CA2325571A1 - 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 i n filamentous fungi.

Description

WO 99/51756 PCT/EP99/02243 _ Promoter and constructions for expression of recombinant g~oteins in filamentous funai 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, Asperaillus awamori has been used for the production of recanbinant proteins such as bovine chymosin and human lactof errin.
Some recombinant proteins, however, have proved to be very difficult to express in filamentous fungi. This is the case for exan~le 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 Thaiunatoccocus~ daniellii Benth (M. Witty, J.D. Higginbotham, Thaumatin ,1994, CRC Press, Boca Rat6n, Florida). Thaumatins can be isolated from these arils in at least five different forms ( I , I I , III , b and c ) , thaiuna,tins I and I I being the most abundant types in the arils. Despite its advantages, industrial use of thaumatins of plant origin is ver~J limited because of the extreme difficulty involved in obtaining the fruit from which it is extracted. Attgnpts 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.

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~ 1 .1 ~~. .. ~~ .~ .1 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 protection 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. p,,.p~'aillus awamori as the promoter and the carrier protein, respectively (P.P. Ward et al., Biotechnolocr~ 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 thau<natin in filamentous fungi.
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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 filamentvus fungi, such as i. _.
-~ 25 thaumatins. This goal is achieved with the new promoter and DNA constructions provided in the present invention, as explained below.
DESCRIPTION OF THE INVENTION

3 0 -.
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 As~ercrillus, particularly, from Asperaillus awamori.
One of the objects of the present invention is a new promoter for the expression of recombinant proteins - in AMENDED SHEET

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Page 2a Insertion for page 2 Gene (1983) , vol. 26, pp. 253-260 discloses the complete nucleotide sequence of the Neurospora crassa NADP-specific glutamate dehydrogenase gene.
Appl. Microbiol. Biotechnol. (1997), Vol. 47, pp 1-11 discloses the efficient production of secreted proteins by Aspergillus. Particular focus is laid on the gene fusion strategies.
EP 0 684 312 A2 relates to a preparation process of a natural protein sweetener, thaumatin.
Said document discloses a new nucleotide sequence encoding thaumatin with optimised codon usage for expression in filamentous fungi.
AMENDED SHEET

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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; and (b) a nucleotide sequence that hy-bridizes under stringent conditions:-to that defined in (a) with the proviso that the sequence is not the promoter of the gdh gene from Aspergillus nidnlans. 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 Asnergillus ~wamori.
Although glutamate dehydrogenase A disclosed herein is the _ first glutamate dehydrogenase identified and described in the filamentous fungus Asperaillus awamori, there may exist other glutamate dehydrogenases in AsQeraillua awamori. The novel nucleotide sequence of the AsnercLillus 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 As~eraillus awamori as well as in other organisms, preferably in filamentous fungi, more preferably in fungi of the genus Asperaillus, still more preferably in Asperaillus awamori and Asperaillus nig~er, and specially in Asneraillus awamori, following the teachings of the present invention. Consequently, the present invention is not limited to .the specific gdhA promoter from AsDerail_1-us awamori disclosed herein but also relates to the promoter of any glutamate dehydrogenase gene from a fungus of the genus As~eraillus with the proviso that it is not from A~peraillus nidulans. Examples of said Asperailli include Asneraillus awamori, Asne_raillus niger, As~eraillus oryzae and As~eraillus o'a ,In a preferred embodiment, the invention relates to a promoter of a glutamate dehydrogenase gene from As~eraillus awamori or Asr~eraillus niQer In a more preferred embodiment, the invention relates to a promoter of a glutamate dehydrogenase gene from Asperqillus awamori. The AMENDED SHEET

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use of the novel nucleotide sequence shown in SEQ ID No. 1 or a portion thereof as probe is also a object oz the present invention. The term "a portion thereof" denotes any part of the nucleotide sequence of SEQ ID No.l 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 trom the group consisting of: (a) the nucleotide sequence numbered 741-2245 in the enclosed SEQ ID No. 1; and (b,~
_ a nucleotide sequence that hybridizes under stringent conditions to that defined in (a) with the proviso that the sequence is not the gdh _ 15 'I gene from Aspergi l lus nidulans . In a pre f erred 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 AsDergillus awamori disclosed herein but also relates to any glutamate dehydrogenase gene from a fungus of the genus Ast~erc~.~illus with the proviso that it is not from As~eraillus ~idulans. In a preferred embodiment, the invention relates to the DNA sequences encoding 25~ glutamate dehydrogenase from Aspergillus awamori or y Asperaillus niQer. In a more preferred embodiment, the invention relates to the DNA sequences encoding glutamate dehydrogenase from Asneraillus awamori.
30! 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 35 dehydrogenase from a fungus of the genus Asperctillus with the proviso that it is not from Asgergillus nidulans, more preferably a glutamate dehydrogenase from Asperaillus awamori or As~erQillus nicer, and still more preferably a glutamate dehydrogenase from As~eraillus ~wamori.
AMENDED SHEET

The invention further relates to the use of the glutamate dehydrogenase promoters above described for the expression of recombinant proteins in . filamentous fungi. Certain 5 glutamate dehydrogenases from several microorganisms are already known and their genes have been disclosed, in particular the glutamate dehydrogenase A (gdhA) gene from Asberaillus 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 fungi. As shown in the examples below, the glutamate dehydrogenase promoter from Asperaillus awamori has proven to be very strong in promoting transcription of heterologous genes. Therefore, this promoter as well as related ghd promoters from Asperailli 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 ~_peraillus for the expression of recombinant proteins in filamentous fungi. Preferably, the gdh promoter is from a fungus of the genus Asperctillus with the proviso that it is not from A.~percLllus nidulans, more preferably it is from Aspercrillus awamori or Aspercrillus ni er, still more preferably it is from Aspergillus-awam~i, 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 Asperaillus;
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 ~speraillus nidulans, more preferably it is from As~ergillus awamori or Ast~eraillus nigger, still more preferably it is from Ast~ercrillus 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, a-amylase and aspartyl proteases from Aspergillus awamori, Asgergillus nicer, Aspergillus oryzae and Asperaillus so'ae, cellobiohydrolase I, cellobiohydrolase II, endoglucanase I
and endoglucanase III from Trichoderma species, glucoamylase from Neurospora and Humicola species, the protein B2 from Acremonium chrvsocrenum 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 Asperaillus awamori, Asperaillus nigger, Asneraillus orvzae or Asperaillus solae; ii) B2 from Acremonium chrvsoaenum;
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 Asperaillus awamori, Asperaillus ni r, As~eraillus oryzae or Asperaillus so' e; ii) B2 from Acremonium chryso e~; and iii) a glutamate dehydrogenase from Asperyillus .awamori or Asperaillus nicer. 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 ahaumatin II coming from plasmid pThIX, 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 Asperaillus 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 Aspe~c~illus with the proviso that it is not from Asberaillus nidulans, more preferably it is from Asberaillus awamori or Asperaillus niger, still more preferably it is from Asperqillus 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, oc-amylase and aspartyl proteases from Asperaillus awamori, Asperaillus ~aer, Asperaillus o~yzae and Asperaillus s_olae, signal sequences from cellobiohydrolase I, cellobiohydrolase II, endoglucanase I and endoglucanase III from Trichoderma species, signal sequences from glucoamylase from Neurosnora and Humicola ,species and the signal sequence from the protein B2 from Acremonium chrysoaenum. 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 constrLictions 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. nicer, A. oryzae or A. s_osae glucoamylase gene, the A. awamori, A. ni er, A.

y orvzae or A. 'ae oc-amylase genes and the Saccharomyces 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 pyre. A
polyadenylation sequence is a nucleotide sequence which when transcribed is recognized by the expression host to add polyadenosine residues to transcribed mRNA. Examples include the polyadenylation sequences from the A. nidulans trpC
gene, the A. awamori, ~ ni_ecL~r, ~ orvzae or A. s_oiae 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: Aspercrillus, Trichoderma, Neurospora, Penicillium, Acremonium, ~e~halosporium, Achlya, Phanerochaete, Podosnora, Endothia, uc , Fusarium, Humicola, ~ochliobolus, Rhizo~us and Pvricularia.
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 Asperaillus awamori, Aspergillus nicer, Asneraillus oryzae, Asperaillus nidulans or Aspergillils .s_Qjae. 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 5 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 10 thaumat~n..
The accompanying examples describe the identification and isolation of the glutamate dehydrogenase A gene and its promoter region from Asperaillus awamori. This was achieved using a probe from ~leurospora 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 Asperg~illus 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 BamFiI fragment that contained the Neuros~ora 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 by 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 Aspercrillus 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 Asperaillus awamori. As shown in these examples, ~anc1 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., Appl. 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 B2KF~ expression cassette.
Figure 2. Restriction map of a 28.7 kb region of A. awamori DNA including the gdhA gene. Map of phages FAN1 and FAN2.
Thick lines indicate the overlapping zone between the two WO 99/51756 PC'T/EP99/02243 phages containing the gdhA gene. pBlO, pB5.5 and PB2.7 indicate the DNA fragments subcloned in the corresponding plasmids . B = BamFiC, S = Sal I .
Fiaure 3. Restriction map of the 2.1 kb XbaI-BamHI fra~nent from pB5.5 plasmid~that was sequenced. The 3' end of the gdhA
gene was contained in the left region of the insert in pBl.7.
B = BamHI, E = EcoRI, EV = EcoRV, P - PstI, S - SalI, X -Xbal .
Fiaure 4, parts A and B. Alignment of the deduced amino acid sequences of NADP-specific glutamate dehydrogenases of A.
awamori, A. nidulans (Genebank accession number P18819), N.
crassa (P00369), S. cerevisiae (P07262), S. occidentalis (P29507), ~ bisporus (P54387), ~ typhimurium (P15111), E.
coli (P00370) and ~ g~lutamicum (P31026). Identical amino acids ~ are shadowed. Motifs a-i with several consecutive conserved residues are overlined.
Fiaure 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.
Fiaure 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 M13 phage from the -40 primer.
Figure 7. Northern blot analysis of the transcripts of the gdhA and .!~-'actin genes . A: hybridization with a probe 3 5 internal to the gdhA gene ( 0 . 694 kb PvuII f ragrnent ) . B
hybridization with the f5-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 1~FA medium with 1~ glucose and 10 mM ammonium sulfate (part A). For comparative purposes, the transcript of the f3-actin gene in the same RNA sample was also studied. Part B:
relative level of the expression of the gdhA to the i3-actin gene. Part C: NADP-dependent glutamate dehydrogenase activity in the same cultures from where the mRNAs were extracted.
Ficrure 9. Slot Blot analysis of the transcript ~of the A.
awamori gdhA gene during the course of a fermentation in I~mFA
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 iS-actin gene was also studied for comparative purposes. Part B: Relative level of expression of the gdhA to the Q-actin gene.
Ficture 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. Schanatic 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 ~ awamori strains TB2b1-44 and TGDTh-4 in fermentor studies. The medium used was NmFA supplemented with the components described below. Empty squares: Strain TB2b1-44; 6.0~ sucrose, pH 6.2, fedbatch with asparagine. Empty circles: TB2b1-44, 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.

1~
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 ir~vention in any way.
A: CONSTRUCTS:
The starting point for all of the constructs that have been prepared in the present patent application is plasmid pThIX, which is described in European patent application EP 684312.
This plasrnid 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 ~speraillus nicer; (iii) the signal sequence ("pre") and the "pro" sequence of the glucoamylase gene (glaA) of Aspercxillus n~, and finally (iv) the promoter region sequence of the glucoamylase gene (glaA) of ~.spergillus niQer.
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 thamnatin 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 COON
end) from Acremonium chrvsoaenum; (iii) the signal sequence of the B2 gene of Acr~nonium chrysocrenum and (iv) three 1~
different promoter regions.
In all the cloning and sub-cloning manipulati~s described in this patent application, Escherichia coli DFi5a 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.
A1. Construction of the expression cassette B2KEX
Protein B2 is an extracellular protease produced by the filamentous fungus Acrgnonium chrvso eq num. This protein is expressed and secreted in the late stages of growth of Acrgnonium_ chrvsoaenum (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 chrysoaenum. 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 prompter region of 477 base pairs.
When Acranonium chrys~ oaen,~m 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 arid NcoI, releasing a 560 by fra~nent 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 NcoI, releasing a large fragment (3740 bp), which was purified from a 0.8~ agarose gel. This fragment was ligated with the 560 by BamHI-NcoI fra~nent from pJElA, yielding plasmid p43)aB2CT
(4300 bp).
Plasmid p43bB2CT was digested with NcoI, treated with the Klenow fragment of DNA polymerase I (in order to obtain blunt ends) and then digested with Stul, yielding a fragment of 3874, by that was also purified from a 0.8~ agarose gel.
The single-stranded oligonucleotides ThS1 and ThS2 (sequences shown below) where used, using plasmid pThIX as a template, to amplify by polymerase chain reaction (PCR) the KEX2-like and thatmnatin sequences present in pThIX. The first 18 nucleotides present in ThSl correspond to the KEX2-like sequence.
ThSl: 5 ' - ~ 'A ~ AAA AC' A~1A A~ ATGGCCACCT'ICGAG - 3 ' Arg Met Lys Arg Lys Arg ThS2: 5'- TTA TTA GGC GGT GGG GCA - 3' A 655 by DNA fragment was obtained by PCR using plasmid pThIX
as the template and ThSl and ThS2 oligonucleotides as primers. This DNA 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 Saccharornyces cerevisiae.
Plasmid p43bB2CTTh was digested with BamI~, treated with calf intestinal- alkaline phosphatase (CIP) and purified from a 0.8~ agarose gel. A 900 by BamKt-BamHI fragment from pJElA
was also isolated. Subsequent ligation of these two DNA
fragments generated plasmid pB2KEX (5430 bp). The 900 by 1'7 BamHI-BamHI fragment from pJElA contains the B2 gene promoter sequence (477 bp), the leader peptide sequence (318 bp) and 107 by of the amino terminal sequence of the B2 gene.
Plasmid pB2KEX was then digested with XbaI, treated with the Klenow fragment of DNA polymerase I (in order to obtain blunt ends) and then digested with Sall, yielding a fragment of 2400 by that was purified in a 0.8~ agarose gel. Plasmid pJL43b was digested with HindIII, also treated with the Klenow fragment of DNA polymerase I, and then digested with XhoI. A fragment of 4500 by 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 pJL43b1 were digested with SacI and StuI, 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 pB2KThb1 (7020 bp; Fig. IC). Plasmid pJL43b1 is a derivative of plasmid pJL43b, where the promoter that drives expression of the phleomycin resistance gene (PpcbC from Penicillium chr~ocrenum) was substituted by the glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter from Asperaillus ~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 chrysoc~enuiin; ( iii) the signal sequence of the B2 gene of Acremonium chrvso eq num and (iv) the promoter region of the B2 gene of Acremonium chryso e~. In this particular construct, expression of the phleomycin resistance gene (phleo) is driven by the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene from Asperaillus nidulans.
A2 Construction of the expression cassette GDHTh A 2.1. Cloning of a DNA fracanent of Asperaillus awamori containing the qdhA gene.
~ awamori ATCC 22342 was used as the source of DNA and RNA. A. nidulans mutants A686 (gdhAl, yA2, methH2, galA1) and ~ nidulans A699 (gdhAl, biA1) (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 A.
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. E. coli NM539 served as host for Lambda GEM12 (Prcgnega Co., Wis) phage derivatives.
Filamentous fungi were routinely maintained on solid Power sporulation medium (F. Fierro et al. , Appl. Microbiol . .
Biotechnol. 1996, vol-. 43, pp. 597-604) at 30pC 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 28gC
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. Antibiot. 1984, vol. 37, pp. 503-511) were incubated with a 15 ~ seed culture and grown at 30gC for 48-72 h in a rotary shaker, as described above.
A.2.1.1. Asperaillus awamori aen~nic 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 reccenbinant phages .
In the next step, and using as probe a 2.6 kb BamHI fragment containing the gdhA gene of Neurosgora 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+ plasnid, 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 ( Prcanega Co . , Wis . ) by digestion with exonuclease III
from appropriate ends, followed by removal of single-stranded DNA with S1 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 autcenatic 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 (ORF1) 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 WO 99/51756 PCT/EP99/02243 _ of pB1.7 , as shown in Figure 3 . The 5 . 5 kb and 1. 7 kb BamFiI
fragments were mapped in detail.
A 2.1 kb Xbal-Xbal fragment corresponding to the right end of 5 plasmid pB5.5 was subcloned in pBluescript SK+ plasmid, creating plasmid pBSGh. More specifically, this 2.1 Kb XbaI-XbaI fragment was generated by digesting pB5.5 at an internal Xbal site and at a second XbaI 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 ORF1 (1380 bp), which started at an ATG located 740 by 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 by into the adjacent 1.7 kb fragment. ORF1 was preceeded by a 740 nucleotide region that contained the necessary signals required for transcription initiation and regulation (see SEQ. ID No. 1).
ORF1 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 IA and I$ for intron I, and IIA and II$ 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 94~C for 5 min, 50~C for 1 min, 72pC for 1 min followed by 30 cycles at 94~C for 1 min, 504C for 1 min, 724C for 1 min and finally one cycle at 72qC for 8 min.
Oligonucleotides:

IA 5 ' TCT AAC CTT CCT CAC 3 ' ATG

IB 5' CTT ACC ACC ACC CAT 3' ACC

IIA5' TTC TGT GTT TCC TTC 3' CGC

IIB5 ' CTT GAA CTT GTT GGC 3 ' GTA

A RF1 encodes a puta tive NADP-dependent glutamate 2.1.2.
~b dehydrocrenas a ORF1 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 ORF1 with other proteins in the SWISS-PROT data base showed that the encoded protein has a high homology with NADP-dependent glutamate dehydrogenases of A. nidulans (84.7 of identical amino acids), N. crassa (74.4 identity), Saccharomyces 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 NADP-dependent glutamate dehydrogenases that catalyze the reductive amination of a-ketoglutarate, in the presence of ATP, to form. L-glutamate. The protein encoded by ORF1 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)-[DNS]-[PL] (PROSITE PS00074). The lysine residue K11° located in the glycine-rich region GGGK11°GG
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 ORF1 was named gdhA.

1 3 The cloned gene complements A nidulans adhA 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 ~ awamori gdhA gene in a 2570 by Xbal-XbaI
fragment. This fragment contains also an upstream promoter region of 740 by and a 322 by region downstream from ORF1 (gdhA gene). The 2570 by XbaI-XBaI fragment was inserted into the XbaI ;site of the fungal vector p43gdh, which contains the phleomycin resistance marker under control of the A.
awamori gdhA prompter as shown later in this patent application.
Seven transformants of A. nidulans A686 with the ~ awamori gdhA gene and 15 transformants of A. nidulans A699 were analyzed on minimal medium supplemented with different concentrations (10, 50 and 100 mM) of ammanium 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 ~ nidulans mutants A686 and A699 grow very poorly in plates with 100 mM ammanium sulfate, whereas three randomly selected transformants grow very well in this medium. The residual growth of A. nidulans gdhA mutants A686 and A699 in ammanium 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 sec and glutamate dehydrogenase activity that allows partial growth of these mutants .
A 2 1 4 Glutamate dehydrog~enase activity in the transformants Nicotinamide adenine dinucleotide phosphate (NADP)-specific glutamate dehydrogenase (NADP-GDH) activity was assayed by following the reductive amination of a-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 nancmol of NADPH per minute.
To confirm the complementation results, the NADP-dependent glutamate. dehydrogenase activity was measured in the nidulans ~dhA mutants A686 and A699, and in three randomly selected transformants complemented with the ~ 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 belay 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, sho~n~ed relatively high levels of glutamate dehydrogenase activity, perhaps due to integration of more than one copy of the gdhA gene in the gencene 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 ~ awamori gdhA gene.
strain t = 24 h t = 48 h t = 72 h A. Awamori 550 0' 0 A686 0 0' 0 A 2 1 5 Characterization of the x~romoter region of the crdhA
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 ~..t,g of mRNA obtained from mycelia gro~nm in 1~FA 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 by 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 by upstream of the ATG initiation codon.
A 2 1 6 The crdhA gene is transcribed as a monocistroni~
transcript of 1 7 kb and its e~p~ession is regulated by nitrocren .
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 iii 1~FA medium with 55.5 mM
glucose and 10 mM ammonium sulfate as carbon and nitrogen sources, respectively. For nitrogen regulation studies, the NmFA 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 l.i.g) was run on a 1.2~
agarose-formaldehyde gel. The gel was blotted onto a nylon 5 filter (NYTRAN 0.45; Schleicher and Schuell) by standard methods. The RNA was fixed by W irradiation using an W-Stratalinker 2400 lamp (Stratagene, La Jolla, Calif.).
For slot blotting, the RNA ~(5 ~,.t,g) was loaded on a filter 10 (NYTRAN~ 0.45) by vacuum in a Bio-Dot SF Microfiltration apparatus:(Slot Blotting, Bio-Rad). The RNA was fixed by W
irradiation as above. The filters were pre-hybridized for 3 h at 42 gC in 50~ form~nicle, 5 x Denhardt' s solution, 5 x SSPE, 0.1~ SDS, 500 ~Lg of denatured salmon-sperm DNA per ml, and 15 hybridized in the same buffer containing 100 ~.g of denatured salmon-sperm DNA per ml at 42~C for 18 h, using as probe an internal DNA fra~nent (0.694 kb PvuII) of the A. awamori gdhA
gene. The filters were washed once in 2 x SSC, 0.1~ SDS at 42~C for 15 min, once in 0.1 x SSC, 0.1~ SDS at 42~C for 15 20 min, and once more in 0.1 x SSC, 0.1~ SDS at 55qC for 20 min and then autoradiographed with Amersham X-ray film. mRNA was purified from total RNA by using the Poly(A) Quick mRNA
isolation kit (Stratagene, La Jolla, Calif.).
25 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 i~-actin gene mRNA, as shown in Figure 7. Since ORF1 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 f~-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. ~wamori, gdhA
hybridizing RNA was compared to f3-actin hybridizing RNA in NmFA medium with ammonium sulfate (Figure 8A) and expressed as the ratio of counts in the gdhA-hybridizing band to the i3-actin hybridizing counts (Figure 8B). Results indicate that expression of both genes (gdhA and f5-actin) is associated with the growth of A awamori but whereas low steady state levels of i3-actin mRNA remained in the cells until 96 hours of growth, the levels of glutamate dehydrogenase mRNA
decreaseddrastically after 48 hours.
The glutamate dehydrogenase enzymatic activity detected when ~ awamori is groHm in NmFA 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 ~ 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 f3-actin gene) were observed in media that contained nitrate, glutamine or nitrite as nitrogen source. These results show that the NADP-dependentglutamate dehydrogenase is subject to a strong nitrogen regulation at the transcriptional level.
The glutamate dehydrogenase activity in 24-hour cultures grown in I~FA medium containing different nitrogen sources, WO 99/51756 PCT/EP99/02243 _ 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. awarnori 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 ~ awamori cultures grown for 24 h in MDFA medium supplemented with different nitrogen sources.
Nitrogen source Total Activity Specific 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 thaiunatin expression cassette similar to 'the one described previously was constructed. Plasmid pBSGh was used as a template to obtain a 750 by 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) .

WO 99/51756 PC'T/EP99/02243 28' gdhl : 5 ' - TTTT GTC TTG CGA CGG CGT ATT GCT - 3 ' Sal I
gdh2 : 5 ' - TTTT CCA~ TCT GAA GGG GAG GAT TGA - 3 ' NCO I
This amplified DNA fragment was digested with SalI and NcoI
and purified in a 0.8~ agarose gel.
Plasmid pJL43 (a derivative of pJL43b, Dr. Jose Luis Barredo, Ph.D. Thesis, Universidad de Lebn, Leon, Spain) was digested with Sall and NcoI and a large fragment (3740 bp) was purified in a 0.8~ agarose gel. This DNA fragment was then ligated with the SalI-NcoI fragment previously amplified, yielding plasmid p43gdh (4500 bp), where the pcbC promoter from Penicillium chrysoaenum has been replaced by the gdhA
promoter from As~aillus awamori.
In the next step, plasmid p43gdh was digested with NcoI, treated first with the Klenow fragment of DNA polymerase I
and then with calf-intestinal phosphatase (CIP). In paralell, a fragment of 1140 by 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 belcaa) . This 1140 by fragment was digested with BamHI and then treated with the Klenow fragment of DNA
polymerase I. From this reaction mix a 425 by DNA fragment containing the amino terminal sequences of the B2 gene was purified from a 1.0 g 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 by 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 thatunatin II gene.
For this part of the work, plasanid pB2KEX was used.
pB2KEX was sequentially digested with XbaI, treated with the Klenow fragment from DNA polymerase I and finally digested with BamHI . A fragment of 4637 by was purif ied from a 0 . 8~
agarose gel. In paralell, plasmid p43gdhB2 was sequentially digested with SalI, treated with the Klenow fragment from DNA
polymerase I and finally digested with BamHI. A fragment of 1173 by was purified from a 0.8~ agarose gel. The ligation of these two fragments yielded plasmid pGDHTh (5810 bp), where a new SalI site was created. This allows for the excision of the complete GDHTh cassette as a 2670 by SalI-SalI fragment.
Starting with plas~ntid pGDHTh, two new plasmids were constructed. The first one was p43GDTh, constructed as follows. Plasmid pJL43 was linearized by digestion with SalI
and ligated to a 2170 by SalI-DraI 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 HindIII, and purified from a 0.8~ agarose gel. In paralell, plasmid pGDHTh was digested with Ec1136II (or SacI*, a variant of SacI from Fermentas that recognizes the standard SacI restriction site but leaves a blunt end), HindIII and DraI. A fra~nent of 2175 by was purified from an agarose gel. Ligation of these two fra~nents yielded plasmid pGD71 (see Fig. 10, part C).
Plasmids p43GDTh and pGD71 contain a cassette to express thatnnatin 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 5 the B2 gene of Acremonium chrysoaenum, (iii) the promoter region from the Aspercrillus 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 10 Pgnicillium chrysog~enum. Plasmid pGD71 contains the hygromyciri B resistance gene driven by the glyceraldehyde-3-phosphate dehydrogenase prompter from Asperaillus nidulans.
A 3. Construction of the expression cassette GPDTh The expression cassette GPDTh is similar to the expression cassette B2K~, exc~t that the B2 promoter from Acr~nonium chrvsogenum has been replaced by the promoter from the glyceraldehyde-3 phosphate dehydrogenase (named "gpd" from now on) gene from As~g~illus nidulans.
The complete promoter region of the gpd gene is present in plasmid pAN52-1 (P.J. Punt et al. , J. Biotecnol. 1990, vol.
17, pp. 19-34). A SacI-NcoI fragment (880 bp) from pAN52-1 has been subcloned, generating pJL43b1.~
Plasmid pJL43b1 was digested with NcoI and treated first with the Klenow fra~nent of DNA polymerase I and then with calf-intestinal phosphatase (CIP), as shown in Figure 11. In parallel, a 1140 by fragment of DNA was obtained by DNA
amplification using the PCR technique, using pJE2A as template and oligonucleotides NTB2b 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 by 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 thatmnatin II gene. For this part of the work, plasmid pB2KEX was once again used.
pB2KEX was sequentially digested with XbaI, treated with the Klenow fragment from DNA polymerase I and finally digested with BamHI. A fra~nent of 4637 by was purified from a 0.8~
agarose gel. In paralell, plasmid pblB2 was sequentially digested with BamHI and Ec1136II (or SacI*) (leaves blunt ends), and a 1300 by fragment was purified from a 0.8~
agarose gel. The ligation of these two fra~nents yielded plasmid pGPDTh (5800 bp).
In the next step, the GPDTh cassette was isolated from pGPIyfh by digestion with Ec1136II (or SacI*), HindIII and DraI, yielding a DNA fragment 2800 by long. In parallel, plasmid pB2KThb1 was sequentially digested with BamHI, treated with the Klenow fra~nent from DNA polymerase I and finally digested with HindIII. A 4500 by 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 prompter from the~B2 gene of Acrgnonium ch~ysocrenum has been replaced by ~ the promoter from the gpd gene from Aspercrillus nidulans .
B. Strains used and transformation protocol Asperaillus awamori strain NRRL312 was obtained from the American Type Culture Collection (ATCC). Using standard mutagenesis tectmiques 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 Asperaillus awamori strain LpR66.
The three expression cassettes that have been described previously were used to transform Asperg'h~lus awam~ri 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 Ei.g/ml of phleomycin.
Therefore, all transformants were selected in plates with 25 ~.g/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 Melton (see above) with some modifications. A plate containing Power medium was inoculated with 10' spores. This plate was incubated for 72 hours at 30~C, 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 28QC. The mycelium obtained from this growth was filtered through a 30 ~.m 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 30QC until protoplasts WO 99!51756 PCT/EP99/02243 _ 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 ~.l of protoplast solution was mixed with 10-20 ~,g of DNA
and left in ice for 20 minutes. After this time interval, 500 ~.1 of PTC were added, and left at room temperature for another 20 minutes . Then, 600 ~,l 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 30$C until the transformants were visualized as individual colonies. TnThen 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 ~.~.g/ml of phleomycin. Confirmation screens were then performed using phleomycin concentrations as high as 200 Ei.g/ml.
Transformants were analyzed by PCR to detect whether the thaumatin II gene had been incorporated into their gencene 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).

WO 99/51756 PCT/EP99/02243 _ ,~ Recombinant strains that produce thaumatin C.1. 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~ (NHa)ZSO4; 0.13 NaHaP04.2Hz0; 0.2~ MgS04.7HZ0; 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 HZS04.
MDFA medium: 1.2~ L-asparagine; 0.8~ of salt solution I [2~
Fe (I~i4) Z (S04) 2. 6HZ0] ; and 14 .4~ of salt solution II [10.4 KZHP04; 10.2 KH2POq; 1.15 NaZCuS04.5Hz0; 0.2~MgS04.7H20; 0.02 ZnS04 . 7H20; 0 . 005 CuS04 . 5Hz0; 0 . 05~ CaCla . 2H20] . 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 anal~rsis 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, exc~t that the pH of the medium 5 was maintained constant at a pre-set value, and adjusted by the automatic addition of either 30~ NaOH or 0.5N HZS04.
C 1 3 Analytical methods 10 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 present.
15 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 20 filtrate was aliquoted and frozen for further analysis.
Thau<natin concentration in the culture broth was determined by an enzyme-linked immunoassay (ELISA) and by immunoblotting (Western blot) analysis, essentially as described (cf . EP
25 684312), using an anti-thaumatin polyclonal antibody. For immunoblotting, samples were sometimes concentrated as follows : 500 ).~,1 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 30 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

WO 99/51756 PCT/EP99/02243 _ DIAG~10STICS kit (Procedure number 510).
Maltose concentration in the culture broth was determined as follows: 250 ~.l 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~ HZSOq) 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 end 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 . Thatunat in nroduc incr s trains r.2.1. Strain TB2b1-44 This strain is a derivative of Lpr66 that was obtained by transformation of the aforementioned LpR66 strain with the expression plasmid pB2KTh-b1. This expression cassette contains the synthetic thaimnatin II gene under the control of the promoter of the B2 protein from Acremonium chrysoaenum.
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 30pC 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=i L-asparagi.ne 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:
Coleccibn Espar~ola de Cultivos Tipo (CELT) Edificio de Investigacibn, 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 prompter of the gdhA gene from Aspercrillus awamori. In shake-flask cultures with NmFA 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 TB2b1-44. 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 Vin. 1~9.~FA 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 Asperaillus awam~ri is more efficient than the B2 protein promoter from Acranonium ~hrvsoQern~m.
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 AsperQll~.~is nidulans . In shake-flask cultures with NB7FA medium this strain secretes 9-10 mg thaumatin/liter.
x.2.4. Double transformants Strains TB2b1-44 and TGP-3 were re-transfornned with expression plasmid pGD7l, 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 shov~m that re-transformation of strain TGP-3 did not result in better producing strains. However, re-transfornnation of TB2b1-44 did result in better producing strains when cultured in shake-flasks under the standard conditions mentioned before.
Table 3: Production of thawnatin in shake flasks by retransformed strains grown in NmFA medium for 96 h.
Quantification by ELISA. All strains were retransformed using hygromicin B resistance as selection marker.
Transformant 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 TB2b1-44-GD1 3.84 TB2b1-44 TB2b1-44-GD2 0. 00 TB2b1-44 TB2b1-44-GD3 9.85 TB2b1-44 TB2b1-44-GD4 11.10 TB2b1-44 TB2b1-44-GD5 11.82 TB2b1-44 TB2b1-44-GD6 10.75 TB2b1-44 TB2b1-44-GD7 10 . 52 TB2b1-44 TB2b1-44-GD8 8.09 TB2b1-44 TB2b1-44-GD9 7 .13 TB2b1-44 ______________ _____________________________--___-____ D ~ Purification of recc~nbinant thaimnatin Two procedures were employed for the purification of recanbinant thaumatin. In the first one the fermentation broth was simply clarified, concentrated and diafiltered, 5 yielding a concentrated and cleaner extract that was used for sensory experiments to ascertain the sweet profile of the recanbinant thaumatin. The second procedure involved a classic purification protocol that yielded pure thaumatin.
10 D 1 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 15 submerged in ice. The clarified broth was then centrifugated at 6000 rpm for 15 minutes at 4qC.
The clarified fermentation broth was further concentrated by ultrafiltration using a ProFluxTM M12 Tangential Filtration 20 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 8-10~C 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 fermentatian broth was concentrated to the desired volume, the solution was diafi.ltered in order to remove low molecular weight solutes (Salts; sugars, etc.).
The system configuration allows the operation in the "pined diafiltration with autcanatic 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 dial~sate supply is exhausted, the system and the secondary pump will shut off autcanatically.
The diafiltered solution is drained from the system, sterilized by filtration (Stericup, 0.22 Eun, Millipore) and stored at 4QC.
D 2 Purification of recanbinant ~haumatin to homogeneitv Reccanbinant 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 7Ø
This mix was then passed through a Sephadex G-25 column (for desalting purposes) and eluted with the same buffer. Fir_ally 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 NaCl linear gradient ( 0-400 mM) . Thaumatin is eluted from this column in WO 99/51756 PCT/EP99/02243 _ almost pure form as detected by Coomassie Blue staining.
Table 4:Purification of thaumatin from the fermentation broth for growing strain TGDTh-4 in MDFA medium SAMPLE VOLLaHE 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.

SEQUENCE LISTING
SEO ID 1. Nucleotide sequence of 2570 by of a DNA fragment present in plasmid~pBSGh, as well as the 5' end from plasnid pBl.7, which contains the gdhA gene of gi . The . numbers to the right indicate the numbering of the sequence.
The promoter region of the gdhA gene of $," precedes the initiating ATG codon at position 742 in the sequence. The initiation of transcription is at T of position -86 with respect to the translation initiation triplet (position 655 in the sequence). The part of the gene encoding the protein begins at position 741. The numbers underneath the amino acids refer to their numbering. There are a total of 460 amine acids. The two introns present are also showil.
TCTAGATTGC GACGGCGTAT TGCTTATCCT TAGfAGGACT 60 GAAAAGACTG T?1GGCGTG? ACCAATOGCT CATAGTACCA 120 CTCGCTZCGA GAAAGCAATC ARAAAAAAAT CCTATCC1'AC 180 CCTACCCTAC CCTAATACTT

2 CCATTGOCAC CCGATTCCTC CCGATAGfAG AGOGGGOGAC 240 CTCAAACTAT CGACTAACAG

CCQ'sAAG2TC GGOGGCCACC GCCAAAOCCG CCOCGGAAGC 360 CGGLCTCATT TGOCGTTTGG

GCGTGCCAGG AAATGCOGCC TGCAGC(A',AG ACTCCCTAG?420 GTOGTC1GTG TTC4rCTGTGT

GTGGATGGCC TGAGGGOGGG

S CTCTACLCTC TTC~CTCTCT

ACATAAATCG GCCTTTCCCC

CCAGTTAAAA AAGTTCTCTC

AATCAATCCT CCOCTTCAGA ATG TCT AAC CTT CC? 770 CAC GAG CCC GAG TTC

Met Ser nen Leu Pro His Giu Pro G1u Phe 1 s . . io , CGAAATTGAT GAT~GAAAAA

Glu Gin Ala Tyr Lys '-ACC CTT GAG AAC TCC

Glu Leu Ala Ser Thr Leu Glu Asn 8er GCC CTT GCT GTC GTC

Thr Leu Phe Gln Lys Asn Pro Glu Tyr Arg Lys Ala Leu Ala Val Val GTC TGG GAG GAT GAT

Ser Val Pro Glu Arg Val Ile Gln Phe Arg Val Val Trp Glu Asp Asp GTC CAG TTC AAG AGC
S

er Ala Gly Aan Val Gln Va1 Asn Arg GIy Phe Arg Val GIn Phe Asn 60 65 ' 70 CAC CCC TCC GTC AAC

ZO Ala Leu Gly Pro Tyr Lys G1y Gly Leu Arg Phe His Pro Ser Vai Asn ATC TTC AAG AAT GCT

Leu Ser I1e Leu Lys Phe Leu Gly Phe G1u Gin Ile Phe Lys Aan Ala GGT TCC GAC TTC GAC

Leu Ths Gly Leu Asn Met Gly Gly Gly Lys Gly Gly Ser Asp Phe Asp ' 110 115 120 105 ' CCC AAG GGC ~JIiAG TCC GAC AAC GAG ATC CGT 1212 CGC TTC TGT GTT TCC TTC

Pro Lya G1y Lys Set Asp Asn G1u Ile Arg Arg Phe Cya Val Ser Phe 125 , 130 135 25 ATG ACC GAG CTC TGC AAG CAC ATC' GGT GCC GAC 1260 ACT GAT GTT CCC GCT

Met Thr Glu Leu Cys Lys His Ile Gly Ala Asp Thr Asp Val Pro A1a TTC CTC TTC GGC CAG

3O Gly Asp Ile Gly Val Thr Gly Arg Glu Val Gly Phe Leu Phe Gly Gln CTC ACC GGT AAG GGT

Tyr Arg Lya Ile Arg Asn Gln Trp Giu Gly vai Leu Thr Gly Lya Gly GCC ACC GGT TAC GGT

G1y Sex Trp Gly Gly Ser Leu Ile Arg Pro Glu Ala Thr Gly Tyr Gly GTT GTC TRC GTATGTCAAT TCCTCT'iCTT ATC~1TTATCT1453 ATGrATNICA

Val Val Tyr GCT CAC GCC ACC AAC

Tyr Val Glu His Met Ile Ala His Ana Thr Asn GGC CAG GAG TCC T?C AAG GGC RAG CGC GTT GCC 1552 ATC TCC GGT TCC GGT

5O Giy G1n Giu Ser Phe Lya Gly Lys Arg Val Aie iie Ser G1y Ser Giy 215 . 220 _, 225 230 GAG CTC GGC GGT TCC

Asn Vai Aia G1n Tyr Ala Ala Leu Lys Val I1e Glu Leu Gly Gly Ser ATC ATC AAC GGC GAG

Yal Val Ser Leu Ser Aap Thr GIn Gly Ser Leu Ile ile Asn Gly Giu O GCT CAG'ACC AAG GTC

Gly Ser Phe Thr Pro Glu Glu Ile Giu Leu Ile Aia Gln Thr Lya Val:, GCT CCC .TTC AGC GAC

Glu Arg Asn Glu Leu A1a Ser Ile Va1 Gly Ala Ala Pro Phe Ser Asp GCC AAC AAG 1'TC,AAG TAC ATT GCT GGT GCC CGC 1792 CCC TGG GTT CAC GTC

7O Ala Asn Lys Phe Lys Tyr Ile Ale Gly Ala Arg Pro Trp Yal His Val GGC AAG GTC GAC GTC GCT CTC CCC ?CC GCT ACC 1840 CAG AAC GAA GTT TCC

Gly Lya Vnl Asp Yal Ala Leu Pro Ser Ala Thr Gln Asn Glu Val Ser TGC AAG TTC ATC GCC

Gly Glu GIu Ale Gln Val Leu Ile Asn Ala Gly Cys Lys Phe Ile Ala 330 335 ~ ~ 340 ATC GAC ACC TTC GAG

Glu G1y Ser Asn Met Gly Cys Thr Gln Glu Ala Ile Asp Thr phe Glu 345 ~ 350 355 ATC TGG TAC GCC CCC

Ala His Arg Thr Ala Asn Ala Gly Ala Ala Ala ile Trp Tyr Ala Pro TCC GGT CTG GAG ATG

Gly Lys Ala Ala Aan Ala Gly Gly Val Ala Val Sar Gly Leu Glu Met 2 375 . . 380 385 390 O

GAG GAG GTT GAT GCC

Aln Gln Asn Ser Ala Arg Leu Ser Trp Thr Ser Glu G1u Yal Asp Ale 2 CGT CTT AAG GAC ATC ATG CGC GAC ; TGC TTC 2128 S AAG AAC GGT C?T GAG ACT

Arg Leu Lya Asp Ile Met Arg Asp Cys Phe Lys Asn Gly Leu Glu Thr 30 CTG CC? TCC CTG GTG

Ala Gin Glu Tyr Ala Thr Pro Ala Glu Gly Vel Leu Pro Ser Leu Val GCT GCC GCC ATG AAG

Thr Gly Ser Asn Ile Ala Gly Phe Thr Lys Val 35 Ala Ala Ala Met Lys GAC CAG GGT GAC TGG TGG TAAATGC~GA AAOCCGCAAA2272 CCOCCGOGGC

Gln Gly Asp Trp Trp 4so .

T?ATGTCATG ACGP1TTATGT AGTTTGATGT TCCCTT'I~CAG2332 CGCGGA1GGA TAGi,GGCGCC

GGTGTTT1'CT TGQ'AG1TTA GA1GGA1GCA TAATGATATC 2392 CTT1'TCTTAA TCCTCAAATT

45 CTTGTA7~fTT GTTGTATCAA TAGI'AGATAA TAUAC1GTA 2452 GT(91ACTACC CT1GCATCTT

CAGTATTTGC AGATGCATTC ATCfCTATTC CGiIGCAf9ITG2512 CACAAAOCCA TCOGACOGCA

CT~1AAC~1AC TATCTAGA

SEO ID 2. Amino acid sequence of the glutamate dehydrogensse A tgdh A) protein from Asnerro;lly~ ~,as deduced from the nucleotide sequence in SEQ ID 1.
Met Ser Asn Leu Pro His Glu Pro Glu Phe Glu Gln Ala Tyr !Lys GIu 1 5 10 . 15 60. Leu Ala Ser Thr Leu Glu Asn Ser Thr Leu Phe Gln Lys Asn Pro Glu Tyr Arg Lys Ala ~Leu Ala Val Val Ser Val Pro Glu Arg Val Ile Gln ss Phe Arg Val Val Trp Glu Asp Asp Ala Gly Asn Val Gln Val Asn Arg 50 55 ~ 60 Gly Phe Arg Val Gln Phe Asn Ser Ala Leu Gly Pro Tyr Lys Gly Gly 65 70 ; 75 80 Leu Arg Phe His Pro Ser Val Asn Leu Ser Ile Leu Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu Thr Gly Leu Asn Met Gly Gly Gly Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Asn Glu Ile Arg krg Phe Cys Val Ser Phe Met Thr Glu Leu Cys Lys His Ile 130 ; 135 190 Gly Ala Asp Thr Asp Val Pro Ala Gly Asp Ile Gly Val Thr Gly Arg 145 150 . 155 160 Glu Val Gly Phe Leu Phe Gly Gln Tyr Arg Lys Ile Arg Asn Gln Trp . 165 170 175 Glu Gly Val Leu Thr Gly Lys Gly Gly Ser Trp Gly Gly Ser Leu Ile _. 180 1B5 190 Arg Pro Glu Ala Thr Gly Tyr Gly Val Val Tyr Tyr Val Glu His Met Ile Ala His Ala Thr Asn Gly Gln Glu Ser Phe Lys Gly Lys Arg Val 210 215 ~ 220 Ala Ile Ser Gly Ser Gly Asn Val Ala Gln Tyr Ala Ala Leu Lys Val Ile Glu Leu Gly Gly Ser Val Val Ser Leu Ser Asp Thr Gln~ Gly Ser Leu Ile Ile Asn Gly Glu Gly Ser Phe Thr Pro Glu Glu Ile Glu Leu Ile Ala Gln Thr Lys Val Glu Axg Asn Glu Leu Ala Ser Ile Val Gly Ala Ala Pro Phe Ser Asp Ala Asn Lys Phe Lys Tyr Ile Ala Gly Ala Arg Pro Trp Val His Val Gly Lys Val Asp Val Ala Leu Pro Ser Ala 305 310 . 315 320 Thr Gln Asn Glu Val Ser Gly Glu Glu Ala Gln Val Leu Ile Asn Ala 325 ~ .'330 ~ 335 Gly Cys Lys Phe Ile Ala GIu Gly Ser Asn Met Gly Cys Thr Gln Glu 390' 345 350 Ala Ile Asp. Thr Phe Glu Ala His Arg Thr Ala Asn Ala Gly Ala Ala WO 99/51756 PCT/EP99/02243 , Ala Ile Trp Tyr Ala Pro Gly Lys Ala Ala Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu Met Ala Gln Asn Ser Ala Arg Leu Ser Trp Thr Ser Glu Glu Val Asp Ala Arg Leu Lys Asp Ile Met Arg Asp Cys Phe Lys Asn Gly Leu Glu Thr Ala Gln Glu Tyr Ala Thr Pro Ala Glu Gly Val Leu Pro Ser Leu Val Thr Gly Ser Asn Ile Ala Gly Phe Thr Lys Val Ala Ala Ala Met Lys Asp Gln Gly Asp Trp Trp

Claims (35)

1

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; and a nucleotide sequence that hybridizes under stringent conditions to that defined in (a), with the proviso, that the sequence is not the promoter of the gdh gene from Aspergillus nidulans.

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 the sequence is not the promoter of the gdh gene 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;
and (b) a nucleotide sequence that hybridizes under stringent conditions to that defined in (a), with the proviso, that the sequence is not the gdh gene from Aspergillus nidulans.

7. A DNA sequence according to claim 6 which has the sequence of nucleotides numbered 741-2242 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 the sequence is not the gdh gene 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 the glutamate dehydrogenase is not the glutamate dehydrogenase 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 oryzae or Aspergillus sojae; ii) B2 from Acremonium chrysogenum;
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 sojae 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 pThIX disclosed in EP 684312.

27. A DNA construction comprising a gdh promoter from a fungus of the genus Aspergillus operatively linked to a DNA
sequence encoding a recombinant protein.

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 sojae.

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
construct 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 to 6 as a probe for the identification and isolation of a glutamate dehydrogenase gene and/or a promoter sequence of a glutamate dehydrogenase gene.