WO2000032797A1

WO2000032797A1 - Transformed thermomyces lanuginosus

Info

Publication number: WO2000032797A1
Application number: PCT/IB1999/002010
Authority: WO
Inventors: Søren Kjærulff; Susan Mampusti Madrid
Original assignee: Danisco A/S
Priority date: 1998-12-01
Filing date: 1999-11-30
Publication date: 2000-06-08
Also published as: AU1511600A; GB9826367D0

Abstract

A transformed T. lanuginosus cell is described which expresses recombinant proteins.

Description

TRANSFORMED THERMOMYCES LANUGINOSUS

FIELD OF THE PRESENT INVENTION

The present invention relates to an organism, in particular a transformed organism.

In particular, the present invention is directed to a new host organism useful in production of recombinant proteins.

More in particular, the present invention discloses a transformation system for fungus T. lanuginosus which can be used in the expression of recombinant proteins, especially enzymes.

BACKGROUND

DE-A-4017522 discloses culturing Thermomyces on lignocellulose to prepare an exocellulase- and endocellulase-free xylanase.

EP-A-0456033 reports on the use of T.lanuginosus to produce xylanase. The xylanase can be used for the enzymatic treatment of plant raw materials containing xylan and lignocellulose.

Production of T. lanuginosus lipase in Aspergillus is disclosed in WO 93/12237.

WO-A-97/35017 discloses a 3,6-phytase from T.lanuginosus - which is said to be useful for liquefying starch, releasing inorganic phosphate from phytate and reducing phytate content of manures.

Schlacher et al (J Biotechnology vol 49 (1996) pages 211-218) disclose a gene for the thermostable xylanase XynA from T.lanuginosus. These workers only ascribe "possible promoter elements" (for example, see the legend to Figure 2 thereof). These workers do not demonstrate the use of those elements to express heterologous nucleotide sequences.

It is desirable to express a nucleotide of interest (NOI) by a transformed organism - such as fungus. For instance, it may be desirable to enhance production of fungus own proteins, such as cellulases, proteases or lipases, or even to produce foreign (heterologous) proteins originating from other species of fungi or bacteria, plants or animals.

By way of example, US-A-5667990 states that:

"The use of recombinant host cells in the expression of heterologous proteins has in recent years greatly simplified the production of large quantities of commercially valuable proteins, which otherwise are obtainable only by purification from their native sources. Currently, there is a varied selection of expression systems from which to choose for the production of any given protein, including prokaryotic and eukaryotic hosts. The selection of an appropriate expression system will often depend not only on the ability of the host cell to produce adequate yields of the protein in an active state, but also to a large extent may be governed by the intended end use of the protein. Although mammalian and yeast cells have been the most commonly used eukaryotic hosts, filamentous fungi have now begun to be recognized as very useful as host cells for recombinant protein production. Among the filamentous fungi which are currently used or proposed for use in such processes are Neurospora crassa, Acremonium chrysogenum, Tolypocladium geodes, Mucor circinelloides and Trichoderma reesei. In addition, certain species of the genus Aspergillus have been used effectively as host cells for recombinant protein production. ... Both Aspergillus niger and Aspergillus oryzae have also been described as being useful in recombinant production of proteins."

The species Aspergillus nidulans has been reported to be transformed with recombinant plasmids (Ballance, et al. Biochem. Biophys. Res. Comm. 112: 284-289, 1983), but transformation was found to occur at fairly low frequency.

Recombinant thermophilic fungi are described in WO 9602653, which reports a range of NOIs expressed in organisms as Acremonium, Corynascus, Thielavia, Myceliophthora, Thermoascus, Sporotrichum, Chaetomium, Ctenomyces, Scytalidium, or Talaromyces.

Alison (Curr. Genet. (1992) 21 :225) discloses transformation of a thermophilic fungi Humicola grisea var. thermoidea and overproduction of Humicolas own glucoamylase, while Jain, Durand and Tiraby describe transformation of another fungus Talaromyces. (Mol. Gen. Genet. (1992) 234:489). There are, however, no earlier teachings on transformation system for 7. lanuginosus or Humicola lanuginosa which was its previous name.

SUMMARY ASPECT OF THE PRESENT INVENTION

The present invention provides a novel recombinant thermophilic organism.

DETAILED ASPECTS OF THE PRESENT INVENTION

Aspects of the present invention are presented in the claims. These and other aspects are now discussed.

According to one aspect of the present invention there is provided a transformed 7. lanuginosus, wherein the transformed 7. lanuginosus comprises an NOI. The NOI may encode a protein of interest (POI).

In another aspect the invention provides a method for producing POI by culturing a 7. lanuginosus host cell comprising a NOi encoding a POI under conditions which allow expression and/or secretion of the POI. The POI is then optionally purified and/or isolated.

Other aspects of the present invention include methods of transforming 7. lanuginosus.

Additional aspects of the present invention include uses of the transformed 7. lanuginosus for expressing NOIs in vitro (e.g. in culture media such as a broth) and/or in vivo (e.g. in the transformed organism).

For ease of reference, these and further aspects of the present invention are now discussed under appropriate section headings. However, the teachings under each section are not necessarily limited to each particular section.

PREFERABLE ASPECTS

Preferably the NOI encodes a POI.

Preferably the NOI codes for a heat stable protein. Preferably the NOI is operably linked to a promoter.

Preferably the promoter is a fungal promoter.

Preferably the promoter is derived or derivable from 7. lanuginosus.

Preferably the POI is secreted to the culture medium.

Preferably the POI is isolated and/or purified.

ADVANTAGES

The present invention is advantageous for a number of reasons.

By way of example, the present invention is advantageous because desirable levels of the product of expression of a NOI (i.e. a POI) can be obtained. Here, the NOI may be, for example, a desired compound of benefit to humans or animals (e.g. a desirable foodstuff or an enzyme having a beneficial effect, such as a foodstuff processing effect or even a pharmaceutical effect. Furthermore, that product may be easily retrievable.

The present invention is also advantageous because it allows transformed Thermocyces lanuginosus to express desirable levels of the product of expression of a NOI.

The present invention is also advantageous because transformation of suitable host cells will facilitate commercial production of valuable enzymes, which otherwise would only be obtainable in minute amounts or by laborious purification from their native sources.

The use of thermophilic fungi as host cells provides other advantages as well. The higher temperature at which they grow, eg 50°C, induces a more rapid growth rate in some species than is seen with non-thermophiles. This in turn leads to a more rapid and efficient production system for proteins.

Other advantages will be apparent from the following commentary. NUCLEOTIDE

The term "nucleotide" in relation to the pomoter for use in the present invention typically means DNA, which may be genomic DNA and/or synthetic DNA.

The term "nucleotide" in relation to the NOI means DNA - which may be genomic DNA and/or cDNA and/or synthetic DNA - or RNA. Preferably it means DNA.

PROMOTER

The term "promoter" is used in the normal sense of the art, e.g. an RNA polymerase binding site and controls the initiation of transcription for the gene product. The promoter for use in the present invention is capable of expressing a NOI.

In addition to the nucleotide sequences described herein, the promoters for use in the present invention could additionally include conserved regions such as a CAAT box and/or a TATA box. The promoters may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the NOI. Other sequences include inducible elements - such as temperature, chemical, light or stress inducible elements. Also, suitable elements to enhance transcription or translation may be present. An example of the latter element is the TMV 5' leader sequence (see Sleat Gene 217 [1987] 217-225; and Dawson Plant Mol. Biol. 23 [1993] 97).

In addition the present invention also encompasses combinations of promoters or other expression elements.

For example, one promoter may be used in combination with another promoter. Other combinations are possible. For example, a promoter may be used in combination with a tissue specific promoter.

Preferably the promoter is a strong promoter.

The promoter sequence can be derived from T. lanuginosus, other fungi or even other eukaryotes or bacteria.

An example of a suitable constitutive promoter is the actin promoter from 7. lanuginosus. Aside from the promoter, one or more additional control sequences may be operably linked to the NOI Examples of such additional control sequences include enhancers, terminator sequences and other expression regulation signals These control sequences may be selected to be compatible with the host organism for which the expression vector is designed to be used in

It may also be advantageous for the promoter for use in the present invention to be inducible so that the levels of expression of the heterologous gene can be regulated during the life-time of a cell Inducible means that the levels of expression obtained using the promoter can be regulated

Non-limiting examples of suitable inducible promoters include the xylanase A promoter, the α-amylase promoter, the glucanase promoter and the lipase promoter from T lanuginosus

For some applications, preferably the promoter has a sequence corresponding to, or derivable from, a xylanase promoter obtainable from Thermomyces Lanuginosus

The term "corresponding" in relation to the present invention means that the promoter sequence need not necessarily be derived from Thermomyces lanuginosus For example, the promoter could be prepared synthetically It may even be derived from another source

In this embodiment, the NOI is preferably not the full length xynA coding region

For some applications, a preferred promoter is a promoter comprising a nucleotide sequence corresponding to the sequence presented as SEQ ID No 1 or a variant, homologue or derivative thereof This promoter may be called the xynA promoter

VARIANT/HOMOLOGUE/DERIVATIVE/FRAGMENT

The terms "variant", "homologue", "derivative" or "fragment" in relation to the specified xynA promoter sequence of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence has promoter activity, preferably having at least the same activity of a promoter comprising the sequence shown as SEQ I D No 1 In particular, the term "homologue" covers identity with respect to structure and/or function providing the resultant nucleotide sequence has promoter activity With respect to sequence identity (i.e. similarity), preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% sequence identity. More preferably there is at least 95%, more preferably at least 98%, sequence identity. These terms also encompass allelic variations of the sequences.

Sequence identity can be determined by a simple "eyeball" comparison (i.e. a strict comparison) of any one or more of the sequences with another sequence to see if that other sequence has, for example, at least 75% sequence identity to the sequence(s).

Relative sequence identity can also be determined by commercially available computer programs that can calculate % identity between two or more sequences using any suitable algorithm for determining identity, using for example default parameters. A typical example of such a computer program is CLUSTAL. Advantageously, the BLAST algorithm is employed, with parameters set to default values. The BLAST algorithm is described in detail at http://www.ncbi.nih.gov/BLAST/blast_help.html, which is incorporated herein by reference. The search parameters can be advantageously set to the defined default parameters.

Advantageously, "substantial identity" when assessed by BLAST equates to sequences which match with an EXPECT value of at least about 7, preferably at least about 9 and most preferably 10 or more. The default threshold for EXPECT in BLAST searching is usually 10.

The xynA promoter is capable of causing effective expression of an NOI in the transformed Thermomyces lanuginosus.

With the xynA promoter of the present invention we have found that under at least some circumstances its promoter activity may depend on the presence of any one or more of a xylose and/or a xylan and a compound containing same (such as oligomers thereof), but wherein the activity may be supressed by the presence of glucose, or even sucrose.

VECTOR

The nucleotide sequences for use in the invention can be incorporated into a recombinant replicable vector. The vector may be used to replicate the nucleic acid in a compatible host organism. The vector may be an expression vector and/or a transformation vector.

The term "expression vector" means a construct capable of in vivo or in vitro expression.

The term "transformation vector" includes a shuttle vetor, i.e. a construct capable of being transferred from one entity to another - such as from E. coli to a fungus or from one fungus to another fungus.

Thus, in a further embodiment, the invention provides a method of preparing quantities of nucleotide sequences comprising introducing a nucleotide sequence into a replicable vector, introducing the vector into a host 7. lanuginosus, and growing the host organism under conditions which bring about expression of the NOI. The POI may then be recovered from the host organism.

Preferably, the vector comprises the NOI operably linked to a promoter, such that the promoter is capable of providing for the expression of the coding sequence by the host 7. lanuginosus, i.e. the vector is an expression vector.

The term "operably linked" means that the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.

The vectors may be, for example, plasmid or virus vectors provided with an origin of replication and optionally a regulator of the promoter.

Vectors may be used, for example, to transfect or transform the host organism either in vitro or in vivo.

Such vectors may be transformed or transfected into the host organsim to provide for expression of a protein of the invention. This process may comprise culturing the host organism transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the protein, and optionally recovering the expressed protein. Vectors of the present invention may introduced into the host 7 lanuginosus using a variety of techniques known in the art, such as transfection, transformation and electroporation Another technique is the protoplast transformation method (Winer et al , Microbiology, 1985, 468 American Society for Microbiology)

Vectors of the invention may be introduced into the host organism for the purpose of replicating the vectors/nucleotide sequences and/or expressing the NOI

The host organism comprising the vector may be used to express the POI In this respect, the host organism may be cultured under suitable conditions which allow expression of the POI In some instances, expression of the POI may be constitutive such that they are continually produced, or inducible, requiring an inducer to initiate expression In the case of inducible expression, protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium

Once the vector has been transformed or transfected into the host 7 lanuginosus then the host organism can be cultivated Here reference can be made briefly to US-A- 5543322 which says that for cultivation of a transformant, a culture medium containing carbon and nitrogen sources assimilable by the transformant and the like can be used Any carbon source assimilable by the transformant can be used Examples thereof include glucose, sucrose, starch, soluble starch, dextrin, glycerin, n-paraffin and the like as well as organic acids (e g , acetic acid, fumanc acid, benzoic acid, etc ), alcohols (e g , methanoi, ethanol, butanol, etc ), fats and oils (soybean oil, lard, etc ) and the like They can be used alone or in combination thereof As the nitrogen sources, there are, for example, peptone, soybean flour, cotton seed flour, meat extract, yeast extract, dried yeast, corn steep liquor, corn gluten meal, urea, ammonium salts (e g , ammonium chloride, ammonium sulfate, etc ), nitrates (e g , potassium nitrate, ammonium nitrate, etc ), other organic or inorganic nitrogen-containing materials and the like They can be used alone or in combination thereof In addition, inorganic salts (e g , phosphates, etc ), trace metal salts (e g , magnesium salt, calcium salt, manganese salt, etc ) can be appropriately added

Thus in a further embodiment, the invention provides a method comprising introducing a NOI into a replicable vector, introducing the vector into Thermomyces lanuginosus, and growing the transformed host organism under conditions which bring about expression of the NOI The POI may then be recovered from the host organism Vectors may be used, for example, to transfect or transform the host organism either in vitro or in vivo.

Such vectors may be transformed or transfected into a suitable host organsim to provide for expression of a protein of the invention. This process may comprise culturing the host organism transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the protein, and optionally recovering the expressed protein.

Vectors of the present invention may introduced into suitable host organisms using a variety of techniques, such as transfection, transformation and electroporation. Another technique is the protoplast transformation method (Winer et al., Microbiology, 1985, 468, American Society for Microbiology).

Host organisms comprising the vectors of the present invention may be used to express the POI. In this respect, host organisms may be cultured under suitable conditions which allow expression of the POI. In some instances, expression of the POI may be constitutive such that they are continually produced, or inducible, requiring an inducer to initiate expression. In the case of inducible expression, protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium.

CONSTRUCT

The present invention also encompasses a transformed 7. lanuginosus cell which transformed fungal cell comprises a construct, which construct comprises at least an NOI, which NOI may be operably linked to a promoter.

Here, the term "construct" - which is synonymous with terms such as "conjugate", "cassette" and "hybrid" - includes a NOI directly or indirectly attached to a promoter. An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the S/77-intron or the ADH intron, intermediate the promoter and the NOI. The same is true for the term "fused" in relation to the present invention which includes direct or indirect attachment. In each case, it is highly preferred that the terms do not cover the natural combination of the wild type gene ordinarily associated with the wild type gene promoter and the wild type promoter and when they are both in their natural environment.

The construct may even contain or express a marker which allows for the selection of the genetic construct in, for example, a fungus into which it has been transferred.

The selectable marker means may reside on an additional vector or may be included in the nucleic acid molecule which contains the expression system. The nature of the selectable marker means may depend on the culture conditions.

US-A-5358864 provides a short list of suitable selectable marker genes that may be used in the present invention - examples of which include fungal selection markers such as those that are the genes for acetamidase (amdS), ATP synthetase, subunit 9 (oliC) and benomyl resistance (benA).

In certain aspects of the present invention, use of the ble marker - which confers resistance to phleomycin/bleomycin/zeocin - is preferred, other selection markers, known in the art, could be used. Examples of auxotrophic markers are pyrG selecting for uridine prototrophs, argB selecting for arginine prototrophs, niaD selecting for nitrate prototrophs, trpC selecting for tryptophan prototrophs, amdS selecting for increased utilization of acetamide as sole nitrogen source. Dominant resistance markers could be chosen from oliC3 conferring resistance to oligomycin, hph conferring resistance to hygromycin B, bar conferring resistance to bialaphos or NPTII conferring resistance to G418.

Nucleotide sequences for subsequent transformation of the Thermomyces lanuginosus cell can be incorporated into a recombinant replicable vector. The vector may be used to replicate the nucleic acid in a compatible host organism.

TRANSFORMED

The term "transformed" is synonymous with the term "transgenic". Typically, the term means use of recombinant DNA techniques to prepare an altered (i.e. transformed) host. By way of example, a transformed host 7. lanuginosus according to the present invention may comprise a promoter and an NOI, wherein said transformed host has been prepared by transforming a host 7. lanuginosus that does not comprise either the promoter and/or the NOI with the promoter and/or the NOI. ORGANISM

The term "organism" as used herein is synonymous where ever appropriate with the words "cell" or "tissue".

With particular reference to transformed fungi according to the present invention, the term "organism" is synonymous the word "cell".

The term "transformed organism" in relation to the present invention means a transformed 7. lanuginosus organism comprising either an expressable construct according to the present invention or a product of such a construct. For example the transformed organism can comprise an exogenous nucleotide sequence (e.g. NOI as herein described) under the control of a promoter; or a native nucleotide sequence under the control of, for example, a variant promoter.

Therefore, the transformed organism of the present invention includes a transformed 7. lanuginosus organism comprising any one of, or combinations of a promoter, constructs as herein mentioned, vectors as herein mentioned, plasmids as herein mentioned, or the products thereof.

The transformed 7. lanuginosus could be used to prepare acceptable quantities of the POI which would be easily retrievable from the organism.

Preferably the expressable construct is incorporated in the genome of the 7. lanuginosus. The term incorporated preferably covers stable incorporation into the genome.

TRANSFORMED FUNGUS

The present invention relates to a transformed fungus.

The term "transformed" is synonymous with the term "transgenic".

To allow for selection of the resulting transformants, the transformation may typically involve the use of a selectable gene marker which may introduced with an expression cassette, either on the same vector or by co-transformation, into a host strain in which the gene marker is selectable. Various marker/host systems are available, including the pyrG, argB and niaD genes which are typically used with auxotrophic strains of Aspergillus nidulans, pyrG and argB genes which are typically used with Aspergillus oryzae auxotrophs, pyrG, trpC and niaD genes which are typically used with Penicillium chrysogenum auxotrophs, and the argB gene which are typically used with Tnchoderma reesei auxotrophs Dominant selectable markers including amdS, oliC, hyg and phleo are

5 also now available which are typically used with filamentous fungi as A niger, A oryzae, A ficuum, P chrysogenum, Cephalosporium acremonium, Cochliobolus heterostrophus, Glomerella cingulata, Fulvia fulva and Leptosphaeπa maculans (for a review see Ward in Modern Microbial Genetics, 1991 , Wiley-Liss, Inc , at pages 455-495) A commonly used transformation marker is the amdS gene of A nidulans which in high copy number allows

10 the fungus to grow with acrylamide as the sole nitrogen source

NQI/POI

The term "NOI" with reference to the present invention means any nucleotide sequence of 15 interest The NOI may encode a protein of interest ("POI")

The NOI may be single-stranded or double-stranded It may also be a polynucleotide which includes synthetic or modified nucleotides A number of different types of modification to oligonucleotides are known in the art These include methylphosphonate 20 and phosphorothioate backbones, addition of acndine or polylysine chains at the 3' and/or 5' ends of the molecule For the purposes of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art Such modifications may be carried out in order to enhance the in vivo activity or life span of the NOIs

__:.

The NOI will typically be or will typically comprise a heterologous gene The term "heterologous gene" typically encompasses any gene that is not naturally associated with the promoter and/or the host with which it is now associated for the transformation/expression uses

30

The heterologous gene may be any allelic variant of a wild-type gene, or it may be a mutant gene The term "gene" is intended to cover nucleic acid sequences which are capable of being at least transcribed Thus, sequences encoding mRNA, tRNA and rRNA, as well as antisense constructs, are included within this definition Nucleic acids 35 may be, for example, nbonucleic acid (RNA) or deoxynbonucleic acid (DNA) or analogues thereof Sequences encoding mRNA will optionally include some or all of 5' and/or 3' transcribed but untranslated flanking sequences naturally, or otherwise, associated with the translated coding sequence It may optionally further include the associated transcriptionai control sequences normally associated with the transcribed sequences, for example transcriptionai stop signals, polyadenylation sites and downstream enhancer elements

Thus, typically, the NOI can be any nucleotide sequence that is either foreign or natural to the organism in question The NOI could even be an anti-sense sequence

Typical examples of a NOI are nucleotide sequences encoding proteins of interest ("POI"s), such as enzymes The enzymes may be capable of modifying metabolic and catabolic processes

The POI may be any suitable prokaryotic or eukaryotic heterologous peptide or protein of interest - in particular a heterologous protein, such as a heterologous fungal enzyme

The POI can be a single-chain polypeptide molecule as well as a multiple-polypeptide complex where individual consituent polypeptides are linked by covalent or non-covalent means

The term "polypeptide" includes peptides of two or more ammo acids in length, typically having more than 5, or more than 10 or more than 20 ammo acids

The NOI may even code for a non-native protein or a compound that is of benefit to animals or humans For example, the NOI could code for a pharmaceutically active protein or enzyme such as any one of the therapeutic compounds insulin, interferon, human serum albumin, human growth factor and blood clotting factors

By way of example, the transformed organism could be used to express heterologous genes like hox from the red algae Chondrus cπspus and lipA from Aspergillus niger More, non-limiting, examples of NOIs are presented herein

Non-limiting examples of POIs encoded by the NOI, which need not be of fungal origin, include acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carboxypeptidases, catalases, cellulases, chitinases, chymosin, cutinase, deoxynbonucleases, epimerases, esterases, α-galactosidases, β-galactosidases, endo-β- glucanases, glucoamylases, glucose oxidases, α-glucosidases, β-glucosidases, glucuronidases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, lipases, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, peroxidases, phenoloxidases, phytases, polygalacturonases, proteases, rhamno- galacturonases, ribonucleases, thaumatin, transferases, transglutaminases, xylanases, or combinations thereof. The NOI may even be an antisense sequence for any of those sequences.

Other non-limiting examples of POIs include, for example, proteins involved in the regulation of cell division, for example growth factors including neurotrophic growth factors, cytokines (such as α-, β- or γ-interferon, interleukins including IL-1 , IL-2, tumour necrosis factor, or insulin-like growth factors I or II), protein kinases (such as MAP kinase), protein phosphatases and cellular receptors for any of the above.

The POI may also be an enzyme involved in cellular metabolic pathways, for example enzymes involved in amino acid biosynthesis or degradation (such as tyrosine hydroxylase), purine or pyrimidine biosynthesis or degradation, and the biosynthesis or degradation of neurotransmitters, such as dopamine, or a protein involved in the regulation of such pathways, for example protein kinases and phosphatases.

The POI may also be a transcription factors or proteins involved in their regulation, for example pocket proteins of the Rb family such as Rb or p107, membrane proteins, structural proteins or heat shock proteins such as hsp70.

The NOI and/or POI may correspond to a mutated or altered or different naturally found NOI and/or POI.

For some applications the NOI may code for a heat stable protein.

In one preferred embodiment, the NOI encodes a fungal POI, such as a fungal enzyme.

The term "fungal enzyme" includes a native fungal enzyme, as well as fungal enzymes which have been modified by amino acid substitutions, deletions, additions, or other modifications which may be made to enhance activity, thermostability, pH tolerance and the like. The POI may even be a fusion protein, for example to aid in extraction and purification.

In this respect, the NOI may be fused to a smaller or a larger part of a fungal gene encoding a stable protein. This can stabilize the POI. In such a system a cleavage site, recognized by a specific protease, can be introduced between the fungal protein and the POI, so the produced fusion protein can be cleaved at this position by the specific protease thus liberating the POI. By way of example, it has been known to introduce a site which is recognized by a KEX-2 like peptidase found in at least some Aspergilli. Such a fusion leads to cleavage in vivo resulting in production of the expressed product and not a larger fusion protein.

Examples of fusion protein partners include the maltose binding protein, glutathione-S- transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptionai activation domains) and β-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion components.

The POI may even be fused to a secretion sequence. Examples of secretion leader sequences are those originating from the amyloglucosidase gene, the α-factor gene, the α-amylase gene, the lipase A gene, the xylanase A gene. Preferably these secretion sequences are obtainable from fungi, more preferably Thermomyces lanuginosus.

Other sequences can also facilitate secretion or increase the yield of secreted POI. Such sequences could code for chaperone proteins as for example the product of Aspergillus niger cyp B gene described in UK patent application 9821198.0.

Thus, the transformed organism could be used to prepare retrievable, acceptable quantities of the expression product of the NOI.

CONTROL SEQUENCES

Aside from the promoter for use in the the present invention, one or more additional control sequences may be operably linked to the NOI. Examples of such additional control sequences include enhancers, terminator sequences and other expression regulation signals. These control sequences may be selected to be compatible with the host organism for which the expression vector is designed to be used in. INDUC1BILITY

It may also be advantageous for the promoter for use in the present invention is inducible so that the levels of expression of the NOI can be regulated during the life-time of a cell. Inducible means that the levels of expression obtained using the promoter can be regulated.

With a preferred promoter for use in the present invention, under at least some circumstances, its activity may depend on the presence of any one or more of a xylose and/or a xylan and a compound containing same (such as oligomers thereof), but wherein the activity may be supressed by the presence of glucose, or even sucrose.

EXTRACTION/PURIFICATION/ISOLATION

The POI can be extracted from the host organisms by a variety of techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption. For some applications, a preferred extraction/purification protocol may involve a centriguation step followed by, if necessary using, column chromatography such as ion-exchange or affinity chromatography.

Thus, after the desired product (e.g. the POI) has accumulated in a culture medium, a supernatant fluid containing the POI can be obtained by centrifugation or filtration. On the other hand, when the POI has accumulated in the organisms, after the cultivation, the organisms can be collected by a known method and the desired product is recovered by an appropriate method. For example, the organisms can be suspended in a buffer containing a protein denaturant such as guanidine hydrochlohde, the suspension is stirred in a cold place, and then the supernatant fluid containing the desired product is obtained by centrifugation or the like.

Alternatively, after the organisms have been suspended in a buffer, the organisms can be ground by glass beads, or broken by French press, sonication, enzymatic treatment or the like, and then the supernatant fluid is obtained by centrifugation or the like.

For separation and purification of the POI from the above supernatant fluid reference can be made to US-A-554332 where it is stated that per se known separation and purification methods can be appropriately combined. As the known separation and purification methods, there are, for example, a method utilizing a difference in solubilities (e.g., salting out, precipitation with a solvent, etc ), a method mainly utilizing a difference in molecular weights (e g , dialysis, ultrafiltration, gel filtration, etc ), a method utilizing a difference in charges (e g , ion exchange chromategraphy, etc ), a method utilizing specific affinity (e g , affinity chromatography, etc ), a method utilizing a difference in hydrophobicities (e g , reverse phase high performance liquid chromatography, etc ), a method utilizing a difference in isoelectnc points (e g , isoelectnc focusing, etc ) and the like

In a preferred aspect, the expressed POI is in a substantially isolated form It will be understood that the said POI may be mixed with carriers or diluents which will not interfere with the intended purpose of the POI and still be regarded as substantially isolated The POI may also be in a substantially purified form, in which case generally more than 90%, e g 95%, 98% or 99% of the protein in the preparation comprises the POI

GENERAL RECOMBINANT DNA METHODOLOGY TECHNIQUES

Although in general the techniques mentioned herein are well known in the art, reference may be made in particular to Sambrook et al , Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al , Short Protocols in Molecular Biology (1999) 4^th Ed, John Wiley & Sons, Inc PCR is described in US-A-4683195, US-A-4800195 and US-A-4965188

SUMMARY

In summation, the present invention relates to a transgenic Thermomyces lanuginosus

In particular the present invention relates to the use of transgenic Thermomyces lanuginosus for the expression of a NOI

DEPOSIT

The following sample has been deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St Machar Drive, Aberdeen, Scotland, AB2 1 RY, United Kingdom, on 25 November 1998

dH5αpSEK18 Deposit No NCIMB 40989 This sample comprises the xynA promoter of the present invention.

EXAMPLES SECTION

The present invention will now be described only by way of examples in which reference is made to the following Figures:

Figure 1 shows a nucleotide sequence; Figure 2 shows a plasmid map;

Figure 3 shows a plasmid map;

Figure 4 shows a plasmid map;

Figure 5 shows a plasmid map;

Figure 6 shows a plasmid map; Figure 7 shows a photograph image;

Figure 8 shows a photograph image;

Figure 9 shows a photograph image;

Figure 10 shows a photograph image;

Figure 11 shows a plasmid map; Figure 12 shows a plasmid map;

Figure 13 shows a plasmid map;

Figure 14 shows a plasmid map;

Figure 15 shows a plasmid map;

Figure 16 shows a plasmid map; Figure 17 shows a plasmid map;

Figure 18 shows a sequence;

Figure 19 shows a map; and

Figure 20 shows two vectors.

Figures 1-20 are now discussed in more detail.

Figure 1. Sequence of the xynA promoter. ATG in bold is the start codon of the xynA gene. Figure 2. Map of pSEK17. Plasmid rescued from λZAPU Thermomyces lanuginosus genomic library. pBluescript containing an approximately 2900 bp insert comprising the xynA gene.

Figure 3. Map of pSEK18. Plasmid rescued from λZAPII Thermomyces lanuginosus genomic library. pBluescript containing an approximately 2700 bp insert comprising the xynA gene.

Figure 4. Map of pSEK25. The DNA segment labeled ble carries the phleomycin resistance gene from S. hindustanus. Promoter (xynAp) and terminator (trpCt) sequences are from T. lanuginosus and A. nidulans, respectively. AP^r is ampicillin resistance gene.

Figure 5. Map of pSEK33. Expression cassette based on the 7. lanuginosus xynA gene. Polylinker (Λ/col, Xho\, Spel, Kpn\) flanked by promoter {xynAp) and terminator (xynAX) sequences. AP^r is ampicillin resistance gene.

Figure 6. Map of pSEK34. Expression cassette based on the T. lanuginosus xynA gene. Polylinker (Spel, Xho\, Λ/col, Kpnl) flanked by promoter (xynAp) and terminator (xynAt) sequences. AP^r is ampicillin resistance gene.

Figure 7. Transcription of the xynA gene is induced by xylose. Northern analysis of expression of the xynA gene from 7. lanuginosus (strain A82). Total RNA was isolated from mycelium before (0) and after transfer of mycelium to medium with 2% xylose and probed with labeled fragments comprising the xynA gene and the actin gene.

Figure 8. Transcription of the xynA gene is repressed by glucose. Northern analysis of expression of the xynA gene from 7. lanuginosus (strain A82). Total RNA was isolated from mycelium grown in medium with 2% glucose, 2% xylose or 2% glucose + xylose and probed with labeled fragments comprising the xynA gene and the actin gene.

Figure 9. PCR to confirm that transformants harbor the phleomycin resistance gene. PCR was performed on genomic DNA from putative transformants (lanes 1-13) and the untransformed parental strain (lane 14) using primers that anneals specifically to the ble gen. A67 + pSEK25 and A82 + pSEK25 are two different strains transformed with pSEK25. Lanes 15 and 16 are positive controls using two different dilutions of the vector pSEK25 as template. M, DNA molecular weight marker VI (Boehringer Mannheim). The arrow indicates the 374 bp fragment corresponding to the ble gene.

Figure 10. Southern analysis to investigate restriction pattern at the XynA locus. Genomic DNA prepared from ble positive transformants (lanes 1-6) and the untransformed parental strain (lane 7) was digested with Smal and probed with a labeled mfel fragment comprising the xynA gene. A67 + pSEK25 and A82 + pSEK25 are two different strains transformed with pSEK25. Position of the DNA molecular weight marker is indicated at the right. The 2000 bp fragment corresponds to the intact xynA gene, whereas the 800 bp and 4000 bp fragments represent the disrupted alelle.

Figure 11. Map of pxynA-amy. XynAp and xynAt are, respectively, the promoter and the terminator of the Thermomyces lanuginosus xynA gene. Amy is the ORF of the α- amyiase gene from Thermomyces lanuginosus. AP^r is ampicillin resistance gene.

Figure 12. Map of pxynA-GUS. XynAp and xynAt are, respectively, the promoter and the terminator of the Thermomyces lanuginosus xynA gene. GUS is the ORF of the glucoronidase gene from E. coli. Ap^ris ampicillin resistance gene.

Figure 13. Map of pxynA-GFP. XynAp and xynAt are, respectively, the promoter and the terminator of the Thermomyces lanuginosus xynA gene. eGFP is the ORF of the gene encoding the green flourescent protein from Aequorea victoria. AP^r is ampicillin resistance gene.

Figure 14. Map of pSEK44. Actin expression vector. Actinp and xynAt are the promoter and the terminator of, respectively, the actin gene and the xynA gene from Thermomyces lanuginosus. AP^r is ampicillin resistance gene.

Figure 15. Map of pactin-amy. Actinp and xynAt are the promoter and the terminator of, respectively, the actin gene and the xynA gene from Thermomyces lanuginosus. Amy is the ORF of the α-amylase gene from Thermomyces lanuginosus. AP^r is ampicillin resistance gene.

Figure 16. Map of pactin-GUS. Actinp and xynAt are the promoter and the terminator of, respectively, the actin gene and the xynA gene from Thermomyces lanuginosus. GUS is the ORF of the glucoronidase gene from E. coli. AP^r is ampicillin resistance gene. Figure 17. Map of pactin-GFP. Actinp and xynAt are the promoter and the terminator of, respectively, the actin gene and the xynA gene from Thermomyces lanuginosus. eGFP is the ORF of the gene encoding the green flourescent protein from Aequorea victoria. AP^r is ampicillin resistance gene.

Figure 18: Sequence of cDNA encoding Thermomyces lanuginosus OMP decarboxylase.

Figure 19: pyrG locus of Thermomyces lanuginosus

Figure 20: Vectors for expressing pyrG in Thermomyces lanuginosus.

EXPERIMENTAL

Media

Initial experiments were conducted to determine which media were suitable for cultivation and sporulation of Thermomyces lanuginosus.

As a complete medium Emerson YpSs (YPS) was finally selected:

YPS:

Yeast extract 4 g soluble starch 15 g KH₂P0₄ 1 9

MgS0₄ - 7 H₂0 0.5 g

H₂0 1000 ml

Plates: agar 20 g As minimal synthetic medium the following medium, designated MT (Minimal Thermomyces), was developed for cultivating Thermomyces lanuginosus:

MT: KH₂P0₄ 1.5 g

MgS0₄ - 7 H₂0 0.5 g

KCI 0.5 g

L-arginine* 2 g

Vishniac^* 1 ml Biotin^* 1 ml

Carbon source# 20 g

H₂0 1000 ml

Plates: agar 20 g

* Added after autoclaving from stock solutions # e.g. glucose or xylose

Vishniac:

EDTA 10 g

ZnS0₄- 7 H₂0 4.4 g

MnCI₂ - 4 H₂0 g

CoCI₂ - 6 H₂0 0.32 g

CuS0₄ - 5 H₂0 0.32 g

(NH₄)₆Mo₇0₂₄ - 4 H₂0 0.22 g

CaCI₂ - 2 H₂0 1.47 g

FeS0₄ - 7 H₂0 1 9

H₂0 1000 ml

Druq sensitivity of Thermomyces lanuαinosus

In order to develop a dominant selection system in Thermomyces lanuginosus the sensitivity of various strains to hygromycin B, phleomycin, zeocin and G418 was determined by plating different densities of spores on YPS/MT medium containing various concentrations of antibiotics. It was found that Thermomyces lanuginosus is very sensitive to phleomycin, even at only 5 μg phleomycin/ml growth is inhibited. However, to inhibit growth completely at high cell density (10⁶ spores pr 8 mm plate) 25 μg phleomycin/ml is required Hence, the bacterial ble gene, conferring resistance to phleomycin, may be used as dominant selection marker in 7 lanuginosus

Table 1 : analysis of phleomycin sensitivity of 7 lanuginosus (strain A67)

Spores were plated at different densities on YpSs/MT medium containing various concentrations of phleomycin (Sigma) Growth was scored after 5 days of incubation at 42° C, + , normal growth, ± , significantly reduced growth, - , no growth

Isolation of the Xyn-4-promoter and -terminater from Thermomyces lanuginosus

In order to have a homologous promoter to control the expression of the selection gene, ble, the xynA promoter was cloned from Thermomyces lanuginosus This was achieved in the following way

A 366 bp DNA fragment comprising the 5' end of the xynA gene (Schlacher et al , 1996) from Thermomyces lanuginosus was amplified by PCR using oligonucleotides

5'-TTTCGAGCTCCGTGGCATTCCC and

5'-CTCACAGACCAGAGCAACTGAGCACC

This PCR fragment was used as probe for screening a λZAPII Thermomyces lanuginosus genomic library made from strain A67 (Michelsen and Rasmussen, 1996)

Approximately 2 x 25 000 pfu were plated on 22 x 22 cm plates containing the following medium (per litre) 10 g NaCI, 10 g peptone, 5 g yeast extract, pH adjusted to 7 4 with NaOH The inoculated plates were incubated overnight at 37° C and two plaque lifts were made from each plate using Hybond-N membranes (Amersham). The DNA was cross-linked to the membranes by UV-light exposure for 4 minutes using a DNA Transfer Lamp (Fotodyne). The membranes were hybridized as described in the following using, as a probe, the PCR fragment specified above labeled with [³²p]-dCTP utilizing the "ready-to-go DNA labeling kit" (Pharmacia) according to manufacturers instructions. The probe was purified on elutip-D columns (Schleicher & Schuell) following the instructions given by the manufacturer.

The membranes were prehybridized for 2 hours at 65° C in 50 ml of hybridization buffer: 5 x SSC (75 mM citrate, 750 mM NaCI), 5 x Denhard solution, 0.5 % SDS and 60 μg/ml herring sperm DNA. Following prehybridization, the radiolabeled PCR-fragment was added and the membranes were incubated overnight at 65° C.

Next day the membranes were washed twice in 2 x SSC + 0,1 % SDS for 15 minutes at 65° C, twice in 1 x SSC + 0,1 % SDS for 15 minutes at 65° C and finally twice in 0.2 x SSC + 0,1 % SDS for 15 minutes at 65° C. The membranes were autoradiographed for 16 hours and three positive plaques were isolated and subjected to a second screening, essential as described above. From this screening two positive plaques were selected and converted into phagemids using the Rapid Excision Kit (Stratagene). The two plasmids were designated pSEK17 and pSEK18.

The two obtained plasmids were sequenced with Cy-5 labeled Universal and Revers primers utilizing the AutoCycle Sequencing kit (Pharmacia) and an ALF automatic DNA sequencer (Pharmacia). pSEK17 and pSEK18 harbored different inserts of the xynA gene. pSEK17 contained the complete ORF of xynA, 731 bp of the region upstream of the initiating ATG and 1323 bp of the region downstream of the Stop codon. pSEK18 harboured a part of the ORF of xynA and 2196 bp of the region upstream of the initiating ATG (the sequence is shown in Figure 1).

Map of pSEK17: - see Figure 2

Map of pSEK18: - see Figure 3 Construction of selection vector

The ble^R transformation vector, designated pSEK25, was constructed by fusing the ble gene from S. hindustanus (Gatignol et al, 1990) to the xynA promoter from Thermomyces lanuginosus and the frpC terminator from A. nidulans (Mullaney et al., 1985) as described in the following text.

A -2200 bp DNA fragment comprising the xynA promoter region was amplified from pSEK18 by PCR using pfu polymerase (Stratagene) and the oligonucleotides:

5'-ATACCATGGCTGCAAGGATTGCAAGACG and

5'-GTAATACGACTCACTATAGGGC,

thereby introducing a Λ/col restriction site in the initiating ATG of xynA. The PCR product was ligated into pCRblunt (InVitrogen) as described by the manufacturer. The obtained plasmid (pSEK22) was digested with Λ/col and Pst\ and the fragment containing the xynA promoter was ligated into Pst\IXba\ digsted pGEM3 (Promega) together with a Nco\-Xba\ fragment obtained from pAN8.1 (Mattern et al, 1988) comprising the ble gene linked to the trpC terminator.

Map of pSEK25 - see Figure 4

Construction of Expression cassettes

Two different expression cassettes, both based on the xynA gene, were constructed by fusing two different polylinkers to the

and -terminator. The two expression vectors, called pSEK33 and pSEK34, were achieved in the following way.

pSEK33: a -1300 bp DNA fragment harboring the xynA terminator region was amplified from pSEK17 by PCR using pfu polymerase and the oligonucleotides:

5'-AATCCATGGCTCGAGACTAGTGGTACCGACGTAACCTGGTGGTGATC and 5'-AATGAATTCGAGCAGGCTGTATACAGGGC.

thereby fusing a polylinker and an EcoR\ site to, respectively, the 5' and 3' end of the xynA terminator. The resulting PCR fragment was ligated into pCRbiunt as described by the manufacturer. The obtained plasmid was digested with Λ/col and EcoRI and the fragment containing the xynA terminator was isolated and ligated into Sa/l/EcoRI digested pGEM3 together with a Sa/l-Λ/col fragment obtained from pSEK22 harboring the xynA promoter.

Map of pSEK33: - see Figure 5

pSEK34:

A -2200 bp DNA fragment comprising the xynA promoter region was amplified from pSEK18 by PCR using pfu polymerase and the oligonucleotides:

5'-ATAACTAGTCACTGCAAGGATTGCAAGACG and

5'-GTAATACGACTCACTATAGGGC,

thereby introducing a Spel restriction site directly upstream of the intiating ATG of xynA. The PCR product was ligated into pCRbiunt as described by the manufacturer, giving pSEK23.

A -1300 bp DNA fragment harboring the xynA terminator region was amplified from pSEK17 by PCR using pfu polymerase and the oligonucleotides:

5'-AATACTAGTCTCGAGCCATGGGGTACCGACGTAACCTGGTGGTGATC and

5'-AATGAATTCGAGCAGGCTGTATACAGGGC,

thereby fusing a polylinker and an EcoRI restriction site to, respectively, the 5' end of and the 3' end of the xynA terminator. The resulting PCR fragment was ligated into pCRbiunt as described by the manufacturer. The obtained plasmid was digested with Spel and EcoRI and the fragment containing the xynA terminator was isolated and ligated into Sa/l/EcoRI digested pGEM3 together with a Sa/I-Spel fragment from pSEK23 containing the xynA promoter. map of pSEK34 - see Figure 6

Analysis of the xynA expression pattern

_.

To investigate under which conditions the xynA promoter is transcribed Northern analysis was made on cells incubated in various media.

2 x 10⁵ spores/ml were inoculated in 100 ml of YPS medium in 250 ml flasks and 0 incubated for 16 hours at 50° C at 200 rpm. Mycelium was harvested on myracloth, washed several times in H₂0 and transferred to either MT + 2% glucose, MT + 2% xylose or MT + 2% glucose + xylose. Mycelium was incubated at 50° C at 200 rpm and samples were withdrawn at various time points and transferred to liquid nitrogen. RNA was extracted using the RNeasy Plant mini kit (Qiagen) according to manufacturers 5 instructions except that the cells were disrupted using a FastPrep FP120 machine (BIO 101 ) instead of grinding. 10 μg aliquots of total RNA were run on 1.5% Formaldehyde agarose gels in 1 X MOPS (Sambrook et al, 1989) and transferred to Hybond-N membranes (Amersham) as instructed by the manufacturer. The membranes were hybridized to radiolabeled xynA and actin DNA probes and washed as described 0 previously (see Isolation of the -XynA-promoter and -terminater from Thermomyces lanuginosus). The membranes were autoradiographed electronically for 5-120 minutes using an Instant Imager (Packard). The XynA and actin probes were made by labeling, respectively, an /Wei fragment from pSEK17 and a PCR fragment comprising the actin gene with [³²p]-dCTP utilizing the "ready-to-go DNA labeling kit". The probes were purified on elutip-D columns following the instructions given by the manufacturer. The PCR fragment containing the actin gene was amplified from Thermomyces lanuginosus genomic DNA using taq polymerase and the oligonucleotides:

5'-TCATGATCGGTATGGGTCAG and

5'-ACGATGTTGCCGTACAGATC.

The Northern analysis revealed that the xynA promoter is repressed by glucose and induced by xylose. When cells are grown in medium with xylose as sole carbon source 5 the xynA gene is highly transcribed (see Figures 7 and 8), whereas no transcription is taken place in medium containing glucose as single carbon source (see Figure 8). In presence of both substrates the xynA promoter is repressed until the glucose is consumed (figure 8: lanes 8-10). Furthermore, it was demonstrated that induction of the xynA promoter with xylose results in a very quick response as the xynA messenger appeared within 15 minutes after shift to xylose medium (see Figure 7). After approximately 60 minutes the xynA messenger reached a constant maximum level. Hence, the xynA gene can be used to drive the expression of the selection gene, ble, by incubating mycelium on MT medium containing xylose as carbon source.

Transformation

To test whether pSEK25, carrying the ble gene under the control of the xynA promoter, conferred phleomycin resistance to Thermomyces lanuginosus a transformation procedure was developed based on the lithium acetate protocol described for yeast (Ito et al., 1983).

To produce conidia for transformation mycelium was inoculated on YPS plates, incubated at 50° C for 5-10 days and spores were harvested by rinsing the culture with 5 ml of 0.9 % NaCI and 0.05 % Tween-20.

7 x 10⁵ spores/ml from Thermomyces lanuginosus (strains A67 and A82) were inoculated in 150 ml of YPS and incubated at 50° C for 3.5 hours. Germinated spores were collected by centrifugation and washed once in TE (10 mM tris, 1 mM EDTA, pH 7.5) and once in 0.1 M LiOac (pH 7.0). For one transformation 2 X 10⁷ germinated spores were used. Spores were resuspended in 400 μl LiOac and incubated for 30 minutes at 45° C with gently shaking. 20 μg of tranforming DNA, pSEK25, was added and incubated further 30 minutes at 45° C. Next 4 ml of 40 % PEG 4000 in 0.1 M LiOac was added and incubation was continued for another hour. Finally, cells were heat shocked at 60° C for 12 minutes, washed twice in H₂0 and plated on MT + 2 % xylose + 25 γ phleomycin. Transformants usually appeared after 4-5 days of incubation at 42° C. Typically, 0.1-0.3 transformants per μg of DNA were obtained using pSEK25 and 2 x 10⁷ spores. Of those typically less than 50 % were true transformants.

The transformants were stable with respect to maintenance of phleomycin resistance. After two rounds of sporulation on non-selective medium (YPS) all transformants tested were found to retain resistance to phleomycin. Analysis of phleomycin resistant transformants

Integration of transforming DNA sequences into the genome of the putative Thermomyces lanuginosus transformants was confirmed by PCR and Southern blot. Genomic DNA was prepared in the following way:

Stable transformants were inoculated in YPS and incubated overnight at 50° C and 200 rpm. 100 mg of mycelium was harvested from each culture, grinded and resuspended in 900 μl 5 x TE and 90 μl 10 % SDS. Proteins and cell debris were precipitated by addition of 100 μl 5 M KAc. After centrifugation the supernatant was transferred to a qiagen tip-20 (Qiagen) and DNA was purified following the instructions given by the manufacturer.

To investigate whether the selecting gene, ble, was integrated in the genome PCR was performed on 10 ng of the genomic DNA extracted from the transformants using the oligonucleotides:

5'-CCATGGCCAAGTTGACCAGTGCCG and

5'-TCAGTCCTGCTCCTCGGCCACGAAG.

These oligonucleotides anneal to the ble gene and give rise to a 374 bp fragment. PCR was carried out with Tag polymerase (Bόhringer Manheim), buffer F (Invitrogen), 1 μM of each primer and 250 μM of each dNTP using a MasterCycler Gradient (Eppendorf). The cycle parameters were as follows: 30 cycles of: denaturation 1 min. 94° C; annealing 1 min. 55° C; extension 1 min. 72° C. Hot Start PCR was applied.

In Figure 9 is shown the result of the PCR analysis. Several of the stable transformants harboured the ble gene as a 374 bp was amplified by the PCR reaction showing that the fungi was transformed with pSEK25 (see Figure 9; lanes 1 , 3, 4, 6, 11 , 12). No PCR product appeared when genomic DNA from the untransformed parental strains was used as template (Figure 9; lane 14, data not shown).

To investigate whether pSEK25 was integrated in the xynA locus by homologous recombination a Southern blot was performed. 2 μg of genomic DNA from positive transformants, as tested by PCR, was digested with Smal, electrophoresed through a 1

% agarose gel and blotted onto Hybond-N membranes (Amersham). The membranes were hybridized to a radiolabeled xynA DNA probe and washed as described previously (see Isolation of the yn \-promoter and -terminater from Thermomyces lanuginosus) The membranes were autoradiographed electronically for 6 hours using an Instant Imager (Packard) The XynA probe was made by labeling an Mfe\ fragment from pSEK17 with [³²p]-dCTP utilizing the "ready-to-go DNA labeling kit" The probes were purified on elutip-D columns following the instructions given by the manufacturer

In most of the transformants the restriction pattern at the xynA locus was identical to the untransformed parental strain indicating that pSEK25 had integrated randomly into the chromosome (see Figure 10, lanes 2, 3, 4, 5, 6,) However, in one of the transformants pSEK25 had integrated in the genome by homologous recombination as the xynA locus was distrupted (see Figure 10, Lane 1) These observations indicate that transforming DNA sequences most often is integrated randomly into the chromosomes as observed in other filamentous fungi

In conclusion, Thermomyces lanuginosus can be made permeable to transforming DNA by treating germinated spores with LiOac Stable transformants can be obtained using a vector expressing the bacterial ble gene from the homologous promoter, xynA, and selecting on media containing phleomycin

Further Examples of promoter/NOI combinations for the preparation of transformed Thermomyces lanuginosus:

Example A Expression of Thermomyces lanuginosus α-amylase (54 kD) from the xynA promoter

The ORF of the α-amylase gene is amplified by PCR using two oligonucleotides

5'-ACTAGTATGAAGTCTCTCGCCGCAATTGC and δ'-CTCGAGTCACGCCGACGCACACAGACC

specific for the amylase gene

The resulting PCR product is digested with Spel and Xho\ restriction enzymes and is ligated into Spe\/Xho\ digested pSEK34, giving pxynA-amy (see Figure 1 1) This vector is co-tranformed with pSEK25 (selecting plasmid) into various Thermomyces lanuginosus strains (preferably A67 and A82), as described previously. Assays are then conducted to determine whether active α-amylase is produced on medium supplemented with xylose.

Example B

Expression of E. coli glucoronidase (68 kD) from the xynA promoter.

The ORF of the glucoronidase gene is amplified by PCR using two oligonucleotides:

5'-ACTAGTATGTTACGTCCTGTAGAAAC and

5'-CTCGAGTCATTGTTTGCCTCCCTGC

specific for the glucoronidase gene. The resulting PCR product is digested with Spel and Xho\ restriction enzymes and is ligated into SpeUXhol digested pSEK34, giving pxynA- GUS (see Figure 12).

This vector is co-tranformed with pSEK25 (selecting plasmid) into various Thermomyces lanuginosus strains (preferably A67 and A82), as described previously. Assays are then conducted to determine whether active glucoronidase is produced on medium supplemented with xylose.

Example C

Expression of GFP (green flourescent protein, 27 kD) of Aequorea victoria from the xynA promoter.

The ORF of GFP is isolated by digestion of pEGFP (Clontech) with Λ/col and Xbal and the resulting restriction fragment is ligated into Λ/col/Spel digested pSEK33, giving pxynA- GFP (see Figure 13).

This vector is co-tranformed with pSEK25 (selecting plasmid) into various Thermomyces lanuginosus strains (preferably A67 and A82), as described previously. Assays are then conducted to determine whether active GFP is produced on medium supplemented with xylose. Example D

Vector construction

In order to investigate if the same proteins can be expressed from a constitutive promoter an expression vector based on the actin promoter from Thermomyces lanuginosus is constructed

The promoter region of the actin gene is amplified by PCR using two oligonucleotides

5'-TTAGTCGACATTCGTATTTGTACTCGTAGACTGGCG and

5'-GGGGGAGGAGCAGGACAAGC

specific for the actin gene The resulting PCR product is digested with Sa/I and Λ/col restriction enzymes and is ligated into Sa/l/Λ/col digested pSEK34, giving pSEK44 (see Figure 14)

Example E Expression of Thermomyces lanuginosus α-amylase (54 kD) from the actin promoter

The ORF of the α-amylase gene is amplified by PCR using two oligonucleotides

5'-CCATGGAGTCTCTCGCCGCAATTGC and

5'-CTCGAGTCACGCCGACGCACACAGACC

specific for the amylase gene The resulting PCR product is digested with Λ/col and Xho\ restriction enzymes and is ligated into Nco\IXho\ digested pSEK44, giving the construct pactin-amy (see Figure 15)

This vector is co-tranformed with pSEK25 (selecting plasmid) into various Thermomyces lanuginosus strains (preferably A67 and A82), as described previously Assays are then conducted to determine whether active α-amylase is produced constitutively Example F

Expression of E. coli glucoronidase (68 kD) from the actin promoter.

The ORF of the glucoronidase gene is amplified by PCR using two oligonucleotides

5'-CCATGGTACGTCCTGTAGAAAC and

5'-CTCGAGTCATTGTTTGCCTCCCTGC

specific for the glucoronidase gene. The resulting PCR product is digested with Λ/col and Xho\ restriction enzymes and is ligated into Nco\IXho\ digested pSEK44, giving pactin- GUS (see Figure 16).

This vector is co-tranformed with pSEK25 (selecting plasmid) into various Thermomyces lanuginosus strains (preferably A67 and A82), as described previously. Assays are then conducted to determine whether active glucoronidase is produced constitutively.

Example G

Expression of GFP (green flourescent protein, 27 kD) of Aequorea victoria from the actin promoter.

The ORF of GFP is isolated by digestion of pEGFP (Clontech) with Λ/col and Xba\ and the resulting restriction fragment is ligated into Λ/col/Spel digested pSEK44, giving pactin- GFP (see Figure 17).

This vector is co-tranformed with pSEK25 (selecting plasmid) into various Thermomyces lanuginosus strains (preferably A67 and A82), as described previously. Assays are then conducted to determine whether active GFP is produced constitutively.

Example H

Homologous transformation system for Thermomyces lanuginosus

In order to develop a homologous transformation system the gene encoding the OMP decarboxylase (pyrG) was cloned from Thermomyces lanuginosus. The sequence is presented in Figure 18. This gene complements pyrG mutants defective in the uridine anabolism and can therefore be used for selection of prototrophic transformants. Using a cDNA library for expression in E. coli we isolated a pyrG homolog by complementing an E. coli pyrF mutant, as described as follows.

An E. coli pyrF mutant (CN1685: F^" ara Δ(gpt pro lac) thi pyrF::TN5) was transformed with a Thermomomyces lanuginosus cDNA library made in the E. coli expression vector pSPORT ~ 1.5 X 10⁶ transformants were obtained and they were plated directly on minimal medium + IPTG to induce the lac promoter. - 1.000 colonies were recovered on selecting medium. In order to distinguish between revertants and "true transformants" 50 colonies were picked and plated on minimal medium +/÷ IPTG. 10 colonies, that only grow on plates containing IPTG, were picked for colony hybridisation using the A. niger pyrG gene as probe. 7 positive clones hybridised to the probe and were chosen for sequencing. They all contained the same gene, showing 67 % identity to the pyrG gene from A. niger. Using a PCR/adaptor based genomic walking approach 3000 bp and 1500 bp of, respectively, the leader and the trailer region was cloned (figure 19). Thus, we have cloned a pyrG cassette for transforming Thermomyces lanuginosus strains defective in uridine anabolism. Based on this cassette two vectors were constructed comprising, respectively, 4721 bp and 2398 bp of the pyrG locus (figure 20).

Isolation of mutants defective in uridine anabolism

Pyrimidine auxotrophic mutants of Thermomyces lanuginosus were isolated as described in the following.

Vegetative spore suspensions (10⁷ spores/ml) of strains A82 and A67 were exposed to UV-light. Aliquots of the sporesuspensions were plated on rich medium (YPS + 10 mM uridine). After sporulation these recycled spores were harvested and plated on minimal medium supplemented with 10 mM uridine and 1 g/l 5-FOA. Colonies appearing after 5 days of incubation at 42°C were tested for uridine requirement. Approximately 100 5- FOA resistent colonies were obtained from each of the strains. Among these 1-4 % were true uridine auxotrophic mutants. A total of 5 uridine auxotrophs, four derived from strain A82 and one derived from strain A67, were isolated and are listed in the Table below.

To identify the block in pyrimidine biosynthesis in the uridine auxotrophs, crude extracts were prepared and assayed for OMP decarboxylase and OPRTase activities. As shown in the Table four of the five auxotrophs is deficient in OMP decarboxylase activity and harbour a mutation in the gene we call pyrG, whereas the last auxothrophs lacks OPRTase activity. The four pyrG strains can be used for transformation using the Thermomyces lanuginosus pyrG gene, we previously isolated, as selection marker.

Table: Orotate phosphoribosyl transferase (OPRTase) and orotidine-5'- monophosphate decarboxylase (OMPase) activities. AB6.4: Aspergillus niger pyrG strain. AB6.4::70.3: AB6.4 transformed with pyrG gene. A82: Thermomyces lanuginosus wild type strain. A82-22, -27, -38, -41 and A76-pyrG: Thermomyces lanuginosus uridine auxothrophic strains. SUMMARY

The present invention discloses a transformation system for the fungus 7. lanuginosus which can be used in the expression of recombinant proteins, especially enzymes. In this respect, we were able to successfully transform the thermophillic fungi Thermomyces lanuginosus using a phleomycin resistance gene, as a dominant selectable marker, linked to the homologous xynA promoter and the trpC terminator from Aspergillus nidulans. Transformation was achieved using the lithium acetate method on germinated spores, and transformants were obtained in a frequency of 0,1-0,3 per μg of plasmid DNA. Transforming DNA was predominantly integrated randomly in the genome and transformants were mitotically stable.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

By way of example, any one or more of the vectors or components thereof presented herein may be used to transform a suitable fungus, in particular a thermophilic fungus. Examples of suitable fugal hosts include any member belonging to the genera Thermomyces, Acremonium, Aspergillus, Penicillium, Mucor, Neurospora, Trichoderma and the like - such as Thermomyces lanuginosis, Acremonium chrysogenum, Aspergillus niger, Aspergillus oryzae, Aspergillus awamori, Penicillinum chrysogenem, Mucor javanious, Neurospora crassa, Trichoderma viridae and the like. References

1. Gatignol, A., Durand, H. and Tiraby, G. (1990) FEBS Lett. 230: 171.

2. Ito, H., Fukuda, Y., Murata, K. and Kimura, A. (1983) J. Bactehol. 153: 163-168.

3. Mattern, IE., Punt, PJ. and van den Hondel CAMJJ. (1988) Fungal Gen. Newslett. 35: 25.

4. Michelsen, B. and Rasmussen P. (1996) WO9601323.

5. Mullaney, EJ., Hamer, JE., Roberti, KA., Yelton, MM. and Timberlake, WE. (1985) Mol. Gen. Genet. 199: 37-45.

6. Sambrook, J., Fritsch, E. and Maniatis, T. (1989) Molecular cloning: a laboratory manual, 2^nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NN.

7. Schlacher, A., Holzmann, K., Hayn, M., Steiner, W. and Schwab, H. (1996). J. Biotech. 49: 211-218.

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

Claims

1. A transformed T. lanuginosus cell comprising a NOI.

2. A transformed 7. lanuginosus cell according to Claim 1 wherein the NOI codes for a heat stable protein.

3. A transformed T lanuginosus cell according to Claim 1 or Claim 2 wherein the NOI is operably linked to a promoter.

4. A transformed T. lanuginosus cell according Claim 3 wherein the promoter is a fungal promoter.

5. A transformed T. lanuginosus cell according to Claim 4 wherein the promoter is derived or derivable from T. lanuginosus.

6. A method for producing a POI which comprises culturing a transformed T. lanuginosus cell comprising a NOI encoding a POI operably linked to a promoter under conditions which allow expression of the POI.

7. A method according to Claim 6 wherein the POI is secreted to the culture medium, and optionally wherein said POI is isolated and/or purified.

8. A method according to Claim 6 or Claim 7 wherein the NOI is an NOI as defined in any one of claims 2 to 7.

9. A process for transforming Thermomyces lanuginosus comprising contacting germinated spores of Thermomyces lanuginosus with lithium acetate.

10. A transformed Thermomyces lanuginosus comprising any one or more of: an NOI, the promoter having the sequence presented as SEQ ID No.1 or a variant, derivative or fragment thereof, a trpC terminator, and the ble gene or a fragment thereof capable of imparting resistance to phleomycin.