of a mutant repressor or activator is dependent on e. g. temperature. Another possibility is the use of a constitutive promoter and instead regulating the expression of the gene at the level of translation.

Summary of the Invention Genes comprising introns within the open reading frame (ORF) of the gene are dependent on the proper splicing of the RNA transcript for expression of the gene product. It has now been found that gene expression, of a gene of interest, can be controlled by inserting an intron sequence within the ORF of the gene, wherein the proper splicing of the intron from the RNA transcript can be controlled in a temperature dependent manner by designing the intron to comprise inverted repeats allowing for secondary structure formation of the transcript at certain temperatures.

In a first aspect the present invention relates to a polynucleotide comprising conserved intron sequences required for RNA splicing in a host cell, wherein the said splicing, resulting in a correctly spliced mRNA, is temperature dependent due to secondary structure formation or hybridization of an antisense RNA.

In a second aspect the present invention relates to a host cell comprising the polynucleotide of the invention, wherein the polynucleotide is inserted in a gene or in a control sequence.

In a third aspect the present invention relates to a method for temperature dependent expression of a polypeptide of interest in a host cell comprising, a) providing a poly nucleotide comprising conserved intron sequences required for RNA- splicing, wherein the said splicing, resulting in a correctly spliced mRNA, is temperature dependent due to secondary structure formation or hybridization of an antisense RNA; b) inserting the polynucleotide into a gene or a control sequence; c) introducing the gene or control sequence comprising the polynucleotide into a host cell d) culturing the host cell under conditions promoting expression of the gene; e) optionally recovering the gene product.

In a fourth aspect the present invention relates to a use of the polynucleotide according to the invention for providing temperature dependent expression of a polypeptide of interest in a mammalian cell, an insect cell, a plant cell or a fungal cell.

Brief Description of the Drawings Figure 1 shows the sequence of the second intron (iAMG2) of the glucoamylase gene from A. niger as well as two variants (iAMG2*) comprising inverted repeats.

Figure 2 shows a diagram of the plasmid pMT2293. The DNA sequence is shown in SEQ ID No 3.

Figure 3 shows a heat sensitive (temperature sensitive) intron according to the invention (pMT2591), comprising two internal consensus sequences (branch points) and two 3'acceptor sites.

Definitions Prior to discussing the present invention in further details, the following terms and conventions will first be defined: Substantially pure polynucleotide : The term"substantially pure polynucleotide"as used

herein refers to a polynucleotide preparation, wherein the polynucleotide has been removed from its natural genetic milieu, and is thus free of other extraneous or unwanted coding sequences and is in a form suitable for use within genetically engineered protein production systems. Thus, a substantially pure polynucleotide contains at the most 10% by weight of other polynucleotide material with which it is naively associated (lower percentages of other polynucleotide material are preferred, e. g. at the most 8% by weight, at the most 6% by weight, at the most 5% by weight, at the most 4% at the most 3% by weight, at the most 2% by weight, at the most 1% by weight, and at the most 1/2% by weight). A substantially pure polynucleotide may, however, include naturally occurring 5'and 3'untranslated regions, such as promoters and terminators. It is preferred that the substantially pure polynucleotide is at least 92% pure, i. e. that the polynucleotide constitutes at least 92% by weight of the total polynucleotide material present in the preparation, and higher percentages are preferred such as at least 94% pure, at least 95% pure, at least 96% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99%, and at the most 99.5% pure. The polynucleotides disclosed herein are preferably in a substantially pure form. In particular, it is preferred that the polynucleotides disclosed herein are in"essentially pure form", i. e. that the polynucleotide preparation is essentially free of other polynucleotide material with which it is natively associated. Herein, the term"substantially pure polynucleotide"is synonymous with the terms "isolated polynucleotide"and"polynucleotide in isolated form".

Control sequence : The term"control sequences"is defined herein to include but not to be limited to, a leader, polyadenylation sequence, propeptide encoding sequence, and a signal peptide encoding sequence.

Codin. q sequence : When used herein the term"coding sequence"is intended to cover a nucleotide sequence, which directly specifies the amino acid sequence of its protein product.

The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon. The coding sequence typically include DNA, cDNA, and recombinant nucleotide sequences.

Expression : In the present context, the term"expression"includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, cellular localization or secretion.

Expression vector : In the present context, the term"expression vector"covers a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide, and which is

operably linked to additional segments that provide for its transcription.

Host cell : The term"host cell", as used herein, includes any eukaryotic cell such as a mammalian cell, an insect cell, a plant cell or a fungal cell.

Polvnucleotide : The term"polynucleotide"denotes a single-or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5'to the 3'end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules.

Secondary structure formation : The term"secondary structure"denotes the two- dimensional and/or three dimentional configuration of a polynucleotide chain in terms of interactions between nucleotides relatively close to one another in the linear sequence.

Antisense RNA : The term"antisense RNA"denotes an RNA complementary to at least part of the normal RNA transcript of a gene, which is not snRNA, and which can block or inhibit expression of the gene by hybridizing to the RNA transcript and preventing its translation or which can prevent or interfere with splicing by hybridizing to the normal RNA transcript either at any of the conserved intron sequences or at a position preventing or interfering with the secondary structure formation according to the invention.

Hybridization : The term"hybridization"means the association or base-pairing of two complementary DNA or RNA strands. The stability of such hybridization complex is dependent largely on factors such as temperature, salt concentration, number and type of bases involved in the hybridization, and the melting temperature of such a complex can be estimated according to several formulas well known in the art.

Stem-loop : The term"stem-loop"denotes the secondary structure that results from the hybridization of two adjacent inverted repeats thereby forming the"stem"and the intervening sequence not involved in hybridization forming the"loop". The stability of such stem-loop structures depends on many factors such as temperature, the number and type of bases involved etc, and is well known in the art (see e. g. Nature New Biology (1973) 246: 40-41 the content of which is incorporated by reference).

Inverted repeat : The term"inverted repeat"in a nucleic acids sequence, denotes two adjacent or nearby sub-sequences of the same length, wherein one of the sub-sequences is

the complementary sequence of the other and in the reverse orientation thereby allowing the formation of a stem-loop structure. The sub-sequences are preferably 100% complementary to each other. However, mismatches can occur and still result in the formation of the secondary structure of the invention. The only requirement being that the stem-loop will be able to form at the desired temperature.

Intron : The term"intron"denotes a non-coding nucleotide sequence, one or more of which interrupt the coding sequences in many eukaryotic genes, and which are transcribed into RNA and subsequently removed by RNA splicing to leave a functional mRNA or other RNA. Introns are sometimes also called intervening sequences.

Conserved intron sequence : Stretches of DNA/RNA sequences that are identical or very similar in different genes and the presence of which are necessary in order to define the boundaries of the intron. Also comprises an internal conserved sequence. In S. cerevisiae each intron contains three conserved sequences, GUAPyGU (Py, pyrimidine) at the 5'donor site, UACUAAC as the internal consensus sequence (the branch point), and PyAG at the 3' acceptor site. The 5'-GU is 100 % conserved in the 5'donor site, whereas some variation can occur at the remaining positions. In the 3'acceptor site the AG is 100 % conserved and the internal consensus sequence (the branch point) is 100 % conserved in S. cerevisiae. In filamentous fungi the internal consensus sequence is less conserved and requires the sequence: CTPuAC (Pu, purine).

Splicina or RNA splicin. a : The term"splicing or RNA splicing"denotes the process by which introns are removed from primary RNA transcripts of eukaryotic genes, in which the introns are cut out at precisely defined splice points and the ends of the remaining RNA rejoined to form a continuous mRNA, rRNA or tRNA.

Correctly spliced mRNA : The term"correctly spliced mRNA"denotes an mRNA molecule, which after splicing of the primary RNA transcript will not comprise any part of the polynucleotide of the invention.

Interfering with splicin : The term"interfering with splicing", as used herein, means that the splicing at the conserved splice sites, any of which are involved in a secondary structure or a hybridization with an antisense RNA, is occurring less efficiently than under normal conditions thereby resulting in a reduced splicing efficiency.

3=splice site : The term"3'-splice site"is identical to the above 3'-acceptor site, which defines the 3'boundary of the intron.

Temperature sensitive : The term"temperature sensitive", as used herein, means that within the temperature range allowing growth of the host cell the production of a functional gene product is inhibited to some extent or completely at the high end of the temperature range. By the term temperature sensitive is also meant heat sensitive.

Cold sensitive : The term"cold sensitive", as used herein, means that within the temperature range allowing growth of the host cell the production of a functional gene product is inhibited to some extent or completely inhibited at the low end of the temperature range.

Detailed Description of the Invention It is known, that genes originating from eukaryotic organisms often contain introns, which are subsequently spliced out in order to get the mature mRNA molecule which can then be translated. The correct removal of the intron sequences from a pre-mRNA transcript is essential for the formation of a functional translation product from the mRNA. Introns contain consensus sequences at their 5'-end and at their 3'-end, as well as an internal consensus sequence. During the splicing reaction, the intron consensus sequences participate in base paring with snRNA's (small nuclear RNA's).

Conserved intron sequences are known from several organisms. Among the different organisms the requirements for what constitutes a functional intron sequence differs.

S. cerevisiae differs from other eukaryotes in that very few of its genes contain introns, and these introns contain an internal consensus sequence which is much more conserved than in other eukaryotes. Since S. cerevisiae is very stringent regarding the intron consensus sequences (Gurr et al., 1987, The structure and organization of nuclear genes of filamentous fungi, pp 93-139 in: Gene Structure in Eukaryotic Microbes ed. Kinghorn, J. R. IRL press, Oxford and Washington D. C. ) it should be expected that S. cerevisiae is more sensitive to interference with one or more of the consensus sequences than would be the case in other eukaryotes like e. g. filamentous fungi.

In yeast each intron contains three conserved sequences, GUAPyGU (Py, pyrimidine) at the 5'donor site, UACUAAC as the internal consensus sequence (the branch point), and PyAG at the 3'acceptor site. In the filamentous fungi the internal consensus sequence is less conserved and requires the sequence CTPuAC (Pu, purine). During RNA splicing these conserved sequences are involved in interactions, either directly or indirectly, with small

nuclear RNA's (snRNA) in a complex termed the splisosome.

The present invention relates to an isolated polynucleotide comprising the necessary conserved intron sequences for RNA splicing without the need for flanking exon sequences, which can be inserted into e. g. a gene as a cassette and subsequently this cassette can be cleaved out by RNA splicing leaving the intact ORF to be translated into an active protein.

Furthermore, according to the present invention RNA splicing, resulting in a correctly spliced mRNA, should be temperature dependent. The temperature dependent splicing is achieved by interfering with the snRNA/splisosome interaction at the conserved intron sequences, by engaging one or more of the conserved intron sequences in base pairing with complementary RNA sequences, e. g. inverted repeats, thereby preventing or interfering with the interaction involving the snRNA's. This in turn will interfere with splicing resulting in a reduced splicing efficiency compared to the normal situation where there is no interference. The reduction in splicing efficiency is preferably at least 60 %, more preferably at least 70 %, even more preferably at least 80 %, and most preferably at least 90 %.

According to the invention the temperature dependent phenotype can be manipulated by varying the length of the sequences involved in base pairing, thereby obtaining a stable hybridization complex at low temperature and an unstable hybridization complex at high temperature, since increasing the temperature would at some point result in the destabilization of the hybridization complex, that would in turn allow interaction of snRNA's/splisosome at the conserved intron sequences. The hybridization complex could be provided in several ways. In one embodiment the polynucleotide of the invention comprises at least two internal subsequences, inverted repeats, one of which should comprise at least one conserved intron sequence, which subsequences will be able to form a hybridization complex or secondary structure. The same result should also be obtainable by e. g. synthesizing an antisense RNA that will be able to hybridize to a complementary sequence comprising at least one conserved intron sequence.

In one embodiment, the present invention therefore relates to a polynucleotide comprising conserved intron sequences required for RNA splicing in a host cell, wherein the said splicing, resulting in a correctly spliced mRNA, is temperature dependent due to secondary structure formation or hybridization of an antisense RNA.

The polynucleotide of the invention can be used in a method of temperature dependent expression of a polynucleotide of interest in a host cell, which method comprises,

a) providing a polynucleotide comprising conserved intron sequences required for RNA splicing, wherein the said splicing, resulting in a correctly spliced mRNA, is temperature dependent due to secondary structure formation or hybridization of an antisense RNA; b) inserting the polynucleotide into a gene or a control sequence; c) introducing the gene or control sequence comprising the polynucleotide into the host cell d) culturing the host cell under conditions promoting expression of the gene; e) optionally recovering the gene product.

In one embodiment the secondary structure formation or the antisense RNA hybridization involve at least one conserved intron sequence. In a particular embodiment the at least one conserved intron sequence is the internal conserved sequence (the branch point).

In another embodiment the secondary structure formation or the antisense RNA hybridization involve both the internal conserved sequence and the 3'acceptor site. In another embodiment the secondary structure formation involve the 3'acceptor site or the 5'donor site.

In a further embodiment the secondary structure formation involves stem-loop formation between inverted repeats comprised in the polynucleotide of the invention.

The polynucleotide of the invention can be designed to provide either cold sensitive expression of the gene product or temperature sensitive expression of the gene product.

Cold sensitivity is e. g. provided in one embodiment wherein the secondary structure formation or the antisense RNA hybridization interferes with correct splicing. This will be the case when the polynucleotide of the invention only comprises one 5'donor site, one internal consensus sequence and one 3'acceptor site. In this case, secondary structure formation involving the internal consensus sequences, the 3'acceptor site or both will interfere with the splicing reaction at temperatures that will allow the stable formation of such a secondary structure. The stability of the said secondary structure will depend on many factors, such as the number and type of bases involved in the base pairing. Melting temperatures for A-T rich complexes will be lower than for comparable G-C rich complexes, and the more base involved the higher the melting temperature. This will allow a person skilled in the art to design a polynucleotide of the invention with the desired properties.

The same basic principle as above can be utilized in order to obtain the opposite effect.

This can be achieved according to the present invention, by a polynucleotide comprising more than one 3'acceptor site, or more than one 5'donor site. A polynucleotide according to the invention can be constructed as shown in figure 3 comprising, in addition to the polynucleotide described above, an adjacent nucleotide sequence comprising one more internal consensus sequence (branch point) and one additional 3'acceptor site. This additional 3'acceptor site, which will be at the boundary of the cassette, will only be used for the RNA splicing reaction in the event that the first 3'acceptor site (which is closer to the 5'donor site) and/or its corresponding branch point consensus sequence are not available for the splicing reaction.

This will be the case when the first 3'acceptor site and/or its corresponding branch point consensus sequence are engaged in the said secondary structure formation. As above the secondary structure will be stable at low temperature, leading to splicing of the complete cassette at low temperature, and unstable at high temperature leading to splicing of an incomplete cassette at high temperature, when the first 3'acceptor site and/or its corresponding branch point consensus sequence are not involved in the secondary structure formation due to instability at the higher temperature.

In the terminology used above, the first 3'acceptor site denotes the 3'acceptor site proximal to the 5'donor site and the corresponding branch point consensus sequence denotes the branch point sequence placed between the 5'donor site and the first 3'acceptor site.

The polynucleotide of the present invention can be used for providing temperature dependent expression of a polypeptide of interest in a mammalian cell, an insect cell, a plant cell or a fungal cell.

In one embodiment use of the present invention provides cold sensitive expression of a polypeptide of interest and in another embodiment use of the present invention provides temperature sensitive expression of a polypeptide of interest.

In one embodiment of the present invention the polynucleotide is an isolated or substantially pure polynucleotide.

The host cell may be a eukaryote, such as a mammalian cell, an insect cell, a plant cell or a fungal cell. Useful mammalian cells include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number of other immortalized cell lines

available, e. g. , from the American Type Culture Collection.

In a particular embodiment, the host cell is a fungal cell."Fungi"as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et a/., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra). Representative groups of Ascomycota include, e. g., Neurospora, Eupenicillium (=Penicillium), Emericella (=Aspergillus), Eurotium (=Aspergillus), and the true yeasts listed below. Examples of Basidiomycota include mushrooms, rusts, and smuts.

Representative groups of Chytridiomycota include, e. g., Allomyces, Blastocladiella, Coelomomyces, and aquatic fungi. Representative groups of Oomycota include, e. g., Saprolegniomycetous aquatic fungi (water molds) such as Achlya. Examples of mitosporic fungi include Aspergillus, Penicillium, Candida, and Altemaria. Representative groups of Zygomycota include, e. g., Rhizopus and Mucor.

In a further embodiment, the fungal host cell is a yeast cell."Yeast"as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). The ascosporogenous yeasts are divided into the families Spermophthoraceae and Saccharomycetaceae. The latter is comprised of four subfamilies, Schizosaccharomycoideae (e. g. , genus Schizosaccharomyces), Nadsonioideae,<BR> Lipomycoideae, and Saccharomycoideae (e. g. , genera Pichia, Kluyveromyces and Saccharomyces). The basidiosporogenous yeasts include the genera Leucosporidim, Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella. Yeasts belonging to the Fungi Imperfecti are divided into two families, Sporobolomycetaceae (e. g. , genera Sorobolomyces and<BR> Bullera) and Cryptococcaceae (e. g. , genus Candida). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A. , Passmore, S. M. , and Davenport, R. R. , eds, Soc.

App. Bacteriol. Symposium Series No. 9,1980. The biology of yeast and manipulation of yeast genetics are well known in the art (see, e. g., Biochemistry and Genetics of Yeast, Bacil, M. ,<BR> Horecker, B. J. , and Stopani, A. O. M., editors, 2nd edition, 1987; The Yeasts, Rose, A. H. , and<BR> Harrison, J. S. , editors, 2nd edition, 1987; and The Molecular Biology of the Yeast Saccharomyces, Strathern et a/., editors, 1981).

In a more particular embodiment, the yeast host cell is a cell of a species of Candida,

Kluyveromyces, Saccharomyces, Schizosaccharomyces, Pichia, or Yarrowia. In a most particular embodiment, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell. In another embodiment, the yeast host cell is a Kluyveromyces lactis cell. In yet another embodiment, the yeast host cell is a Yarrowia lipolytica cell.

In a particular embodiment, the fungal host cell is a filamentous fungal cell.

"Filamentous fungi"include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et a/., 1995, supra). The filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.

In an even more particular embodiment, the filamentous fungal host cell is a cell of a species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, or Trichoderma.

In a most particular embodiment, the filamentous fungal host cell is an Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. In another most particular embodiment, the filamentous fungal host cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum cell. In still another embodiment, the filamentous fungal parent cell is a Fusarium venenatum (Nirenberg sp. nov.) cell. In another particular embodiment, the filamentous fungal host cell is a Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se.

Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81: 1470-

1474. Suitable methods for transforming Fusarium species are described by Malardier et aL, 1989, Gene 78: 147-156 and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. l., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et aL, 1983, Journal of Bacteriology 153: 163; and Hinnen et al, 1978, Proceedings of the National Academy of Sciences USA 75: 1920.

In the production methods of the present invention, the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fementors performed in a suitable medium and under conditions allowing the polypeptide, encoded by e. g. a gene comprising the polynucleotide according to the invention, to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to <BR> <BR> published compositions (e. g. , in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysats.

The polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide.

The resulting polypeptide may be recovered by methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

The polypeptides may be purified by a variety of procedures known in the art including, <BR> <BR> but not limited to, chromatography (e. g. , ion exchange, affinity, hydrophobic,<BR> chromatofocusing, and size exclusion), electrophoretic procedures (e. g. , preparative<BR> isoelectric focusing), differential solubility (e. g. , ammonium sulfate precipitation), SDS-PAGE,<BR> or extraction (see, e. g., Protein Purification, J. -C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

The polypeptide may be an anti-microbial peptide, a regulatory protein such as a transcriptional activator or repressor, or an enzyme.

In one embodiments the polypeptide is an enzyme such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, e. g. , a laccase, and/or a peroxidase.

The enzyme classification employed in the present specification and claims is in accordance with Recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, Academic Press, Inc., 1992.

Accordingly the types of enzymes which may appropriately be produced according to the method of the invention include oxidoreductases (EC 1.-.-.-), transferases (EC 2.-.-.-), hydrolases (EC 3.-.-.-), lyases (EC 4. -.-.-), isomerases (EC 5. -.-.-) and ligases (EC 6.-.-.-).

Preferred oxidoreductases in the context of the invention are peroxidases (EC 1.11. 1) such as haloperoxidase, laccases (EC 1.10. 3.2) and glucose oxidases (EC 1.1. 3.4), while preferred transferases are transferases in any of the following sub-classes : a) Transferases transferring one-carbon groups (EC 2.1) ; b) Transferases transferring aldehyde or ketone residues (EC 2.2) ; acyltransferases (EC 2.3) ; c) Glycosyltransferases (EC 2.4) ; d) Transferases transferring alkyl or aryl groups, other than methyl groups (EC 2.5) ; and e) Transferases transferring nitrogenous groups (EC 2.6).

A most preferred type of transferase in the context of the invention is a transglutaminase (protein-glutamine y-glutamyltransferase ; EC 2.3. 2.13).

Further examples of suitable transglutaminases are described in WO 96/06931 (Novo Nordisk A/S).

Preferred hydrolases in the context of the invention are: Carboxylic ester hydrolases (EC 3.1. 1. -) such as lipases (EC 3.1. 1.3) ; phytases (EC 3.1. 3. -), e. g. 3-phytases (EC 3.1. 3.8) and 6-phytases (EC 3.1. 3.26) ; glycosidases (EC 3.2, which fall within a group denoted herein as "carbohydrases"), such as a-amylases (EC 3.2. 1.1) ; peptidases (EC 3.4, also known as proteases); and other carbonyl hydrolases.

In the present context, the term"carbohydrase"is used to denote not only enzymes capable of breaking down carbohydrate chains (e. g. starches) of especially five-and six-membered ring

structures (i. e. glycosidases, EC 3. 2), but also enzymes capable of isomerizing carbohydrates, e. g. six-membered ring structures such as D-glucose to five-membered ring structures such as D-fructose.

Carbohydrases of relevance include the following (EC numbers in parentheses): a-amylases (3.2. 1.1), (3-amylases (3.2. 1.2), glucan 1, 4-a-glucosidases (3.2. 1.3), cellulases (3.2. 1.4), endo-1,3 (4)-, 8-glucanases (3.2. 1.6), endo-1, 4-p-xylanases (3.2. 1.8), dextranases (3.2. 1.11), chitinases (3.2. 1.14), polygalacturonases (3.2. 1.15), lysozymes (3.2. 1.17), ß- glucosidases (3.2. 1.21), a-galactosidases (3.2. 1.22), ß-galactosidases (3.2. 1.23), amyl-1, 6- glucosidases (3.2. 1.33), xylan 1, 4-p-xylosidases (3.2. 1.37), glucan endo-1, 3-ß-D-glucosidases (3.2. 1.39), a-dextrin endo-1, 6-a-glucosidases (3.2. 1.41), sucrose a-glucosidases (3.2. 1.48), glucan endo-1, 3-a-glucosidases (3.2. 1.59), glucan 1, 4-p-glucosidases (3.2. 1.74), glucan endo-1, 6-ß-glucosidases (3.2. 1.75), arabinan endo-1, 5-a-L-arabinosidases (3.2. 1.99), lactases (3.2. 1.108), chitosanases (3.2. 1.132) and xylose isomerases (5.3. 1.5).

Examples of commercially available carbohydrases include Alpha-GaITM, Bio-FeedTM Alpha, Bio-Feed Beta, Bio-Feed Plus, Bio-FeedTM Plus, NovozymeTM 188, CelluclastTM, Cellusoft, CeremylTM, CitrozymTM, DenimaxTM, DezymeTM, DextrozymeTM, FinizymTM,<BR> FungamylTM, GamanaseTM, GlucanexTM, LactozymTM, MaltogenaseTM, PentopanTM, Pectine, Promozyme, PulpzymeTM, Novamyl, Termamyl, AMG (Amyloglucosidase Novo), MaltogenaseTM, Sweetzyme and AquazymTM (all available from Novo Nordisk A/S).

Proteases: Suitable proteases include those of animal, vegetable or microbial origin.

Microbial origin is preferred. Chemically modified or protein engineered mutants are included.

The protease may be a serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e. g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like proteases are trypsin (e. g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27,36, 57,76, 87,97, 101,104, 120,123, 167,170, 194, 206,218, 222,224, 235 and 274.

Examples of commercially available proteases (peptidases) include Kannase, Prìmase, EveriaseTM, EsperaseTM, AlcalaseTM, Neutrase, DurazymTM, Savinase, Pyrase, Pancreatic Trypsin NOVO (PTN), Bio-FeedTM Pro and Clear-Lens Pro (all available from Novozymes A/S, Bagsvaerd, Denmark).

Other commercially available proteases include Maxatase, Maxacal, Maxapem, OpticleanTM, ProperaseTM, PurafectTM, Purafect OxP, FN2TM, and FN3TM (available from Genencor International Inc. or Gist-Brocades).

Lipases: Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e. g. from H. lanuginosa (T lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e. g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372, 034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e. g. from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131,253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).

Other examples are lipase variants such as those described in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.

Examples of commercially available lipases include Lipoprime Lipolase, Lipolase Ultra, LipozymeTM, Palatase, Novozym 435 and Lecitase (all available from Novozymes A/S). Other commercially available lipases include Lumafast (Pseudomonas mendocina lipase from Genencor International Inc.) ; Lipomax (Ps. pseudoalcaligenes lipase from Gist- Brocades/Genencor Int. Inc. ; and Bacillus sp. lipase from Solvay enzymes.

Amylases : Suitable amylases (a and/or ß) include those of bacterial or fungal origin.

Chemically modified or protein engineered mutants are included. Amylases include, for example, a-amylases obtained from Bacillus, e. g. a special strain of B. licheniformis, described in more detail in GB 1,296, 839.

Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15,23, 105,106, 124,128, 133,154, 156,181, 188,190, 197,202, 208,209, 243,264, 304,305, 391,408, and 444.

Commercially available amylases are Duramyl, TermamylT"", Fungamyl" and BANT"" (Novozymes A/S), Rapidase and Purastar (from Genencor International Inc.).

Cellulases : Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e. g. the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435, 307, US 5,648, 263, US 5,691, 178, US 5,776, 757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral celfulases having colour care benefits. Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457, 046, US 5,686, 593, US 5,763, 254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Commercially available cellulases include CelluzymeTM, and Carezyme (Novozymes A/S), ClazinaseTM, and Puradax HATS (Genencor International Inc.), and KAC-500 (B) TM (Kao Corporation).

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprins, e. g. from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Examples of commercially available oxidoreductases (EC 1.-.-.-) include GluzymeTM (enzyme available from Novo Nordisk A/S).

In one embodiment the polynucleotide of the invention can be inserted directly in a gene encoding a polypeptide of interest, e. g. a cloned gene to be over expressed in order to obtain an enzyme or other protein. In case the production of the protein is detrimental to the host cell, it will now be possible to control the expression level by selecting a suitable growth temperature. Alternatively, culture biomass may be produced at the permissive temperature where the protein is not expressed and expression may subsequently be turned on by changing the growth temperature.

In another embodiment the polynucleotide of the invention is inserted in a gene encoding a transcription factor, which regulates the expression of the gene encoding a protein of interest. The transcription factor can be an activator or a repressor. An example of a transcriptional activator is the amyR transcription factor positively regulating amylase and glucoamylase promoters in Aspergillus sp. such as A. oryzae and A. niger (Petersen et al.,

1999, MGG 262: 668-676). An Aspergillus host strain in which a polynucleotide according to the invention has been inserted in the amyl gene will serve to allow temperature dependent expression of any subsequently introduced endogenous or exogenous coding sequence expressed from an Aspergillus amylase promoter.

In yet another embodiment the polynucleotide of the invention is inserted into a marker gene, like e. g. an antibiotic resistance marker, on an expression vector. By selecting transformants at the appropriate temperature the selection pressure can be adjusted to allow selection of only those transformants in which the copy number of the marker gene is high enough to allow sufficient expression of the marker gene product. This way it is possible to select for high copy number transformants or transformants where the marker gene has integrated in the chromosome of the host cell at loci resulting in high transcription levels.

Alternatively, the expression vector, carrying the polynucleotide of the invention inserted in a marker gene, may first be introduced into the host cell at a relatively permissive temperature, i. e. allowing relatively efficient RNA splicing, and the copy number of the marker gene may subsequently be amplified by varying the temperature to increase the selection pressure.

Either way of selecting for increased expression from the marker gene also selects for higher expression of the desired product encoded by the same expression vector.

In a still further embodiment the polynucleotide of the invention can be inserted in any gene the function of which is essential in a particular growth phase or condition, but unwanted or deleterious in other growth phases or conditions.

EXAMPLES Example 1: Design and synthesis of a functional intron.

The second intron (iAMG2) of the glucoamylase gene from A. niger (Boel et al., (1984), EMBO J. 3: 1581) shown in Figure 1, was chosen as the starting point for constructing an intron with varying lengths of an inverted repeat. First it had to be demonstrated that this intron was indeed functional when inserted in another gene. Two oligonucleotides named, Primer A and Primer B (SEQ ID NO 1 and SEQ ID NO 2; purchased from Life Technologies/GIBCO BRL) were designed as a Hind3-EcoR1 adapter containing the entire iAMG2 sequence which had been modified at two base positions to introduce a BsrG1 (SspB1) site early in the intron sequence. Furthermore the adapter had been designed such that the modified iAMG2 could be precisely excised as a 55 bp SnaB1-Pvu2 blunt ended fragment. Oligonucleotides Primer A

and Primer B at 50 pmoles/pl each in nick translation buffer (e. g. Sambrook and Russell, 2001) were boiled and allowed to anneal. The annealed product was gel purified on a 4% agarose gel (NuSieveGTG; BioWhittaker). The isolated fragment was ligated to the Hind3- EcoR1 fragment of pBluescriptSK2 (-) (Stratagene) and transformed into E. coli. Plasmids were prepared from a number of transformants and the insert sequence determined on an ABI sequencer using standard methods. One plasmid, pMT2374, containing the desired iAMG2 sequence was chosen. The 55 bp SnaB1-Pvu2 fragment was isolated from pMT2374 and inserted in the unique Xmn1 in the A. oryzae pyrG gene of plasmid pMT2293 (Figure 2; SEQ ID NO 3). For this pMT2293 was digested with Xmn1, dephosphorylated with alkaline phosphatase and the fragment gel purified before being ligated to the iAMG containg SnaB1- Pvu2 fragment. After transformation into a pyrF negative E. coli (E. coli DB6507 (DSM 6201/ ATCC 35673) ) and selecting for growth in the absence of uridine, transformants were obtained and plasmids prepared. Plasmids were sequenced and one plasmid, pMT2377, was identified in which the AMG2 had been inserted in the correct orientation. Next, pMT2377 was transformed into a pyrG negative strain of A. oryzae, JaL355 (JaL355 is a derivative of A. oryzae A 1560 wherein pyrG has been inactivated as described in W09801470) and transformants were selected for prototrophy at both 30 degrees C and at 37 degrees C. The transformants obtained with pMT2377 were tested for growth in the absence of uridine at various temperatures between 30 and 42 degrees C and in all cases showed as good growth as did parallel transformants obtained with the plasmid pMT2293. 1t was concluded that the synthetic iAMG2 intron in pMT2377 was fully functional in the temperature range tested.

Example 2: Design and synthesis of introns containing inverted repeats.

A series of iAMG2 derived introns containing inverted repeats of varying length were constructed in pMT2374 by substituting the BsrG1-Nde1 fragment with synthetic adapters.

Adapters were annealed and ligated as described above and in all cases the sequence of the modified, inverted repeat containing iAMG2 sequences were verified by sequencing. All the modified introns could still be excised as SnaB1-Pvu2 fragments. The following inverted repeat containing iAMG2 inserts were made as shown in Table 1.

Table 1.

Plasmid Adapter oligos SEQ ID Aspergillus vector pMT2396 Primer C SEQ ID NO 4 pMT2407 Primer D SEQ ID NO 5

pMT2397 Primer E SEQ ID NO 6 pMT2408 Primer F SEQ ID NO 7 pMT2398 Primer G SEQ ID NO 8 pMT2409 Primer H SEQ ID NO 9 pMT2437 Primer J SEQ ID NO 10 pMT2443 Primer K SEQ ID NO 11 pMT2438 Primer L SEQ ID NO 12 pMT2444 Primer M SEQ ID NO 13 pMT2439 Primer N SEQ ID NO 14 pMT2445 Primer O SEQ ID NO 15 From each of the above plasmids pMT2396-98 and pMT2437-39 the sequence verified BsrG1- Nde1 fragment was transferred to the Aspergillus vector pMT2377 (see example 1). This transfer was made as three part ligations of the 1.9 kb fragment Alw44. 1-Nde1 and the 3.5 kb Alw44. 1-SspB1 from pMT2377 to the short Nde1-SspB1 fragment from each of the above intron containing plasmids. The resulting corresponding Aspergillus vectors pMT2407-09 and pMT2443-45 have also been listed in the right hand column of the above table. The modified iAMG2 sequence, of all of these vectors have been confirmed by sequencing.

Example 3: Characterization of tranformants containing introns with inverted repeats.

Each of the plasmids pMT2407-09 and pMT2443-45 were transformed into the uridine requiring A. oryzae JaL355 as described above (Example 1). Transformants were selected at 37 degrees C in the absence of uridine. For each plasmid approximately 20 individual transformants were chosen for two rounds of sporulation and re-isolation at 37 degrees and without uridine. The re-isolated transformants were then tested for growth at different temperatures in the absence of uridine. The following was noted: Transformants with plasmids pMT2407 and pMT2408 appeared to grow as wild type, Aspergillus oryzae (strain BECh2), at 30,37 and at 42 degrees C. For both wild type and these transformants, 42 degrees C seems to be the absolute highest growth temperature and occasional poor growth was observed probably due to insufficient thermo control in the incubator; at 30 and at 37 degrees C growth was always good. Regarding transformants with

pMT2409 the growth pattern varied. Some transformants grew normally at 37and 42 degrees C and showed only slightly reduced growth at 30 degrees. Other transformants showed near normal growth at 37 and at 42 degrees C while showing very poor growth indeed at 30 degrees C.

The table below, table 2, shows the growth pattern of two such transformants, JaL355/pMT2409#14 (later named MT2430) and JaL355/pMT2409#17 (later named MT2431), as well as the wild type Aspergillus oryzae strain BECh2. Plates were incubated at 30 degrees C or at 42 degrees C as indicated for six days.

Table 2. Growth pattern of transformants MT2430 and MT2431, and wild type BECh2. Strain Temperature Temperature 30 degrees C 42 degrees C BECh2 +++ +++ MT2430 (+) ++ MT2431 (+) ++ Symbols : +++ normal vigorous growth, ++ slightly reduced growth, (+) very poor, barely detectable growth From these results it was concluded that the iAMG2 intron version present in the pyrG gene of pMT2409 leads to a cold sensitive phenotype. Addition of uridine to the growth medium abolished the cold sensitive phenotype of strains MT2430 and MT2430 consistent with the cold sensitivity being due to the lack of a functional pyrG at low temperature.

Transformants were also made with plasmids pMT2443-45 in the same way as described above for plasmids pMT2407-2409. For plasmids pMT2443 and pMT2444 transformants were obtained that showed somewhat reduced growth rate at 30 degrees in the absence of uridine and normal growth at 37 and 42 degrees C. For pMT2445 transformants were obtained that showed essentially the same degree of cold sensitivity as the transformants obtained with pMT2409.

Example 4: Estimation of plasmid copy number by Southern blots.

Total DNA was prepared from 15 transformans of A. oryzae JaL355 with plasmid pMT2409. The DNA was digested with Asp718 and BssH2, separated on a 0.7% agarose gel and transferred to a membrane filter for Southern blotting using essentially the protocols described by the manufacturer (Roche) for the non radioactive DIG based detection system.

The probe was a 877 bp Asp718-EclX1 fragment of pMT2409 which was DIG labelled by

hexamer priming as stipulated by the manufacturer (Roche). The probe overlaps with part of the pyrG promoter left in strain JaL355 which has a deletion of the pyrG coding sequences.

The overlap to the remaining pyrG promoter is 364 bp in length and is located on an approximately 4.8 kb Asp718-BssH2 fragment of the JaL355 host DNA. The probe overlaps, naturally, with all 877 bp of pMT2409 and the Asp718-BssH2 fragment of this plasmid has a size of 1.6 kb. Since the probe hybridizes to both the single copy of the host DNA comprising the pyro locus (on the 4.8 kb fragment) and to plasmid DNA it is possible to make a very rough estimate of plasmid copy numbers from the Southern, and to correlate plasmid copy number to growth characteristics of the transformants. Generally it is seen that those JaL355/pMT2409 transformants that fail to grow at low temperature has got few (maybe only a single) copies of the plasmid integrated (as seen by a faint signal on the southern blot in lanes representing MT2430 and 2431, respectively), while those transformants that showed only a slight growth impairment at 30 degrees C have a very high number of plasmid copies (as seen by a strong signal; corresponding strains preserved as MT2427 and 2429, respectively).

Example 5: Design of heat sensitive introns (ts intron).

In order to modify the iAMG2 sequence such that base pairing and stem-loop formation alters the choice of splice sequence (as illustrated in Figure 3), i. e. the sequence recognised as the 3'acceptor site of the intron, pMT2374, pMT2396 and pMT2439 were cut with Nde1 and EcoR1 and the large fragments isolated. These fragments were ligated to the Nde1- EcoR1 adapter formed by annealing of the two oligonucleotides, Primer P and Primer Q (SEQ ID NO 16 and SEQ ID NO 17, respectively). After tranformation into E. colithe plasmids pMT2589,2590 and 2591, derived from pMT2374, pMT2396 and pMT2439, respectively, were obtained and their respective correct sequences verified by sequencing. pMT2589 is a control plasmid where no significant stem loop structure can be formed, while in pMT2590 and 2591 stem-loops of increasing length can be formed.

Example 6: Transfer of temperature sensitive introns to Aspergillus vectors.

The modified introns of pMT2589 to pMT2591 are transferred to the Xmn1 site of a pyrG selection based Aspergillus expression vector essentially as described in example 2. The resulting Aspergillus vectors are transformed into JaL355 selecting for prototrophy at low temperature (e. g. 30 degrees C). Re-isolated transformants are tested for growth characteristics in the absence of uridine a different temperatures. Transformants obtained with pMT2589 shows wt growth characteristics in the temperature range 30-42 degrees C.

Transformants obtained with plasmids pMT2590 and 2591 shows near normal growth at 30 degrees but shows growth which to different degrees dependent on the particular transformant

is poor at higher temperature (e. g. 37 or 42 degrees). In the presence of uridine all transformants shows normal growth at all temperatures, consistent with the temperature sensitive (ts) phenotype being a result of the modified iAMGs inserted in the pyrG gene of plasmids pMT2590 and pMT2591.