WO1999051756A2 - Promoter and constructions for expression of recombinant proteins in filamentous fungi - Google Patents

Abstract

The invention relates to novel promoters of the glutamate dehydrogenase (gdh) genes from Aspergillus awamori and related aspergilli as well as new DNA sequences encoding glutamate dehydrogenases from Aspergillus awamori. The invention also relates to the use of the promoters of the gdh genes from fungus of the genus Aspergillus for the expression of recombinant proteins in filamentous fungi.

Description

Promoter and constructions for expression of recombinant proteins in filamentous funσi

This invention relates to improvements in the expression of proteins, particularly of fusion proteins, by recombinant DNA technology, using filamentous fungi as the host. These improvements refer mainly to the use of a new promoter and new DNA constructions containing it.

DESCRIPTION OF THE PRIOR ART

Filamentous fungi are known to produce in nature a wide range of homologous proteins in large amounts. For this reason, filamentous fungi have been regarded as attractive hosts for the expression of recombinant proteins. For instance, Aspergillus awamori has been used for the production of recombinant proteins such as bovine chymosin and human lactoferrin.

Some recombinant proteins, however, have proved to be very difficult to express in filamentous fungi. This is the case for example of interleukin-6 and thaumatin. The thaumatins are proteins with a very sweet taste and the ability to increase the palatability of food. In industry they are currently extracted from the arils of the fruit of the plant Thaumatoccocus daniellii Benth (M.Witty, J.D. Higginbotham, Thaumatin ,1994, CRC Press, Boca Raton, Florida). Thaumatins can be isolated from these arils in at least five different forms (I, II, III, b and c) , thaumatins I and II being the most abundant types in the arils. Despite its advantages, industrial use of thaumatins of plant origin is very limited because of the extreme difficulty involved in obtaining the fruit from which it is extracted. Attempts have been made to produce thaumatins by genetic engineering in different hosts such as bacteria, yeasts and transgenic plants, but until now the results have been considered disheartening and thus the thaumatin available to industry is very scarce and expensive. European patent EP 684312 describes a process for preparing recombinant thaumatin in filamentous fungi. One problem of this process is that the yields obtained are low in comparison with those needed for industrial production of thaumatins.

It is known in the art that yields of recombinant proteins can be improved when the recombinant protein of interest is expressed as a fusion with another protein, and when expression of this cassette is driven by a strong fungal promoter. This other protein, named "carrier protein", is usually a highly expressed protein of fungal origin. Up to now, the most frequently used expression system involves the glucoamylase promoter and gene from Aspergillus awamori as the promoter and the carrier protein, respectively (P.P. Ward et al., Biotechnology 1995, vol. 13, pp. 498-502). However, in some cases the use of this expression system does not lead to high levels of the desired recombinant protein. One of these specially problematic cases is the expression of recombinant thaumatin in filamentous fungi.

In view of the above, it is clear that there is the need to provide new and more efficient expression systems that allow the production of higher concentrations of those proteins that are difficult to express in filamentous fungi, such as thaumatins. This goal is achieved with the new promoter and DNA constructions provided in the present invention, as explained below.

DESCRIPTION OF THE INVENTION

The present invention provides a new expression system that makes use of the promoter from the glutamate dehydrogenase (gdh) gene from filamentous fungi of the genus Aspergillus, particularly from Aspergillus awamori .

One of the objects of the present invention is a new promoter for the expression of recombinant proteins in filamentous fungi that comprises a nucleotide sequence - or a complementary strand thereof - selected from the group consisting of: (a) the nucleotide sequence numbered 1-740 in the enclosed SEQ ID No. 1; (b) a nucleotide sequence that is analogous to that defined in (a) ; and (c) a nucleotide sequence that hybridizes under stringent conditions to that defined in (a) . Particularly preferred is the promoter comprising the sequence defined in (a), i.e. the nucleotide sequence numbered as 1-740 in SEQ ID No. 1, which corresponds to the gdhA promoter of the glutamate dehydrogenase A gene from Aspergillus awamori .

Although glutamate dehydrogenase A disclosed herein is the first glutamate dehydrogenase identified and described in the filamentous fungus Aspergillus awamori , there may exist other glutamate dehydrogenases in Aspergillus awamori . The novel nucleotide sequence of the Aspergillus awamori gdhA promoter and/or gene shown in SEQ ID No . 1 or a portion thereof can be used as a probe for the identification and isolation of other homologous promoters/genes of glutamate dehydrogenases in Aspergillus awamori as well as in other organisms, preferably in filamentous fungi, more preferably in fungi of the genus Aspergillus , still more preferably in Aspergillus awamori and Aspergillus niger , and specially in Aspergillus awamori , following the teachings of the present invention. Consequently, the present invention is not limited to the specific gdhA promoter from Aspergillus awamori disclosed herein but also relates to the promoter of any glutamate dehydrogenase gene from a fungus of the genus Aspergillus with the proviso that it is not from Aspergillus nidulans . Examples of said Aspergilli include Aspergillus awamori , Aspergillus niger, Aspergillus oryzae and Aspergillus soiae In a preferred embodiment, the invention relates to a promoter of a glutamate dehydrogenase gene from Aspergillus awamori or Aspergillus niger. In a more preferred embodiment, the invention relates to a promoter of a glutamate dehydrogenase gene from Aspergillus awamori . The use of the novel nucleotide sequence shown in SEQ ID No. 1 or a portion thereof as probe is also a object of the present invention. The term "a portion thereof" denotes any part of the nucleotide sequence of SEQ ID No .1 that is functional as a probe.

Another object of the present invention is a new DNA sequence, purified and isolated, that encodes a glutamate dehydrogenase protein and that comprises a nucleotide sequence - or a complementary strand thereof - selected from the group consisting of: (a) the nucleotide sequence numbered 741-2245 in the enclosed SEQ ID No. 1; (b) a nucleotide sequence that is analogous to that defined in

(a) ; and (c) a nucleotide sequence that hybridizes under stringent conditions to that defined in (a) . In a preferred embodiment, the nucleotide sequence encoding a glutamate dehydrogenase is the sequence defined in (a), i.e. the nucleotide sequence numbered as 741-2245 in SEQ ID No. 1. The present invention is not limited, however, to the specific gdhA gene from Aspergillus awamori disclosed herein but also relates to any glutamate dehydrogenase gene from a fungus of the genus Aspergillus with the proviso that it is not from Aspergillus nidulans . In a preferred embodiment, the invention relates to the DNA sequences encoding glutamate dehydrogenase from Aspergillus awamori or Aspergillus niger . In a more preferred embodiment, the invention relates to the DNA sequences encoding glutamate dehydrogenase from Aspergillus awamori .

Another object of the invention are the novel proteins encoded by any of the DNA sequences defined above . In a preferred embodiment, this protein has the amino acid sequence shown in the enclosed SEQ ID No. 2. But are also included in the present invention any glutamate dehydrogenase from a fungus of the genus Aspergillus with the proviso that it is not from Aspergillus nidulans , more preferably a glutamate dehydrogenase from Aspergillus awamori or Aspergillus niger. and still more preferably a glutamate dehydrogenase from Aspergillus awamori . The invention further relates to the use of the glutamate dehydrogenase promoters above described for the expression of recombinant proteins in filamentous fungi . Certain glutamate dehydrogenases from several microorganisms are already known and their genes have been disclosed, in particular the glutamate dehydrogenase A (gdhA) gene from Aspergillus nidulans (A.R. Hawkins et al . , Mol. Gen. Genet. 1989, 218(1), pp. 105-111). However, to the best of our knowledge, there has been no disclosure up to now of the expression of a recombinant protein making use of the gdhA promoter from A_^. nidulans nor has it ever been mentioned that it might be useful for improving the expression of recombinant proteins in filamentous f ngi. As shown in the examples below, the glutamate dehydrogenase promoter from Aspergillus awamori has proven to be very strong in promoting transcription of heterologous genes. Therefore, this promoter as well as related ghd promoters from Aspergilli are expected to drive high-level transcription of genes and thus are expected to be of use in the expression of recombinant proteins in filamentous fungi . It is thus a further object of the present invention the use of a promoter from a glutamate dehydrogenase gene from a fungus of the genus Aspergillus for the expression of recombinant proteins in filamentous fungi. Preferably, the gdh promoter is from a fungus of the genus Aspergillus with the proviso that it is not from Aspergillus nidulans . more preferably it is from Aspergillus awamori or Aspergillus niger , still more preferably it is from Aspergillus awamori, and particularly preferably it is one of the novel gdh promoters described above .

There is in principle no limitation on the desired recombinant protein to be expressed. Examples of such desired proteins (which term, as used herein, includes proteins and smaller polypeptides) include, but are not limited to, enzymes, hormones, cytokines, growth factors, structural proteins, plasma proteins and others. A non- limiting list of examples of proteins that can be expressed includes human proteins such as interferons, interleukins, tissue plasminogen activator, serum albumin, growth hormone, and growth factors . Other proteins can be of non-human origin such as lipases of both fungal and non-fungal origin, proteases, thaumatins, bovine chymosin, etc. Polypeptides, which can be of human and non-human origin, include calcitonin, glucagon, insulin, nerve growth factor, epidermal growth factor, the anticoagulant Hirudin and analogs such as R3-hirulog.

A further object of the present invention are the DNA constructions that comprise: a) a promoter from a glutamate dehydrogenase gene from a fungus of the genus Aspergillus ; b) a DNA sequence encoding a protein normally expressed from a filamentous fungus or a portion thereof; c) a DNA sequence encoding a cleavable linker peptide; and d) a DNA sequence encoding a desired protein. In a preferred embodiment, the promoter under a) comprises a gdh promoter from a fungus of the genus Aspergillus with the proviso that it is not from Aspergillus nidulans , more preferably it is from Aspergillus awamori or Aspergillus niger, still more preferably it is from Aspergillus awamori , yet more preferably it comprises any of the new promoters described above, and more particularly it comprises the nucleotide sequence 1-740 in SEQ ID No . 1. The DNA sequence under b) encodes a protein normally expressed from a filamentous fungus or a portion thereof that is functional, i.e. that is capable of producing increased secretion of the desired protein. Examples of such protein under b) include glucoamylase, oc- amylase and aspartyl proteases from Aspergillus awamori , Aspergillus niger, Aspergillus oryzae and Aspergillus soiae, cellobiohydrolase I, cellobiohydrolase II, endoglucanase I and endoglucanase III from Trichoderma species, glucoamylase from Neurospora and Humicola species, the protein B2 from Acremonium chrysogenum and a glutamate dehydrogenase from a filamentous fungi. In a preferred embodiment, the DNA sequence under b) encodes a protein or portion thereof selected from the group consisting of: i) glucoamylase from Aspergillus awamori , Aspergillus niger, Aspergillus orvzae or Aspergillus soiae; ii) B2 from Acremonium chrvsogenum; and iii) a glutamate dehydrogenase from a filamentous fungi; more preferably, the DNA sequence under b) encodes a protein or portion thereof selected from the group consisting of: i) glucoamylase from Aspergillus awamori , Aspergillus niger, Aspergillus orvzae or Aspergillus soiae; ii) B2 from Acremonium chrvsogenum,- and iii) a glutamate dehydrogenase from Aspergillus awamori or Aspergillus niger . The DNA sequence under c) encodes a cleavable linker peptide; as used herein, cleavable linker peptide means a peptide sequence which under certain circumstances allows the separation of the sequences bordering the cleavable linker, for example sequences that are recognized and cleaved by a protease or cleaved after exposure to certain chemicals . In a preferred embodiment, the DNA sequence under c) contains a KEX2 processing sequence. As mentioned above, the desired protein under d) can be in principle any recombinant protein. In a preferred embodiment, the DNA sequence under d) encodes thaumatin; particularly preferred constructions for the preparation of thaumatin include those wherein the DNA sequence encoding thaumatin under d) is the synthetic gene encoding thaumatin II coming from plasmid pThlX, which is disclosed in EP 684312.

Although in the context of the present invention it is preferred, when expressing a desired protein, to use the gdh promoters in fusion constructions, it is also possible to use a gdh promoter to express directly a desired protein. Therefore, it is a further object of the present invention the new DNA constructions that comprise a gdh promoter from a fungus of the genus Aspergillus operatively linked to a DNA sequence encoding the protein that it is desired to express. In a preferred embodiment, the gdh promoter is from a fungus of the genus Aspergillus with the proviso that it is not from Aspergillus nidulans , more preferably it is from Aspergillus awamori or Aspergillus niger, still more preferably it is from Aspergillus awamori, yet more preferably it is one of the new promoters described above, and more particularly it comprises the nucleotide sequence 1-740 in SEQ ID No. 1.

As will be obvious to those skilled in the art of recombinant DNA technology, all the above DNA constuctions may additionally contain other elements which include, but are not limited to, signal sequences, termination sequences, polyadenylation sequences, selection sequences, sequences that allow the replication of the DNA, etc. There is no limitation on the number and nature of these additional sequences and any of the known sequences for exerting these functions can in principle be used in the constructions according to the present invention. For example, as a signal sequence functional as a secretory sequence we can mention the signal sequences from glucoamylase, -amylase and aspartyl proteases from Aspergillus awamori , Aspergillus niger, Aspergillus orvzae and Aspergillus soiae, signal sequences from cellobiohydrolase I, cellobiohydrolase II, endoglucanase I and endoglucanase III from Trichoderma species, signal sequences from glucoamylase from Neurospora and Humicola species and the signal sequence from the protein B2 from Acremonium chrvsogenum. In general it is preferred to use as signal sequence those derived from proteins secreted by the filamentous fungus used as expression host to express and secrete the recombinant protein or, in case fusion constructions are used, also those derived from the protein used as carrier protein. A termination sequence is a nucleotide sequence which is recognized- by the expression host to terminate transcription. Examples include the terminators from the A. nidulans trpC gene, the A_^ awamori , A. niger, A. orvzae or A. soiae glucoamylase gene, the A_^ awamori , A. niger , A. orvzae or A_^ soiae α-amylase genes and the Saccharomvces cerevisiae cycl gene. A selection sequence is a sequence useful as selection marker to allow the selection of transformed host cells. In principle any known selection marker for the filamentous fungus that is intended to be used as host can be employed. Examples of such selection markers include genes confering resistance to a drug such as an antibiotic (e.g. hygromycin or phleomycin) as well as auxotrophic markers such as argB, trpC, niaD and pyrG. A polyadenylation sequence is a nucleotide sequence which when transcribed is recognized by the expression host to add polyadenosine residues to transcribed mKNA. Examples include the polyadenylation sequences from the A__ nidulans trpC gene, the _^ awamori , A. niger, A. orvzae or A_^. soiae glucoamylase genes and the Mucor miehei carboxyl protease gene.

The present invention also relates to the filamentous fungus cultures capable of producing a recombinant protein that have been transformed with plasmids that contain any of the DNA constructions mentioned above. Examples of species of filamentous fungi that may be used as expression hosts include the following genera: Aspergillus , Trichoderma, Neurospora , Penicillium, Acremonium, Cephalosporium, Achlva , Phanerochaete , Podospora, Endothia , Mucor, Fusarium, Humicola, Cochliobolus , Rhizopus and Pyricularia. Particularly preferred are those cultures wherein the filamentous fungus is selected from a fungus of the genus Aspergillus , and more preferably it is selected from Aspergillus awamori, Aspergillus niger , Aspergillus oryzae, Aspergillus nidulans or Aspergillus soiae. In another preferred embodiment, the recombinant protein produced is thaumatin.

A further object of the present invention is to provide a process for producing a recombinant protein in a filamentous fungus that comprises the following steps: a) preparation of an expression plasmid that contains a DNA construction as defined above; b) transformation of a strain of filamentous fungus with said expression plasmid; c) culture of the transformed strain under appropriate nutrient conditions to produce the desired protein, either intracellularly, extracellularly or in both ways simultaneously; and d) depending on each case, separation and purification of the desired protein from the fermentation broth. Preferred is the process wherein the recombinant protein produced is thaumatin..

The accompanying examples describe the identification and isolation of the glutamate dehydrogenase A gene and its promoter region from Aspergillus awamori . This was achieved using a probe from Neurospora crassa. The selection of a suitable DNA fragment from the glutamate dehydrogenase gene in Neurospora crassa to be used as a probe to get the homologous gene in Aspergillus awamori is not, however, straightforward. In this case, there were no clear homology sequences that could be detected, and therefore what was used was a 2.6 kb BamHI fragment that contained the Neurospora crassa gdh gene. This is a large fragment of DNA, and is certainly not the optimal size fragment. Ideally, one wants to use as a probe a highly homologous fragment of DNA, no more than 200-300 bp long. Here a much larger fragment (2600 bp) with undefined homology was used. Yet the present inventors managed to clone a sequence that was later on proven to be the gdh from Aspergillus awamori .

The accompanying examples also describe the application of the above described novel promoters ^' and DNA constructions to the expression of the recombinant protein thaumatin in the filamentous fungus Aspergillus awamori . As shown in these examples, and as illustrated graphically in Figure 12, the expression system of the present invention offers several advantages over the prior art systems. On the one hand, it allows to reach concentrations of expressed protein of about 100 mg/1, which are one order of magnitude higher than the best described (for example, using the process described in EP 684312, concentrations of about 5-10 mg/1 are attained; see I. Faus et al . , Appi . Microbiol . Biotechnol . , 1998, vol. 49, pp. 393-398) . On the other hand, for a same carrier protein and a same fermentation time, the use of the promoter of the present invention leads to higher concentrations of expressed protein. And last but not least, with the constructions of the present invention it is possible to use a more economical nitrogen source (ammonium sulfate) than the one that is commonly used (asparagine) .

DEFINITIONS

The term "promoter" means a DNA sequence operative in a filamentous fungus capable of promoting transcription of a coding region when operatively associated therewith.

The term "recombinant protein" means a protein that is not expressed under standard normal conditions by the host, and that is only expressed by the host as a result of the introduction into said host of a DNA sequence that allows for the expression of said recombinant protein. This recombinant protein can be fungal or non-fungal, and it can even be found in the non-recombinant host .

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 , parts A, B and C. Schematic representation of the steps involved in the construction of the B2KEX expression cassette.

Figure 2. Restriction map of a 28.7 kb region of A_^ awamori DNA including the gdhA gene. Map of phages FANl and FAN2. Thick lines indicate the overlapping zone between the two phages containing the gdhA gene. pBlO, pB5.5 and PB1.7 indicate the DNA fragments subcloned in the corresponding plasmids. B = BamHI, S = Sal I.

Figure 3. Restriction map of the 2.1 kb Xbal-BamHI fragment from pB5.5 plasmid that was sequenced. The 3' end of the gdhA gene was contained in the left region of the insert in pB1.7. B = BamHI, E = EcoRI, EV = EcoRV, P = Pstl, S = Sail, X = Xbal.

Figure 4 , parts A and B. Alignment of the deduced amino acid sequences of NADP-specific glutamate dehydrogenases of A. awamori, I _ nidulans (Genebank accession number P18819), N. crassa (P00369), £ _ cerevisiae (P07262), £L occidentalis (P29507), . bisporus (P54387) , S_^_ tvphimurium (P15111) , E_^ coli (P00370) and C _ glutamicum (P31026) . Identical amino acids are shadowed. Motifs a-i with several consecutive conserved residues are overlined.

Figure 5. Complementation of the gdhA mutation in two strains of A_^. nidulans with the gdhA gene of A_^ awamori . Part A: 1, . nidulans A686 mutant; 2, transformant A686-4; 3, transformant A686-6; 4, transformant A686-7. Part B. 1, A. nidulans A699 mutant; 2, transfomant A699-2; 3, transformant A699-3; and 4, transformant A699-4.

Fi^gure 6. Primer extension identification of the 5' end of the gdhA gene transcript. One protected band (arrow) is observed in the lane corresponding to the extension reaction (lane Pe) . G, A, T, C lanes correspond to the sequencing reactions of Ml3 phage from the -40 primer.

Figure 7. Northern blot analysis of the transcripts of the gdhA and .^β-^'actin genes. A: hybridization with a probe internal to the gdhA gene (0.694 kb PvuII fragment). B: hybridization with the β-actin gene of A_^ nidulans as control . Figure 8. Slot Blot analysis of the trancript of the A. awamori gdhA gene, during the course of a fermentation in MDFA medium with 1% glucose and 10 mM ammonium sulfate (part A) . For comparative purposes, the transcript of the β-actin gene in the same RNA sample was also studied. Part B: relative level of the expression of the gdhA to the β-actin gene. Part C: NADP-dependent glutamate dehydrogenase activity in the same cultures from where the mRNAs were extracted.

Figure 9. Slot Blot analysis of the transcript of the A. awamori gdhA gene during the course of a fermentation in MDFA medium with different nitrogen sources (part A) . The medium contained ammonium sulfate 10 mM as a control and glutamic acid, glutamine, sodium nitrite, sodium nitrate and asparagine as nitrogen source, all of them at a concentration of 10 mM. The transcript of the β-actin gene was also studied for comparative purposes. Part B: Relative level of expression of the gdhA to the β-actin gene.

Figure 10, parts A, B and C. Schematic representation of the steps involved in the construction of the GDH expression cassette.

Figure 11, parts A and B. Schematic representation of the steps involved in the construction of the GPD expression cassette.

Figure 12. Production (expressed as concentratin CT of secreted protein in mg/1) of thaumatin from A_^_ awamori strains TB2bl-44 and TGDTh-4 in fermentor studies. The medium used was MDFA supplemented with the components described below. Empty squares: Strain TB2bl-44; 6.0% sucrose, pH 6.2, fedbatch with asparagine. Empty circles: TB2 -44, 6.0% sucrose, pH 6.2, fedbatch with ammonium sulfate. Filled triangles: Strain TGDTh-4; 6.0 % sucrose, pH 6.2, fed-batch with ammonium sulfate. DETAILED DESCRIPTION OF ONE MODE OF CARRYING OUT THE INVENTION

This section describes the application of the new promoter and constructions described in the present invention to the preparation of recombinant thaumatin. The teachings of the examples below can be applied to the expression and production of any other recombinant protein and thus these examples should not be construed as limiting the scope of the present invention in any way.

A: CONSTRUCTS :

The starting point for all of the constructs that have been prepared in the present patent application is plasmid pThlX, which is described in European patent application EP 684312.

This plasmid contains: (i) a sulfanilamide resistance marker;

(ii) a DNA sequence which encodes a fusion protein comprising in his turn (a) the synthetic gene encoding thaumatin II, (b) a spacer sequence which in turn contains a KEX2 processing sequence, and (c) the complete glucoamylase gene (genomic) of Aspergillus niger; (iii) the signal sequence ("pre") and the "pro" sequence of the glucoamylase gene (glaA) of Aspergillus niger, and finally (iv) the promoter region sequence of the glucoamylase gene (glaA) of Aspergillus niger.

In the context of the present invention three new expression cassettes were prepared, which contained: (i) a drug resistance marker (most of the times it was a phleomycin resistance marker) ; (ii) a DNA sequence which encodes a fusion protein comprising in his turn (a) the synthetic gene of thaumatin II, (b) a spacer sequence which in turn contains a KEX2 processing sequence, and (c) a cDNA sequence that encodes most of the B2 protein (except sequences in the COOH end) from Acremonium chrvsogenum; (iii) the signal sequence of the B2 gene of Acremonium chrvsogenum and (iv) three different promoter regions.

In all the cloning and sub-cloning manipulations described in this patent application, Escherichia coli DH5a served as the recipient strain for high-frequency plasmid transformation. E. coli WK6 was used as host for obtaining single-stranded DNA from pBluescript plasmids for sequencing purposes .

Al . Construction of the expression cassette B2KEX

Protein B2 is an extracellular protease produced by the filamentous fungus Acremonium chrvsogenum. This protein is expressed and secreted in the late stages of growth of Acremonium chrvsogenum (between 120 and 144 hours after the start of growth) .

Plasmid pJElA (Laboratory of Prof. Juan-Francisco Martin, Universidad de Leon, Leon, Spain) contains the promoter region, leader peptide (including the signal sequence) and coding region of the B2 gene from Acremonium chrvsogenum. The gene itself has 1298 base pairs and two introns . These two introns are not present in the sequence that has been subcloned in pJElA, since these subcloned sequences were obtained from a cDNA. Upstream from the ATG start point of translation there is a promoter region of 477 base pairs. When Acremonium chrvsogenum is grown in a defined medium which contains sucrose and glucose as carbon sources and asparagine as nitrogen source, the gene is expressed at its highest levels between 72 and 96 hours of growth.

The steps involved in the construction of the B2KEX cassette are detailed in Figure 1 (parts A-C) . Plasmid pJElA was digested sequentially with BamHI and Ncol, releasing a 560 bp fragment that was purified from a 0.8% agarose gel. This fragment contains most of the coding region of the B2 gene, but excludes the active center of the protein. Similarly, plasmid pJL43b (J.L. Barredo, Ph.D. Thesis, Universidad de Leon, Leon, Spain) was also digested with BamHI and Ncol, releasing a large fragment (3740 bp) , which was purified from a 0.8% agarose gel. This fragment was ligated with the 560 bp BamHI-ΝcoI fragment from pJElA, yielding plasmid p43bB2CT (4300 bp) .

Plasmid p43bB2CT was digested with Νcol, treated with the Klenow fragment of DΝA polymerase I (in order to obtain blunt ends) and then digested with Stul, yielding a fragment of 3874. bp that was also purified from a 0.8% agarose gel. The single-stranded oligonucleotides ThSl and ThS2 (sequences shown below) where used, using plasmid pThlX as a template, to amplify by polymerase chain reaction (PCR) the KEX2-like and thaumatin sequences present in pThlX. The first 18 nucleotides present in ThSl correspond to the KEX2-like sequence .

ThSl: 5 ' - CGA ATG AAA AGG AAA AGG ATGGCCACCTTCGAG - 3 ' Arg Met Lys Arg Lys Arg

ThS2 : 5 ' - TTA TTA GGC GGT GGG GCA

A 655 bp DΝA fragment was obtained by PCR using plasmid pThTX as the template and ThSl and ThS2 oligonucleotides as primers. This DΝA fragment was ligated with the previously obtained fragment from p43bB2CT, yielding plasmid p43bB2CTTh. This plasmid (aprox. 4530 bp) contains part of the B2 protein gene fused to a KEX-2 sequence and to the synthetic gene encoding thaumatin II. The transcription termination signal present in this construct is the terminator sequence from the cycl gene of Saccharomvces cerevisiae.

Plasmid p43bB2CTTh was digested with BamHI, treated with calf intestinal - alkaline phosphatase (CIP) and purified from a 0.8% agarose gel. A 900 bp BamKE-BamHI fragment from pJElA was also isolated. Subsequent ligation of these two DΝA fragments generated plasmid pB2KEX (5430 bp) . The 900 bp BamHI-BamHI fragment from pJElA contains the B2 gene promoter sequence (477 bp) , the leader peptide sequence (318 bp) and 107 bp of the amino terminal sequence of the B2 gene.

Plasmid pB2KEX was then digested with Xbal, treated with the Klenow fragment of DNA polymerase I (in order to obtain blunt ends) and then digested with Sail, yielding a fragment of 2400 bp that was purified in a 0.8% agarose gel. Plasmid pJL43b was digested with Hindlll, also treated with the Klenow fragment of DNA polymerase I, and then digested with Xhol. A fragment of 4500 bp was purified as before. Finally, the two gel-purified fragments described above were ligated, generating plasmid pB2KTh (6900 bp; Fig. 1C) .

On the final sub-cloning step, both plasmids pB2KTh and pJL43bl were digested with Sad and Stul, yielding fragments of 5714 and 1305 bp, respectively, which were purified in a 0.8% agarose gel. These two fragments were then ligated, thus obtaining plasmid pB2KThbl (7020 bp; Fig. 1C) . Plasmid pJL43bl is a derivative of plasmid pJL43b, where the promoter that drives expression of the phleomycin resistance gene

(PpcbC from Penicillium chrvsogenum) was substituted by the glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter from

Aspergillus nidulans (P. Punt et al., Gene 1990, vol. 93, pp.101-109).

This plasmid contains a cassette to express thaumatin that comprises: (i) a phleomycin resistance marker; (ii) a DNA sequence which encodes a fusion protein comprising in his turn (a) the synthetic gene of thaumatin II, (b) a spacer sequence which in turn contains a KEX2 processing sequence, and (c) a cDNA sequence that encodes most of the B2 protein (except sequences in the COOH end) from Acremonium chrysoqenum; (iii) the signal sequence of the B2 gene of Acremonium chrvsogenum and (iv) the promoter region of the B2 gene of Acremonium chrvsogenum. In this particular construct, expression of the phleomycin resistance gene (phleo) is driven by the promoter of the glyceraldehyde-3- phosphate dehydrogenase gene from Aspergillus nidulans .

A2. Construction of the expression cassette GDHTh

A.2.1. Cloning of a DNA fragment of Aspergillus awamori containing the gdhA gene.

A. awamori ATCC 22342 was used as the source of DNA and RNA. . nidulans mutants A686 (gdhAl, yA2 , methH2 , galAl) and . nidulans A699 (gdhAl, biAl) (J.R. Kinghorn, J.A. Pateman, J. Gen. Microbiol . 1973, vol. 78, pp. 39-46) were obtained from the Fungal Genetics Stock Center, and were used for complementation studies with the gdhA gene from awamori . The partial glutamate auxotrophy of these two strains was confirmed by growth on media with glutamic acid or high ammonium sulfate concentrations (100 mM) as nitrogen source . Both gdhA mutants grow very poorly under high ammonium sulfate concentrations but show normal growth when glutamic acid is used as nitrogen source. ] _ coli NM539 served as host for Lambda GEM12 (Promega Co., Wis) phage derivatives.

Filamentous fungi were routinely maintained on solid Power sporulation medium (F. Fierro et al . , Appi. Microbiol. Biotechnol . 1996, vol. 43, pp. 597-604) at 30²C for 3 days. A. awamori and A_^_ nidulans seed cultures in CM medium (containing 20 g/1 malt extract; 5 g/1 yeast extract; 5 g/1 glucose) were inoculated with 10⁶ spores/ml and grown at 28^aC in a rotary G10 incubator (New Brunswick Scientific, New Brunswick, N.J.) for 48 h. For gdhA transcript isolation and characterization studies, A_^ awamori cultures in MDFA medium (Y.Q. Shen et al . , J. Antbiot . 1984, vol. 37, pp. 503-511) were incubated with a 15 % seed culture and grown at 30²C for 48-72 h in a rotary shaker, as described above.

A.2.1.1. Aspergillus awamori genomic library A genomic library of total DNA of A_^ awamori ATCC 22342 was constructed in a Lambda GEM12 phage vector. Total DNA was extracted and partially digested with Sau3AI to obtain DNA fragments of between 17 and 23 kb. This DNA was purified by sucrose-gradient centrifugation, ligated to Lambda GEM12 phage arms, and packaged in vitro using a Gigapack III Gold packaging system (Stratagene) resulting in a total of 8x10⁴ recombinant phages .

In the next step, and using as probe a 2.6 kb BamHI fragment containing the gdhA gene of Neurospora crassa (J.H. Kinnaird, J.R.S. Fincham, Gene 1983, vol. 26, pp. 253-260), two phages, FANl and FAN2, that gave a clear hybridization signal were isolated and purified by three rounds of infection. Restriction mapping of these two phages showed that they overlap in 7.2 kb. The total DNA region cloned in the two phages extended for 28.7 kb.

BamHI fragments of 1.7, 5.5 and 10 kb were subcloned in pBluescript KS+ plasmid, giving rise to plasmids pBl.7, pB5.5 and pBlO, as shown in Figure 2. They were then sequenced by generating ordered sets of deletions with the Erase-a-base system (Promega Co., Wis.) by digestion with exonuclease III from appropriate ends, followed by removal of single-stranded DNA with SI exonuclease. Sequencing of fragments of the gdhA gene was performed by the dideoxynucleotide chain termination method. For sequencing the cDNA clones containing the intron- exon junctions, reactions were performed with 90 ng of dsDNA using the GeneAmp PCR 2400 system coupled to the ABI-PRISM 310 automatic sequencer (Perkin Elmer) . Computer analysis of nucleotide and amino-acid sequences were made with the DNASTAR software (DNASTAR, Inc., UK) .

Initial sequencing showed that an open reading frame (ORFl) occurred in the right end of the 5.5 kb insert of pB5.5 extending into the left region of the 1.7 kb BamHI fragment of pBl.7, as shown in Figure 3. The 5.5 kb and 1.7 kb BamHI fragments were mapped in detail.

A 2.1 kb Xbal-Xbal fragment corresponding to the right end of plasmid pB5.5 was subcloned in pBluescript SK+ plasmid, creating plasmid pBSGh. More specifically, this 2.1 Kb Xbal- Xbal fragment was generated by digesting pB5.5 at an internal Xbal site and at a second Xbal site in the polylinker of pBSKS÷ (and close to the BamHI site shown in Fig. 3) .

A region Of 2570 nt was sequenced in both strands by the dideoxynucleotide chain termination method. This region contained ORFl (1380 bp) , which started at an ATG located 740 bp downstream from the left end of the insert in pBSGh and extended until the end of the 5.5 kb BamHI fragment, with 60 additional bp into the adjacent 1.7 kb fragment. ORFl was preceeded by a 740 nucleotide region that contained the necessary signals required for transcription initiation and regulation (see SEQ. ID No. 1) .

ORFl contained two putative introns at positions 785-850 and 1414-1471 (following the numbering in SEQ ID No. 1) that showed lariat and 5 ' and 3 ' splicing sequences similar to those of other fungal introns (D.J. Ballance, Yeast 1986, vol. 2, pp. 229-236) . The presence of the two introns was confirmed by sequencing the DNA regions corresponding to introns I and II obtained by PCR from a A,, awamori cDNA library using as primers oligonucleotides I_A and I_B for intron I, and II_A and II_B for intron II (sequences shown below) .

cDNA for these experiments was obtained from total RNA extracted as described above, from mycelia grown for 48 h in MDFA medium. The first and second cDNA strands were synthetized using a cDNA synthesis kit from Stratagene (La Jolla, Ca) . This cDNA was used for PCR amplification of the fragments containing the exon-exon junctions by the following program: 1 cycle at 94²C for 5 min, 50²C for 1 min, 72²C for 1 min followed by 30 cycles at 94²C for 1 min, 50²C for 1 min, 72 ^SC for 1 min and finally one cycle at 72²C for 8 min.

Oligonucleotides :

I_A 5 ' ATG TCT AAC CTT CCT CAC 3 '

I_B 5 ' ACC CTT ACC ACC ACC CAT 3 '

II_A 5 ' CGC TTC TGT GTT TCC TTC 3 '

II_B 5 ' GTA CTT GAA CTT GTT GGC 3 '

A.2.1.2. ORFl encodes a putative NADP-dependent glutamate dehydrogenase

ORFl encoded a protein of 460 amino acids (see SEQ ID No . 2) with a deduced molecular mass of 49.4 kDa and a pi value of 5.62. Comparison of the protein encoded by ORFl with other proteins in the SWISS-PROT data base showed that the encoded protein has a high homology with NADP-dependent glutamate dehydrogenases of _^_ nidulans (84.7% of identical amino acids), IL. crassa (74.4% identity), Saccharomvces cerevisiae (66.5% identity) and Schwanniomvces occidentalis (66.9% identity), as shown in Figure 4. The homology is extensive throughout the entire protein. All these proteins are ΝADP- dependent glutamate dehydrogenases that catalyze the reductive amination of α-ketoglutarate, in the presence of ATP, to form L-glutamate. The protein encoded by ORFl contains nine conserved motifs when compared with other fungal and yeast glutamate dehydrogenases . One of the conserved domains (amino acids 108-121) corresponds to a region implicated in the catalytic mechanism of the enzyme. The consensus sequence of this region is [LIV] -X (2) -G-G- [SAG]-K-X-[GV]-X(3)-[DΝS]-[PL] (PROSITE PS00074) . The lysine residue K¹¹⁴ located in the glycine-rich region GGGK¹¹GG corresponds to the lysine in the active center of Glu/Leu/Phe/Val (GLFV) dehydrogenases. Therefore, following standard fungal gene nomenclature, the gene encoded by ORFl was named gdhA. A.2.1.3. The cloned gene complements A. nidulans gdhA mutants

A. nidulans A686 and A699 strains were transformed by a known method (M.M. Yelton et al . Proc. Natl. Acad. Sci. USA 1984, vol. 81, pp. 1470-4) with plasmid pGDHaw (7.1 kb) , which contains the j_ awamori gdhA gene in a 2570 bp Xbal-Xbal fragment . This fragment contains also an upstream promoter region of 740 bp and a 322 bp region downstream from ORFl (gdhA gene) . The 2570 bp Xbal-XBal fragment was inserted into the Xbal 'site of the fungal vector p43gdh, which contains the phleomycin resistance marker under control of the A. awamori gdhA promoter as shown later in this patent application.

Seven transformants of A_^. nidulans A686 with the A_^_ awamori gdhA gene and 15 transformants of _^_ nidulans A699 were analyzed on minimal medium supplemented with different concentrations (10, 50 and 100 mM) of ammonium sulfate as nitrogen source, and their growth was compared with that of wild type A_^ nidulans . As a control, growth was also tested on medium containing 10 mM glutamic acid. As shown in Figure 5, the untransformed A_^. nidulans mutants A686 and A699 grow very poorly in plates with 100 mM ammonium sulfate, whereas three randomly selected transformants grow very well in this medium. The residual growth of A_^. nidulans gdhA mutants A686 and A699 in ammonium sulfate as nitrogen source is known (J.R. Kinghorn, J.A. Pateman, Heredity 1973, vol. 31, pp. 427) and is due to the presence of a second glutamate dehydrogenase activity that allows partial growth of these mutants .

A.2.1.4. Glutamate dehydrogenase activity in the transformants

Nicotinamide adenine dinucleotide phosphate (NADP) -specific glutamate dehydrogenase (NADP-GDH) activity was assayed by following the reductive amination of α-ketoglutarate in the presence of ammonium and NADPH and expressed as units of enzyme activity per mg protein. The initial reaction velocity was estimated from the change in optical density at 340 nm in a Hitachi U-2001 spectrophotometer. One unit of glutamate dehydrogenase was defined as the activity that catalyzes the oxydation of one nanomol of NADPH per minute.

To confirm the complementation results, the NADP-dependent glutamate dehydrogenase activity was measured in the A. nidulans gdhA mutants A686 and A699, and in three randomly selected transformants complemented with the A_^ awamori gdhA gene. Results are shown in Table 1 and they clearly indicated that while the glutamate dehydrogenase activity in strains A686 and A699 was clearly below the detection levels, significant levels of glutamate dehydrogenase activity were obtained in the transformants with the A_^ awamori gdhA gene, particularly at 24 and 48 h of growth. Some of the transformants, like A699-4, showed relatively high levels of glutamate dehydrogenase activity, perhaps due to integration of more than one copy of the gdhA gene in the genome of this transformant .

Table 1: NADP-dependent glutamate dehydrogenase activity (U/mg of protein) , in the A_^ nidulans gdhA mutants A686 and A699, and in three transformants of each of these mutants with the A_^. awamori gdhA gene.

strain t = 24 h t = 48 h t = 72 h

A. Awamori 550 0 0

A686 0 0 0

A686-4 350 280 100

A686-6 340 200 80

A686-7 310 160 90

A699 0 0 0 A699-2 270 240 100

A699-3 410 420 400

A699-4 500 670 580

A.2.1.5. Characterization of the promoter region of the gdhA gene

Analysis of the nucleotide sequence upstream from the ATG translation initiation codon revealed the presence of GTATA, CTATA and.! TCAATC sequences at positions -316, -61 and -17, respectively, with respect to the translation initiation codon, which may correspond to putative TATA and CAAT boxes involved in regulation of gene expression (see SEQ ID No. 1) .

Identification of the transcription start point was performed by "primer extension" with 2 μg of mRNA obtained from mycelia grown in MDFA for 48 h, as shown in Figure 6.

Primer extension analysis using as primer the oligonucleotide "Pe" 5 ' -GGGGTTCTTCTGGAAGAGGGT-3 ' (corresponding to the nucleotide sequence 70 bp downstream from the ATG) revealed a single band in the extension reaction (Fig. 8). The 5 ' -end of the mRNA corresponds to a thymine (T) located 86 bp upstream of the ATG initiation codon.

A.2.1.6. The gdhA gene is transcribed as a monocistronic transcript of 1.7 kb, and its expression is regulated by nitrogen.

In order to perform expression studies, total RNA of A. awamori was obtained by the phenol-SDS method from mycelia grown for 12, 24, 48, 60 or 72 h in MDFA medium with 55.5 mM glucose and 10 mM ammonium sulfate as carbon and nitrogen sources, respectively. For nitrogen regulation studies, the MDFA base medium (without ammonium sulfate) was supplemented with glutamic acid, L-glutamine, sodium nitrite, sodium nitrate and L-asparagine at 10 mM final concentrations.

For Northern analysis, total RNA (5 μg) was run on a 1.2% agarose-formaldehyde gel. The gel was blotted onto a nylon filter (NYTRAN 0.45; Schleicher and Schuell) by standard methods. The RNA was fixed by UV irradiation using an UV- Stratalinker 2400 lamp (Stratagene, La Jolla, Calif.).

For slot blotting, the RNA (5 μg) was loaded on a filter (NYTRAN. 0.45) by vacuum in a Bio-Dot SF Microfiltration apparatus.'. (Slot Blotting, Bio-Rad) . The RΝA was fixed by UV irradiation as above . The filters were pre-hybridized for 3 h at 42²C in 50% formamide, 5 x Denhardt's solution, 5 x SSPE, 0.1% SDS, 500 μg of denatured salmon-sperm DΝA per ml, and hybridized in the same buffer containing 100 μg of denatured salmon-sperm DΝA per ml at 42²C for 18 h, using as probe an internal DΝA fragment (0.694 kb PvuII) of the A_^. awamori gdhA gene. The filters were washed once in 2 x SSC, 0.1% SDS at 42²C for 15 min, once in 0.1 x SSC, 0.1% SDS at 42²C for 15 min, and once more in 0.1 x SSC, 0.1% SDS at 55²C for 20 min and then autoradiographed with Amersham X-ray film. mRΝA was purified from total RΝA by using the Poly(A) Quick mRΝA isolation kit (Stratagene, La Jolla, Calif.).

Northern analysis of the transcription of the gdhA gene revealed that it is strongly expressed as a 1.7 kb transcript (mRNA) with a size slightly larger than that of the β-actin gene mRNA, as shown in Figure 7. Since ORFl contains 1380 nt, this size of the transcript indicates that the gdhA gene is expressed as a monocistronic transcript.

Since the same amount of total RNA was used in all lanes of Fig. 7, it was concluded that the gdhA steady state transcript ^'levels in the cell are higher than those of the β- actin gene (arrows) indicating that the glutamate dehydrogenase A is expressed from a very efficient promoter. To determine the pattern of expression of the gdhA gene during the time-course of growth of A_^ awamori , gdhA hybridizing RNA was compared to β-actin hybridizing RNA in MDFA medium with ammonium sulfate (Figure 8A) and expressed as the ratio of counts in the gdhA-hybridizing band to the β- actin hybridizing counts (Figure 8B) . Results indicate that expression of both genes (gdhA and β-actin) is associated with the growth of Aj_. awamori but whereas low steady state levels of β-actin mRNA remained in the cells until 96 hours of growth, the levels of glutamate dehydrogenase mRNA decreased^drastically after 48 hours.

The glutamate dehydrogenase enzymatic activity detected when A. awamori is grown in MDFA medium with ammonium sulfate (10 mM) as nitrogen source at different times of the culture is shown in Fig. 8C . There is a sharp decrease in glutamate dehydrogenase activity between 24 and 48 h after start of growth, which is in good agreement with the decrease in transcript levels at this time of the culture, as shown in Fig. 8B.

Since glutamate dehydrogenase plays a central role in nitrogen utilization by A., awamori , it was also of interest to study if expression of gdhA was regulated by different nitrogen sources. As shown in Figure 9, very high gdhA transcript (mRNA) levels were obtained in media containing NH₄ ⁺, or asparagine as sole nitrogen sources. Glutamic acid repressed transcription of the gdhA gene, whereas intermediate levels of expression (normalized with respect to the β-actin gene) were observed in media that contained nitrate, glutamine or nitrite as nitrogen source. These results show that the NADP-dependent glutamate dehydrogenase is subject to a strong nitrogen regulation at the transcriptional level .

The glutamate dehydrogenase activity in 24-hour cultures grown in MDFA medium containing different nitrogen sources, all at a concentration of 10 mM, is shown in Table 2. The highest activity (per ml of culture) was observed in cultures with NH₄ ⁺ or asparagine as nitrogen sources. Moreover, these two nitrogen sources favoured a strong growth of A_^. awamori. When the results were expressed per mg of protein in the cell extracts, the highest specific activity was observed in MDFA medium with nitrate as the sole nitrogen source. This is due to the fact that in the presence of nitrate, A___ awamori grows very slowly. The lowest activity was observed in MDFA medium with glutamate as nitrogen source, confirming the results observed previously at the transcription level .

Table 2 : NADP-dependent glutamate dehydrogenase activity in A_^ awamori cultures grown for 24 h in MDFA medium supplemented with different nitrogen sources.

Nitrogen source Total Activi -ty Speci .fie Activity

(lOmM) (U/ml) (U/mg protein)

ammonium 1450 800 glutamic acid 330 280 glutamine 1100 600 nitrite 990 660 nitrate 1150 1680 asparagine 1300 720

A.2.2. Construction of the expression cassette GDHTh

Once the promoter region of the gdhA gene was located, a thaumatin expression cassette similar to the one described previously was constructed. Plasmid pBSGh was used as a template to obtain a 750 bp DNA fragment corresponding to the promoter region of the gdhA gene. This fragment was obtained by DNA amplification using the oligonucleotides gdhl and gdh2 and the Pfu enzyme (Stratagene) . gdhl: 5 ' - TTTT GTCGAC TTG CGA CGG CGT ATT GCT - 3 '

Sal I

gdh2 : 5 ' - TTTT CCATGG TCT GAA GGG GAG GAT TGA - 3 '

Nco I

This amplified DNA fragment was digested with Sail and Ncol and purified in a 0.8% agarose gel.

Plasmid pJL43 (a derivative of pJL43b, Dr. Jose Luis Barredo, Ph.D. Thesis, Universidad de Leon, Leon, Spain) was digested with Sail and Ncol and a large fragment (3740 bp) was purified in a 0.8% agarose gel. This DNA fragment was then ligated with the Sall-Ncol fragment previously amplified, yielding plasmid p43gdh (4500 bp) , where the pcbC promoter from Penicillium chrvsogenum has been replaced by the gdhA promoter from Aspergillus awamori .

In the next step, plasmid p43gdh was digested with Ncol, treated first with the Klenow fragment of DNA polymerase I and then with calf-intestinal phosphatase (CIP) . In paralell, a fragment of 1140 bp containing the B2 protein gene was amplified via the PCR technique, using plasmid pJElA as the template and oligonucleotides NTB2b and CTB2b as primers (sequences given below) . This 1140 bp fragment was digested with BamHI and then treated with the Klenow fragment of DNA polymerase I. From this reaction mix a 425 bp DNA fragment containing the amino terminal sequences of the B2 gene was purified from a 1.0 % agarose gel. This fragment of DNA was ligated by blunt-end ligation to the fragment of DNA from p43gdh previously described, resulting in plasmid p43gdhB2, where the BamHI site that is shown in Figure 10 has been regenerated. This plasmid is 4925 bp long and contains the gdhA promoter fused "in frame" to the amino terminal portion of the B2 gene. The next step in the construction of the complete expression cassette was the addition of the second portion of the B2 gene, the KEX2 sequence and the synthetic thaumatin II gene. For this part of the work, plasmid pB2KEX was used.

pB2KEX was sequentially digested with Xbal, treated with the Klenow fragment from DNA polymerase I and finally digested with BamHI. A fragment of 4637 bp was purified from a 0.8% agarose gel. In paralell, plasmid p43gdhB2 was sequentially digested with Sail, treated with the Klenow fragment from DNA polymerase I and finally digested with BamHI. A fragment of 1173 bp was purified from a 0.8% agarose gel. The ligation of these two fragments yielded plasmid pGDHTh (5810 bp) , where a new Sail site was created. This allows for the excision of the complete GDHTh cassette as a 2670 bp Sall-Sall fragment.

Starting with plasmid pGDHTh, two new plasmids were constructed. The first one was p43GDTh, constructed as follows. Plasmid pJL43 was linearized by digestion with Sail and ligated to a 2170 bp Sall-Dral fragment from pGDHTh (see Fig. 10, part B) .

Similarly, plasmid pGD71 was constructed as follows: plasmid PAN7-1 (P.J. Punt et al . , J. Biotecnol . 1990, vol. 17, pp. 19-34) was sequentially digested with Xbal, treated with the Klenow fragment from DNA polymerase I, and finally digested with Hindlll, and purified from a 0.8% agarose gel. In paralell, plasmid pGDHTh was digested with Ecll36ll (or Sad*, a variant of Sad from Fermentas that recognizes the standard Sad restriction site but leaves a blunt end), Hindlll and Dral. A fragment of 2175 bp was purified from an agarose gel . Ligation of these two fragments yielded plasmid pGD71 (see Fig. 10, part C) .

Plasmids p43GDTh and pGD71 contain a cassette to express thaumatin that comprises: (i) a DNA sequence which encodes a fusion protein comprising in his turn (a) the synthetic gene of thaumatin II, (b) a spacer sequence which in turn contains a KEX2 processing sequence, and (c) a cDNA sequence that encodes most of the B2 protein (except sequences in the COOH end) from Acremonium chrvsogenum; (ii) the signal sequence of the B2 gene of Acremonium chrvsogenum, (iii) the promoter region from the Aspergillus awamori glutamate dehydrogenase A gene, and (iv) a drug resistance gene that can be used as a transformation marker. Plasmid p43GDTh has the phleomycin resistance gene (phleo) driven by the the pcbC promoter from Penicillium chrvsogenum. Plasmid pGD71 contains the hygromyciή B resistance gene driven by the glyceraldehyde-3- phoεphate dehydrogenase promoter from Aspergillus nidulans .

A.3. Construction of the expression cassette GPDTh

The expression cassette GPDTh is similar to the expression cassette B2KEX, except that the B2 promoter from Acremonium chrvsogenum has been replaced by the promoter from the glyceraldehyde-3-phosphate dehydrogenase (named "gpd" from now on) gene from Aspergillus nidulans.

The complete promoter region of the gpd gene is present in plasmid pAN52-l (P.J. Punt et al . , J. Biotecnol. 1990, vol. 17, pp. 19-34). A Sacl-Ncol fragment (880 bp) from pAN52-l has been subcloned, generating pJL43bl.

Plasmid pJL43bl was digested with Νcol and treated first with the Klenow fragment of DΝA polymerase I and then with calf-intestinal phosphatase (CIP) , as shown in Figure 11. In parallel, a 1140 bp fragment of DΝA was obtained by DΝA amplification using the PCR technique, using pJElA as template and oligonucleotides ΝTB2b and CTB2b as primers. This fragment of DNA was digested with BamHI and treated with the Klenow fragment from DNA polymerase I, yielding a fragment of 425 bp that was purified from a 0.8% agarose gel. The final ligation reaction yielded plasmid pblB2 (see Fig. 11) . NTB2b: 5 ' - ATG CGT GCT GCT ACT CTC - 3 ' CTB2b: 5' - CTG GCC GTT GTT GAT GAG - 3'

As with the GDHTh cassette, the next step in the construction of a complete expression cassette was the addition of the second portion of the B2 gene, the KEX2 sequence and the synthetic thaumatin II gene. For this part of the work, plasmid pB2KEX was once again used.

pB2KEX was sequentially digested with Xbal, treated with the Klenow fragment from DNA polymerase I and finally digested with BamHI. A fragment of 4637 bp was purified from a 0.8% agarose gel. In paralell, plasmid pblB2 was sequentially digested with BamHI and Ecll36II (or Sad*) (leaves blunt ends), and a 1300 bp fragment was purified from a 0.8% agarose gel. The ligation of these two fragments yielded plasmid pGPDTh (5800 bp) .

In the next step, the GPDTh cassette was isolated from pGPDTh by digestion with Ecll36ll (or Sad*) , Hindlll and Dral, yielding a DNA fragment 2800 bp long. In parallel, plasmid pB2KThbl was sequentially digested with BamHI, treated with the Klenow fragment from DNA polymerase I and finally digested with Hindlll. A 4500 bp fragment was isolated from a 0.8% agarose gel. The plasmid resulting from the ligation of these two fragments was named pGPThbl.

This plasmid contains a cassette for the expression of thaumatin that is identical to the expression cassette B2KEX except that the promoter from the B2 gene of Acremonium chrvsogenum has been replaced by the promoter from the gpd gene from Aspergillus nidulans .

B. Strains used and transformation protocol

Aspergillus awamori strain NRRL312 was obtained from the American Type Culture Collection (ATCC) . Using standard mutagenesis techniques with nitrosoguanidine (NTG) , a derivative of this strain was obtained, and was named LpR66. This mutant strain secretes into the growth medium an inactive exoprotease aspergillopepsin A (named "pepA" from now on) . In all of the transformation experiments that are described below the strain that was used was Aspergillus awamori strain LpR66.

The three expression cassettes that have been described previously were used to transform Aspergillus awamori strain LpR66.

In all single transformation experiments, the antibiotic phleomycin was used as the selection marker. Strain LpR66 can grow in plates that contain 20 μg/ml of phleomycin. Therefore, all transformants were selected in plates with 25 μg/ml of the antibiotic . The regeneration medium that was used is TSAS, which contains 30 g/1 of Triptone-Soja (Difco) , 103 g/1 of sucrose and 1.5% agar (Difco) .

The transformation protocol was similar to the one described by Yelton (see above) with some modifications. A plate containing Power medium was inoculated with 10⁷ spores. This plate was incubated for 72 hours at 30²C, at which point the spores were scraped from the plate and were inoculated in 100 ml of CM medium (500 ml shake flask) . Incubation was for 16- 18 hours at 250 rpm and 28^aC. The mycelium obtained from this growth was filtered through a 30 μm nylon filter (Nytal) and washed with 10 mM sodium phosphate buffer (pH 5.8) which also contained 0.6 M magnesium sulfate. One gram of mycelium was re-suspended in "protoplast buffer" (10 mM sodium phosphate buffer (pH 5.8) which also contained 1.2 M magnesium sulfate) . An equal volume of buffer containing the enzyme "Lysing" (Sigma) was added, yielding a final concentration of 3 mg/ml of the enzyme. The mycelium solution was left to incubate for 3-4 hours at 100 rpm and 30²C until protoplasts were formed. Protoplast formation was monitored by visual inspection using a light microscope. Protoplasts were filtered, washed and finally resuspended in STC solution, to a final concentration of 10⁸ protoplasts/ml.

100 μl of protoplast solution was mixed with 10-20 μg of DNA and left in ice for 20 minutes. After this time interval, 500 μl of PTC were added, and left at room temperature for another 20 minutes. Then, 600 μl of STC medium were added and the transformation mix was aliquoted in different test tubes. Finally, the phleomycin antibiotic solution and TSAS medium that contained agar were added. The contents of the tubes were gently homogenized and added to TSAS plates that contained phleomycin. Plates were incubated at 30²C until the transformants were visualized as individual colonies. When hygromycin B was used as selection marker, a similar protocol was used.

The linearization of all the plasmids that have been described in this work gave a 4-fold increase in the efficiency of transformation as compared to transformations performed with plasmids that had not been linearized. Therefore, in most transformation experiments the plasmids were used linearized.

Several transformants were obtained and analyzed. Initial screens were performed in plates containing 25 μg/ml of phleomycin. Confirmation screens were then performed using phleomycin concentrations as high as 200 μg/ml.

Transformants were analyzed by PCR to detect whether the thaumatin II gene had been incorporated into their genome essentially as described (cf . EP 684312) . Those transformants that were positive were then further analyzed for expression of thaumatin by immunoblot analysis and ELISA (enzyme-linked immunoassay) also as described (cf. EP 684312). C: Recombinant strains that produce thaumatin

C.l. Materials and methods

C .1.1. Culture media

CM medium: malt extract, 5 g/1; yeast extract, 5 g/1; glucose, 5 g/1.

SMM medium: 8% sodium citrate; 1.5% (NH₄)₂S0₄; 0.13%

NaH₂P0₄.2H_?0; 0.2% MgS0₄.7H₂0; 0.1% Tween 80; 0.1% uridine,

0.1% antifoam AF and 7% soya milk. The carbon source

(glucose, sucrose, maltose, etc.) is present at a final concentration of 15%. The pH of the medium is adjusted to 6.2 with H₂S0₄.

MDFA medium: 1.2% L-asparagine; 0.8% of salt solution I [2% Fe(NH₄)₂(S0₄)₂.6H₂0] ; and 14.4% of salt solution II [10.4% K₂HP0₄; 10.2% KH₂P0₄; 1.15% Na₂CuS0₄.5H₂0 ; 0.2%MgS0₄.7H₂0; 0.02% ZnS0₄.7H₂0; 0.005% CuS0₄.5H₂0; 0.05% Cad-,.2110] . The carbon source used was either maltose (usually 6.5%) or a mix of sucrose (3.6%) and glucose (2.7%) . Other amounts of carbon source are indicated in each experiment that is described. The initial pH of this medium is 6.5.

C.1.2. Fermentation analysis

Growth and expression studies were conducted in SMM and MDFA media, first in shake flasks, and later in several fermentors equipped with measurement and control systems for the following variables: stirring, dissolved oxygen, pH, antifoam and culture level .

Experiments were conducted in 1-liter shake flasks with a working volume of 150 ml. Inoculation was to a final concentration of 3 x 10⁵ spores/ml. Stirring was at 150 rpm, and the incubation temperature was 30°C. The media used was either SMM or MDFA.

The experiments conducted in the fermentor were analogous to the ones in shake flasks, except that the pH of the medium was maintained constant at a pre-set value, and adjusted by the automatic addition of either 30% NaOH or 0.5N H₂S0₄.

C .1.3. Analytical methods

2-10 ml samples were taken at different times from the fermentation culture and processed to determine the dry weight, thaumatin, maltose and glucose concentrations that were presen .

Dry weight was determined by passing a sample through a pre- filter (Nucleopore, Cat .No. 211114) . The biological material retained in the pre-filter was washed with 40 ml of pure ethanol and 50 ml of distilled water. It was then incubated at 90°C until a constant weight could be recorded. The filtrate was aliquoted and frozen for further analysis.

Thaumatin concentration in the culture broth was determined by an enzyme-linked immunoassay (ELISA) and by immunoblotting

(Western blot) analysis, essentially as described (cf. EP 684312), using an anti-thaumatin polyclonal antibody. For immunoblotting, samples were sometimes concentrated as follows: 500 μl of filtrate were mixed with an equal volume of 10% trichloroacetic acid (TCA) , and frozen for 12 h. The sample was then allowed to regain room temperature and centrifuged in a table-top centrifuge (15,000 rpm; 20 min. 4°C ) . The pellet that is recovered contains all the proteins that were present in this sample. The pellet was then resuspended in protein loading buffer, boiled for 5 minutes, and subjected to SDS-PAGE as described (cf EP 684312) .

Approximately 1 ml of filtrate was used for glucose/maltose determination. Glucose levels were determined using a SIGMA DIAGNOSTICS kit (Procedure number 510).

Maltose concentration in the culture broth was determined as follows: 250 μl of sample filtrate were placed in a test-tube that had been previously chilled; 1.250 ml of anthrone solution (prepared by dissolving 2 g anthrone in 50 ml absolute ethanol and then adding 950 ml of 75% H₂S0₄) were then added, and the sample was kept chilled for five minutes. The sample was then transferred to a boiling water bath, and incubated for 10 minutes. Finally the samples were once again chilled .and the absorbance read at 625 nm. Maltose concentrations were determined by comparison to a calibration curve generated by measuring the absorbance of maltose solutions of known concentrations (range: 0 - 0.2 g/1).

C.2. Thaumatin producing strains

C.2.1. Strain TB2bl-44

This strain is a derivative of Lpr66 that was obtained by transformation of the aforementioned LpR66 strain with the expression plasmid pB2_KTh-bl . This expression cassette contains the synthetic thaumatin II gene under the control of the promoter of the B2 protein from Acremonium chrvsogenum. In shake-flask cultures with MDFA medium this strain secretes 6-8 mg thaumatin/1.

Further optimization studies were performed in a 5-liter New Brunswick fermentor. The inoculum was obtained by growing the strain for 40 hours at 30²C in CM medium. 450 ml of this inoculum were then used to seed the 5-liter fermentor (working volume of 4.5 liters). RPMs were between 250 and 500, and varied according to the oxygen status of the system, which was always set at 30%.

Different parameters were tested, such as the pH of the medium and the carbon and nitrogen sources. Representative experiments are described in Figure 12 :

1. Growth in MDFA medium with 6.0% sucrose and L-asparagine as the nitrogen source. The set-point for the pH was set at 6.2, and a fed-batch system was installed. Feedings were done at 36, 48, 60 and 72 hours after the beginning of the fermentation. In each feeding, 45 ml of a 0.5 g/ml sucrose solution were added.

2. The conditions were identical to those described under 1 above, but L-asparagine was replaced by ammonium sulfate (the molar amounts were the same in both experiments) as the nitrogen source.

The best productivity was obtained with the conditions described under 1 above, with asparagine as nitrogen source, and with 6% sucrose as the carbon source, with four "feedings" of sucrose every 12 h after 36 h of fermentation. Under these conditions, yields of 100 mg thaumatin/1 were obtained.

C.2.2. Strain TGDTh-4

This strain was deposited according to the Budapest Treaty with Access No. CECT20241 on March 25, 1998 (25.03.98) in the following institution:

Colecciόn Espanola de Cultivos Tipo (CECT) Edificio de Investigaciόn, planta baja, no. 34 Universidad de Valencia

Campus de Burjasot 46100 Valencia, Spain

It is a derivative of Lpr66 which was obtained by transformation of the aforementioned LpR66 strain with the expression cassette p43GDTh. This expression cassette contains the synthetic thaumatin II gene under the control of the promoter of the gdhA gene from Aspergillus awamori. In shake-flask cultures with MDFA medium (with 6.0% sucrose) this strain secretes 6-8 mg thaumatin/1.

Experiments were also conducted in the controlled environment of a 5-liter New Brunswick fermentor, as described before for strain TB2bl-44. Ammonium sulfate was used in place of asparagine as nitrogen source, at the same molar levels. In this experiment, also shown in Figure 12, the following conditions were tested: strain TGDTh-4 was grown in MDFA medium supplemented with 6% sucrose and ammonium sulfate as nitrogen source. The pH set-point was 6.2. and a fed-batch system was also installed. Feedings were done at 36, 48, 60 and 72 h after the beginning of the fermentation. In each feeding, 45 ml of a 0.5 g/ml sucrose solution were added.

The results (Fig. 12) indicate that the production of thaumatin is also in the order of 100 mg/1, but with the added advantage of having an earlier production and the use of a more economical nitrogen source. Therefore, it is concluded that the glutamate dehydrogenase promoter from Aspergillus awamori is more efficient than the B2 protein promoter from Acremonium chrvsogenum.

C.2.3. Strain TGP-3

This strain is a derivative of Lpr66 which was obtained by transformation of the aforementioned LpR66 strain with the expression cassette pGPThbl . This expression cassette contains the synthetic thaumatin II gene under the control of the promoter of the gpd gene from Aspergillus nidulans . In shake-flask cultures with MDFA medium this strain secretes 9- 10 mg thaumatin/liter .

C.2.4. Double transformants

Strains TB2 -44 and TGP-3 were re-transformed with expression plasmid pGD71, which contains the thaumatin gene under control of the glutamate dehydrogenase promoter from A. awamori and a hygromycin B resistance gene as a selection marker for transformation experiments. A battery of different transformants (see Table 3) was analyzed in shake flask experiments. It was shown that re-transformation of strain TGP-3 did not result in better producing strains. However, re-transformation of TB2bl-44 did result in better producing strains when cultured in shake-flasks under the standard conditions mentioned before.

Table 3 : Production of thaumatin in shake flasks by retransformed strains grown in MDFA medium for 96 h. Quantification by ELISA. All strains were retransformed using hygromicin B resistance as selection marker.

Trans formant Production (mg/1) Original strain

TGP3-GD1 2.08 TGP3

TGP3-GD2 0.40 TGP3

TGP3-GD3 9.44 TGP3

TGP3-GD4 8.25 TGP3

TGP3-GD5 0.40 TGP3

TGP3-GD6 9.71 TGP3

TB2bl-44-GDl 3.84 TB2bl-44

TB2bl-44-GD2 0.00 TB2bl-44

TB2bl-44-GD3 9.85 TB2 -44

TB2bl-44-GD4 11.10 TB2M-44

TB2bl-44-GD5 11.82 TB2bl-44

TB2bl-44-GD6 10.75 TB2bl-44

TB2bl-44-GD7 10.52 TB2bl-44

TB2bl-44-GD8 8.09 TB2 -44

TB2bl-44-GD9 7.13 TB2M-44

D: Purification of recombinant thaumatin Two procedures were employed for the purification of recombinant thaumatin. In the first one the fermentation broth was simply clarified, concentrated and diafiltered, yielding a concentrated and cleaner extract that was used for sensory experiments to ascertain the sweet profile of the recombinant thaumatin. The second procedure involved a classic purification protocol that yielded pure thaumatin.

D.l. Clarification, concentration and diafiltration of the fermentation broth

Biomass was removed by filtration through filter paper. The filtrate was collected in a filtering flask that was submerged in ice. The clarified broth was then centrifugated at 6000 rpm for 15 minutes at 4²C.

The clarified fermentation broth was further concentrated by ultrafiltration using a ProFluxTM Ml2 Tangential Filtration System. The system configuration was: base unit, level switch, 2.5 1 reservoir, cooling coil, inlet and oulet pressure transducers, secondary pump, one Spiral-wound membrane cartridges S1Y3 (Molecular weight cut-off 3,000 Daltons) .

The system was operated as follows: (1) Calibration of the pressure sensors. (2) Adjustment of alarm set points: low inlet pressure 3.0 Bars, high inlet pressure 3.5 Bars, differential pressure 0.3 Bars. (3) Washing of the system and the cartridges with deoinized, distilled water (4) Fill- up of the reservoir with process solution; the solution is kept at 8-10²C by recirculating cold water (HAAKE, DC1-K20 refrigerated circulator) through the cooling coil. (5) Setting of the level switch at the desired concentration volume (1/4 to 1/5 of the initial volume) . (6) Operation of the recirculation pump at 75 %. (7) Adjustment of the Back Pressure Valve to obtain a 3.0 Bar inlet pressure. If necessary, back pressure was reduced during operation.

Once the fermentation broth was concentrated to the desired volume, the solution was diafiltered in order to remove low molecular weight solutes (Salts, sugars, etc.).

The system configuration allows the operation in the "pumped diafiltration with automatic safety stop" mode. The dialysate (five volumes of deionized water) was transferred by the secondary pump in steps as directed by the level switch. Once the dialysate supply is exhausted, the system and the secondary pump will shut off automatically.

The diafiltered solution is drained from the system, sterilized by filtration (Stericup, 0.22 μm, Millipore) and stored at 4²C.

D.2. Purification of recombinant thaumatin to homogeneity

Recombinant thaumatin was purified to homogeneity using a four step purification scheme that is detailed in Table 4. The starting point for the particular purification protocol that is described here are 500 ml of fermentation broth obtained from the growth of strain TGDTh-4, with thaumatin present at a concentration of 50 mg/1.

Proteins from this broth were precipitated with ammonium sulfate (20-50% range) . The precipitate was then re-suspended in 25 mM phosphate buffer, pH 7.0.

This mix was then passed through a Sephadex G-25 column (for desalting purposes) and eluted with the same buffer. Finally the sample was loaded onto a CM-Sepharose column at a flux of 0.5 ml/minute. The column was washed with 25 mM phosphate buffer, pH 7.0 in order to eliminate proteins in the flow- through fraction. Thaumatin was eluted with a NaCI linear gradient (0-400 mM) . Thaumatin is eluted from this column in almost pure form as detected by Coo assie Blue staining.

Table 4 : Purification of thaumatin from the fermentation broth for growing strain TGDTh-4 in MDFA medium

SAMPLE VOLUME CONC. TOTAL YIELD (ml) (mg/1) (mg) (%)

Broth 500 50 25 100

Ammonium sulfate 11 1745 19.2 76.8

Sephadex G-25 30 596 17.9 71.6

CM-Sepharose 24 704 16.9 67.6

While the foregoing illustrative examples are directed to the production of recombinant thaumatin, the production of any other recombinant protein by means of the new methodology provided in the present invention, particularly the new promoter and DNA constructions disclosed herein, is also encompassed by the present invention.

Claims

1. A promoter for the expression of recombinant proteins in filamentous fungi that comprises a nucleotide sequence - or a complementary strand thereof - selected from the group consisting of: (a) the nucleotide sequence numbered 1-740 in the enclosed SEQ ID No. 1; (b) a nucleotide sequence that is analog to that defined in (a) ; and (c) a nucleotide sequence that hybridizes under stringent conditions to that defined in (a) .

2. A promoter according to claim 1 which has the sequence of nucleotides numbered 1-740 in SEQ ID No. 1 or its complementary strand.

3. Isolated promoter of a glutamate dehydrogenase gene from a fungus of the genus Aspergillus with the proviso that it is not from Aspergillus nidulans .

4. Isolated promoter according to claim 3 wherein the fungus is Aspergillus awamori or Aspergillus niger .

5. Isolated promoter according to claim 4 wherein the fungus is Aspergillus awamori .

6. A purified and isolated DNA sequence that encodes a glutamate dehydrogenase protein and that comprises a nucleotide sequence - or a complementary strand thereof - selected from the group consisting of: (a) the nucleotide sequence numbered 741-2245 in the enclosed SEQ ID No. 1; (b) a nucleotide sequence that is analog to that defined in (a) ; and (c) a nucleotide sequence that hybridizes under stringent conditions to that defined in (a) .

7. A DNA sequence according to claim 6 which has the sequence of nucleotides numbered 741-2245 in SEQ ID No . 1, or its complementary strand.

8. An isolated DNA sequence encoding a glutamate dehydrogenase from a fungus of the genus Aspergillus with the proviso that it is not from Aspergillus nidulans .

9. An isolated DNA sequence according to claim 8 wherein the fungus is Aspergillus awamori or Aspergillus niger .

10. An isolated DNA sequence according to claim 9 wherein the fungus is Aspergillus awamori .

11. The protein encoded by any of the DNA sequences according to claim 6.

12. The protein which has the amino acid sequence in SEQ ID No. 2.

13. An isolated glutamate dehydrogenase from a fungus of the genus Aspergillus with the proviso that it is not from Aspergillus nidulans .

14. An isolated glutamate dehydrogenase according to claim

13 wherein the fungus is Aspergillus awamori or Aspergillus niger .

15. An isolated glutamate dehydrogenase according to claim

14 wherein the fungus is Aspergillus awamori .

16. Use of a promoter from a glutamate dehydrogenase gene from a fungus of the genus Aspergillus for the expression of recombinant proteins in filamentous fungi .

17. Use according to claim 16, wherein the promoter is a promoter according to any one of claims 1 to 5.

18. A DNA construction that comprises: a) a promoter from a glutamate dehydrogenase gene from a fungus of the genus Aspergillus ; b) a DNA sequence encoding a protein normally expressed from a filamentous fungus or a portion thereof; c) a DNA sequence encoding a cleavable linker peptide; and d) a DNA sequence encoding a desired protein.

19. A DNA construction according to claim 18, wherein the promoter under a) is a promoter according to any one of claims 1 to 5.

20. A DNA construction according to claim 18, wherein the DNA sequence under b) encodes a protein or portion thereof selected from the group consisting of: i) glucoamylase from Aspergillus awamori , Aspergillus niger, Aspergillus orvzae or Aspergillus soiae; ii) B2 from Acremonium chrvsogenum; and iii) a glutamate dehydrogenase from a filamentous fungus .

21. A DNA construction according to claim 20, wherein the DNA sequence under b) encodes glucoamylase from Aspergillus awamori , Aspergillus niger, Aspergillus oryzae or

Aspergillus soiae, or a portion thereof.

22. A DNA construction according to claim 20, wherein the DNA sequence under b) encodes the protein B2 from Acremonium chrysogenum or a portion thereof.

23. A DNA construction according to claim 20, wherein the DNA sequence under b) encodes a glutamate dehydrogenase from a filamentous fungus or a portion thereof.

24. A DNA construction according to claim 18, wherein the DNA sequence under c) contains a KEX2 processing sequence.

25. A DNA construction according to any one of claims 18 to 24, wherein the DNA sequence under d) encodes thaumatin.

26. A DNA construction according to claim 25, wherein the DNA sequence under d) is the thaumatin II synthetic gene from plasmid pThlX.

27. A DNA construction comprising a promoter from a glutamate dehydrogenase gene from a fungus of the genus

Aspergillus operatively linked to a DNA sequence encoding the protein that it is desired to express.

28. A DNA construction according to claim 27, wherein the promoter is a promoter according to any one of claims 1 to

5.

29. A filamentous fungus culture capable of producing a recombinant protein which has been transformed with a plasmid containing a DNA construction according to any one of claims 18 to 28.

30. A culture according to claim 29, wherein the filamentous fungus is a fungus from the genus Aspergillus.

31. A culture according to claim 29, wherein the filamentous fungus is selected from the group consisting of Aspergillus awamori, Aspergillus niger, Aspergillus oryzae , Aspergillus nidulans and Aspergillus soiae.

32. A culture according to claim 29, wherein the plasmid contains a DNA construction according to any one of claims 25 or 26.

33. A process for producing a recombinant protein in a filamentous fungus comprising the following steps : a) preparation of an expression plasmid containing a DNA construction according to any of claims 18 to 28; b) transformation of a strain of filamentous fungus with said expression plasmid; c) culture of the transformed strain under appropriate nutrient conditions to produce the desired protein, either intracellularly, extracellularly or in both ways simultaneosly; and d) depending on the case, separation and purification of the desired protein from the fermentation broth.

34. A process according to claim 33, wherein the recombinant protein is thaumatin and the expression plasmid contains a DNA construction according to claims 25 or 26.

35. Use' of a DNA sequence derived from a nucleotide sequence according to claims 1 or 6 as a probe for the identification and isolation of a glutamate dehydrogenase gene and/or a promoter sequence of a glutamate dehydrogenase gene.