1. A fungus, wherein the areA gene by recombinant DNA technology has been modified in a way by which it cannot be expressed in a way providing for a functional AreA activator.

2. The fungus of claim 1, wherein said inactivation has been obtained by deletion of all or parts the areA gene.

3. The fungus of claim i, wherein said inactivation has been obtained by interfering with the regulation of the expression signals regulating the expression of the areA gene itself.

4. The fungus of claim i, wherein said inactivation has been obtained by using anti-sense technology.

5. The fungus of clairr, 1, wherain said inactivation has been obtained by inserting extra DNA internally in the areA gens. Li) producing a DNA construct comprising the areA gene wherein an internal part has been substituted, deleted, or extra DNA has been inserted, iii) transforming said fungus with the construct, and iv) selecting traneformants which are areA' .

9. A method for producing a fungus according to claim l, wherein said inactivation has been obtained by using anti-sense technology, which method comprises i) construction of an expression plasmid which gives rise to synthesis of an RNA molecule complementary to the mRNA transcribed from the areA gene, ii) transformation of the hcsz fungus with said expression plssmid and a suitable marker, either on separate plasmids or on ths same plasmid, iii) selection of transforTnants using said marker, and iv) screening selected transfcrmants for strains exhibiting h reduction in the synthesis of the AreA product.

10. A process for the croduction, cf a desired gene product, whereby a fungus according tc may of the claims 1 to 7 is crltivared in a suitable growth medium at appropriate con-ait ions and the desired gene product is recovered and purified.

11. A process for the production of a desired gene product, whereby a fungus according tc any of the claims 1 to 7, which has been tran.sformed tc .integrate a ZiNA sequence coding ror the desired gene product into the genome of the fungus in a functional manner, cultivated in a suitable growth medium at appropriate ate conditions and the desired gene prcOuct is recovercd and curified.

12. A process for producing a desired polypeptide comprising cultivating a fungus in an appropriate growth medium and recovering said polypeptide from said culture, said fungus carrying a recombinant DNA construct capable of causing expression of said polypeptide or a precursor thereof in said fungus, said fungus further being characterized by producing lower amounts of functional AreA than the wild-type of said fungus.

13. A method according to claim 12, wherein said fungus has been modified to produce lower than wild-type amounts of AreA by a process comprising transforming a parent of said fungus with a DNA construct capable of causing reduced production of functional AreA when integrated in the geneome of said fungus.

14. A method according to claim 12, wherein said polypeptide is secreted to the extracellular medium by said fungus.

15. A method according to claim 12, wherein said fungus produces higher amounts of said polypeptide than a similar fungus where said similar fungus produces AreA in amounts similar to those produced by the wild-type ol aaid fungus, said similar fungus being identical to and fungus in all other respects.

19. The process of any of the claims 10 to 116, wherein said desired gene product is a therapeutically active peptide or protein.

20. The process of claim 19, wherein said therapeutically active peptide or protein is selected from the group comprising insulin, growth hormone, glucagon, somatostatin, interferon, PDGF, factor VII, factor VIII, urokinase, tPA, EPO, or TPO.

21. A gene produci: produced in accordance with any of the processes 10 to 20.

22. A DNA sequence coding for the areA gene from A. aryzae (SEQ ID No. 1) or functional alleles thereof.

23. An ArsA activator from A. oryzae (SEQ ID No. 2). (24) A fungus substantially as hereinbefore described with reference to the accompanying drawings. (25) A nethod for producing a fungus substantially as hereinbefore described with reference to the acconpanying drawings. (26) A process for the production of a desired gene product substantially as hereinbefore described with reference to the accompanying drawings. (27) A gene product substantially as hereinbefore described with reference to the accompanying drawings.

The present invencion relates to fungi, which do not produce proteases. The fungi of the invention are useful as hosts for the production of proteins susceptible to protsolytic degrada¬tion by the proteases usually produced, and the invention con¬sequently encorr.passes processes for the production of proceins of interest in high yields by using the fungi of the invention. The invention also comprises methods for producing such fungi and DNA constructs to be used in these methods.


For a number of years it has been known that the regulatory gene areA which mediates nitrogen uietaboiics repression z.n A. nidulans influences the production of extracellular proteases (Arst & Cove, molec. gen. Genet. 126, (1975) 111-141).
The areA gene from A. nxdul^ns has been cloned (Caddick et al. , EM30 Journa.1 5, (1986) 1087-1050) and various modifications r>ade to it to evaluate functions of different regions in the activator protein encoded by this gene (Stankcvitch et: al. Mel. Microbicl . 1, (1993) Sl-87). Furtherrnore the gene coding the corresponding function in A. fimlgatus apparently has been clo¬ned recently (Kensel et ai. 2nd European Conference on Funga] Genetics, April 28 to May 1, 1S94, Book of Abstracts, Ell) .


ii) producing a DNA construct comprising the areA gene wherein an internal part has been substituted, deleted, or extra DNA has been inserted, iii) transforming said fungus with the construct, and iv) selecting transformants which are areA' .
The information obtained from the above mentioned cloning of the areA gene may also be used m connection with the well-known anti-sense technology, to construct an expression plasmid giving rise to synthesis of a RNA molecule complementary to the mRNA transcribed from the areA gene, and to transform the fun¬gus of interest therewith-
The invention furthermore relates to DNA constructs intended for use in the above mentioned methods.



The invention is described in further detail in the following parts of the specification with reference to the Examples and the drawing/ wherein
Fig.l shows the steps involved in the construction of HowBlOl, pIg. 2 shows the steps involved in the construction of pSK5 and
pSK9,
Figs. 3a and 3b show the steps involved in the construction of pToC2S6,
F:Lg. 4 shows the steps involved in the construction of pMTlSOS,
and
fig. 5 shows the steps involved in the construction of pToC56 .
In"the present specification the folloving definitions are used The expression areAA means 3 strain in which the areA gene is
The expz^ession a.reA' means s, strain vhich does not produce a functional Ar^A ac~ivator, Th:?. term ''loss of function" is alsc of ten used for this .
The expression "anti-sense techno log*/" describes methods such as disclosed in US Patent No. 5,190,931.


As indicaced the present invention relates in its first aspect to fungi, wherein the areA gene by recombinant DNA technology-has been modified such that it cannot be expressed in a way providing for a functional AreA activator.
This object may specifically be' obtained by deletion or dis¬ruption of the areA gene.
The cloning of the areA gene is 'described in the Examples.
AjtreA horr.ologs from other fungi could be cloned either by cross hybridization with one of the already known genes or by comple¬mentation of areA mutants; e.g. A. nidula.ns areA-18 or the .4. oryza.e areA deleted strain described in this application.
Methods for deleting or disrupting a gene are specifically described in wo 90/00192 (GenenQor).
.Methods for substituting DNA in a gene are also generally known, and can be accomplished ty substituting one or more continuous parts of the gene, but it may also be obtained ' by site directed mutagenesis generating a DlSiA sequence encoding a AreA activator variant that is not tunctional.
Another method by v;hich such an object may be obtained is by using anti-sense technology'.
The anti-sen.^e technologv' and how to employ it is described in
detail in tha aforementioned US Pazent No_ 5,190,931 ;Univer-
sity of New York).
A further method of obtaining said inactivation is by inserting extra DNA internally in the areA, gene, thereby giving rise to the expression of a dysfunctional activator protein.

In connection with this method information provided by the clo¬ning can be used to make DNA constructs that can be integrated into the areA gene, and even replace it with another gene, such as the pyrG gene.
A further method of avoiding the presence of the areA activator is by interfering with the regulation of the expression signals regulating the expression of the a.reA gene itself.
According to the invention the fungus preferably belongs to a genus selected from the group comprising -Aspergillus, Tricho-derma, Hiunlcola, Candida, Acremonium, Fusarium, and Penicil-lium
Among these genera species selected from the group comprising A. oryzae, A. nigsr, A. awamori, A. phoenicis, A. japcnicus, A.-foetidus, A. nidulans, T, reesei, r. harzianum, H. insulsns, H. lanuginosa, F. grairdrxearum, F. solani, P. chrysogenim, and
others ai^e preferred.
As indicated the invention also is meant to encompass the mezhod for producing the fungi of the first aspect of the invention, and wherein said inactivazion has been obtained by deletion of the areA. gene, which method comprises i} cloning of homologues of the areA gene from a fungus of
interest. ii) producing a DNA construct comprising the areA gene
wherein an internal part has been substituted, deleted,
or extra DNA has been inserted, iii) transforming said fungus with the construct, and iv) selecting transformants which are areA" .
Also included is the method for producing the fungi, wherein the inactivaticn has been obtained by using anti-sense tech¬nology. Such a mechod comprising i) construction of an expression plasmid which gives rise to
synthesi.s oi a RNA molecule corapleraen-ary to the mRNA
transcribed from the areA gene,

ii) transformation of the host fungus with said expression plasmid and a suitable marker, either on separate plasmids or on the same plasmid, iii) selection of transformants using said marker, and iv) screening transformants for strains exhibiting a reduc¬tion in the synthesis of the AreA product, e.g. by-analysis of the growth on various nitrogen sources.
A further aspect of the invention is meant to comprise DNA constructs for use in the above mentioned methods.
In respect of the former method said DNA constructs may com¬prise the areA gene wherein an internal part has been substi¬tuted, deleted, or extra DNA has been inserted.
The DNA construct may furthermore also comprise DNA sequences encoding a protein product of interest, such as those mentioned latez".
in respect of the latter anf.i-sense method r.he DNA construct may comprise an inverted DNA sequence of the areA gene connec¬ted to a functional promoter, whereby the mRNA is at least partially complementary to mRNA produced from the areA gene.
A further aspect of the invention relates co a process' for the production of a desired gene product, preferably a secreted gens product, whereby a fu.ngus according to che invention is cultivated in a suitable growth medium at appropriate con¬ditions and the desired gene product is recovered and purified.
In che case of a gene product expressed by a heterologous gene the DNA -sequence coding for the desired gene product may be a part of the DNA construct used for producing said fungus.
Normally, however, a separate -transformation of the fungus of the invention is performed in order to make the fungus capable of producing the desired product.

Methods for transforming fungi are well known m the art, of. e.g. EP 0 184 438 A2 (Gist-Brocades N.V.) and EP application no. 87103Q06 (Novo Nordisk A/S) and.
For indigenous products this ie of course not necessary, but in order to increase the production it may be an advantage to pro¬vide for multiple copIes of the gene encoding the protein of interest to be incorporated into the host.
The desired gene product is generally a peptide or protein, preferably an enzyme.
Among enzymes it is preferably selected from the group ccnpri-sing proteases, such as trypsin and chymosm; lipases, cutina-ses, cellulases, xylanases, laccases, pectinases, etc.
Another type cf desired gene product is generally a therapeuti¬cally active peptide or protein.
Among the therapeutically active peptide or protein the protein preferably is selected from the group comprising insulin, growth hormone, glucagon, somatostatin, interferons, PDGF, factor VII, factor VIII, urokinase, t-PA, CSF, lactoferrin, TFO etc.
The invention is explained i.n further detail in the Examples given below. These should, however, not in any way be construed as limiting the scope of the invention as defined in the ap¬pended claims.

Materials and Methods
Straing.
A. oryz&e, IF04177: available from Institute for Fermentation
Osaka; 17-25 Juso Hatnmachi 2-Chome Yodoga
wa-Ku, Osaka, Japan.
ToC9l3: The construction of this scrain is descri
bed in the Examples.
Genes
areA: This gene codes tor a regulatory protein controlling
nitrogen catabolism.
pyrQ: This gene codes for orotidine-S'-phosphate decarboxylase, an enzyme involved in the biosynthesis of uridine.
jbar: This gene was originally isolated from Strspto/nycss hycrrcscopIous and cndes lor phosphinothricin acetyltrans-ferase. The enzyme modifies phosphinothricin (=glufosina-te) and "hereby inactivates this compound which is toxic to bacteria, fungi and plants.
pUCllS : Viera and r-lesing J. Meth. En2vmol - 196 7 153 3-11
pSC2 r The construction of this plasmid is descx-ibed in the Examples.
pJers4 : A 2 , G kb subclone of pS02 in pUCllS . oJers4 contain?; a___funct ional /:. orvzaa fvrG aene .^
pS05 : The construction of this plasmid from pSC2 i.; described in the Examples.

pToC56; The construction of this plasmid is described in EP application no. 87103806.
pToC266: The construction of this plasmid is described in the
Examples.
pMTlS06; The construction of this plasmid from pBPlT (B. Straubinger et al. Fungal Genetics Newsletter 39(19921:82-83) and p775 (EP application no-87103806) is described in the Examples.
p777: The construction of this plasmid is described in 3P
application no. 87103806,
pHW470: The construction of chis plasmid is described in the ExaiT.ples.
EXAMPLE: 1
Constrctions of an Aspergillus oryzas RreAa. strain.
The axeA, strain v;as constructed by Che following steps. The P.. oryzae pyrG gene was cloned and an A. oryzae pyrG mutant strain was isolated- The areA gene from A. oryzs-e was cloned. The pyrG mutant was transformed with a plasmid carrying the pyrC gene inserted between DNA fragments upstream and downstream from the areA. gene. The coding region for areA was not present on the plasmid. Transformants were selected for their ability to grow in the absence of uridine and in the presence of chlorate. This dcumle selection selects both for a functional py^G g~ne and for areA minus. Strains obtained by this selection procedtre were finally screened hy Southern analysis to identify those in whxch the chromosomal areA gene was substituted by the pyrG
Cloning of the -A. orvzae pvrG aene .
The A. ory.za.e pyrG gene was cloned by cross hybridization w:th
the A. niger pyrG gene (W. van Hartmgsveldt at al. , Mol . G-n.

Genet 206:71-75 (1987)). A lambda library of partial SaulIIA digested A. oryzae IF04177 DNA was probed at low stringency with a 1 kb DNA fragment from the A. niger pyrQ gene. A 3.8 kb Hindlll fragment from a positive clone was subcloned into a pUCll8 vector. The resultant plasmid, pS02, was shown to contain' the pyrG gene fay complementation of an A. niger pyrG ' mutant.
Construction of an A. oryzae pvIG minus strain. A pyrG deletion plasmid, pSOS, containing about 1 kb of pyrG flanking sequences on each end was constructed from the plasmid pS02. A. oryzae IF04177 was transformed with this construct and transformants were selected by resistance to 5-fluoro-orotic acid, a phenotype characteristic of pyrG mutants. One transfor-mant, HowBlCl, was shown by Southern analysis to have the ex¬pected deletion at the pyrG locus. Being a pyrG mutant HowElOl requires uridine for growth. HowBlGl can be transformed with the wt pyrG gene by selection for ability to grew without uridine.
The steps involved in the coris=cruction of HcwBlOl are illus¬trated in Fig. 1.
Cloning of the are-A aene _
The A. oryzae areA gene was cloned by cross hybridization to the A., nidulans areA gene (E. Kudla et al. , 2M£0 J. 9:1355-1364 (1990)) . A genomic library of A. oryzae IFC4177 v;as prepared by partial digestion of chromosomal DNA with Sa-L-IIIA and cloning of the obtained DNA ira-^gments into the vector XGEM-II (obtained from Promega) . Cross hybridization of the library with th'S A. nidulans areA gene was performed in 40% fcrmamide at S^'-C. Hybridizing X clones were isolated and from these fragments were sub-cloned into the vector pBluescript sK+ (obtained iron-. Stratagene') giving rise to the plasmids p3K5 and pSK9 ilius trated in Fig. 2. The cloned gene was able to complement an A. nidulans areA mutant, proving that it is indeed the A. oryzae areA- homclog. 5643bp of the clone was ssquenced, and comparison of the sequences of the A. oryzae and the A. nidulans areA

genes shows that they are highly homologous. The sequence of the A. oryzae a.reA gene is shown in SEQ ID Mo. 1.
Construction of the areA deletion plasmid.
In order to delete the AreA gene from the A. oryza.e chromosome the plasmid pToC266 was constructed. pToC266 contains a 2.1 kb DNA fragment originating upstream of the areA gene (isolated from pSK5) and a 1.4 kb DNA fragment originating downstream from the areA gene (isolated from pSK9). The two fragments are separated by appr. 3.2 kb in the genome, the coding region is situated in this part of the gene. The A. oryzae pyrG gene from pJers4 was inserted between the areA upstream and downstream DNA fragments. The construction of pToC266 is illustrated in Figs. 3a and 3b. pToC266 has a unique EcoRI site and was li¬nearized by cutting with this restriction enzyme before used in transformations
Selection of A. orvzse areAA strains.
A. oryza.a HowBlOl was transformed with linearized pToC266. Transformants were selected on:minimal plates (Cove Biochem. biophy. Acta {1966) 113 Sl-SS) containing 5% sodium chlorate and 0 . 3 mM ammonium sulfate and ' 1% glucose. Transfcrmar.ts v.'ere thus subject to a double selection, both for having obtained the pyrG gene by being able to grow without addition of uridine and for chlorate resistance. Chlorate resistance is one of the phenotypes of A. nldvlans are a mutants (H. N. Arst and D. J. Cove, MGG 126 : 111-141 (1573));. Weakly growing transfomants were reisolated twice on the; same type of plates. Three independent transforr.iants named! TcC913 , TQC?15 and ToCS20 were subjected to growth test on different nitrogen .sources. They grew well on glutamine, but: weakly on other nitrogen sources tested, including airraonia. Southern analysis shewed chat che three strains have the lost the arsA structural gene, which had bean replaced by the pyrG gene
arsAA. strains can also be obtained by selection of transfor-mants of linearized pTcC266 on minimal plates containing glu-

tamine as nitrogen source. In one such experiment one out of 25
transformants was an areA strain.
EXAMPLE 2
Construction of pMT1606
A plasmid containing the bar gene from streptomyces hygrosco-pIus (C. J. Thompson et. al, EMBO J. 5 : 2519-2523 (1987)) inserted after the A. ozyzae TAKA-amylase promoter and followed by a fragment containing the transcriptional termin-ator and polyadenyXaticn signal from the A. niger gla gene was constructed.
The plasmid, pMTl506, can be used for selection of glufosinate resistant transformants of A. oryzae. pMTlSOS was conscructed by isolating the bar ^er.e from the plasmid pBPlT (B. Straubin-ger et, al, Fungal Genetics Newsletter 39 : 82-83 (1992)) and cloning it into Che fungal expression plasmid p775 described in E? application no. 87103806. Fig. 4 illustra^.es the construc-cion of pMT1606.
EXAMPLE 3
Production of chymosin in ToC913 (A. oryzae IF04177 areAa.)
The- A. oryzae areAA strain ToC913 was transformed with zhe plasmid p7oC55 (Fig. 5), which is a fungal expression plas-.id for the mammalian enzv-me chymosin, by co-transforniation with pMT1506. Cons'cructicn of r.he pla.?mid pToC56 is described in Z? application no. 37103806.
Transformants were selected for growth on minimal medium con¬taining 10 mM ammonium and i mg/ml glufosinate and screened for the presence cf pToC56 by che ability to produce chymosin. Three transformants were grown in shake flasks in minimal me¬dium, containing maltodextrin and glutamine for 4 days at 3G=C.

Two transformants of pToC5 6 in IF04177 (obtained as described in EP 87103806) as well as untransformed IF04177 and ToC9i3 were grown along with the ToC9l3 transformants.
Samples of the fermentation broth were taken every day and applied co SDS-Page and Western blotting. The blotting membrane was incubated with chymosin specific rabbit antibody followed by goat rabbit antibody coupled to peroxidase. Staining of the membrane showed that the supernatants from transformants of IF04177 contained small amounts of chymosin or degradation products thereof on the first and second day of fermentation and nothing later in fermentation.
Transformants of ToCSlS contained at least ten times more full size chymosin. The amount of chymosin in the supernatants in¬creased for the first two-three days and then remained con¬stant .
Supernatants from the third and fourth day of fermentation of IF04177, TOC313, a transformant of pToC56 in ToC913, and a transformant in IF04177 were applied to an isoelectric focus¬sing gel and electrophoresis was performed. The pH gradient was from 3.5 to 9.5. After electrophoresis the gel was rinsed with 3. buffer at pH = 7.0 containing 2 mM 2n2+ and overlaysd with an agar containing 0.5% casein. The gel was incubated at 45C untill protease activity was visible.
En samples from IFC4177 three bands with protease activity :ould be seen; cne with an alkaline pI and two with acidic
Cn samples from the pToC56 transformant of IF04177 a faint reaction from chyrr.osin could be" seen, which partially over--apped with one of the acidic bands found in untraroformed IF04177, the protease with most acidic pI was barely visible, while the prctease with the alkaline pI was clearly visible along with one or more band with' an almost neutral pI.

In the samples from ToC9l3 no protease activity was detected,
while the sample from the pToC56 transformant of ToC913 showed a strong chymoein signal. No other proteases were detected in samples from this transformant.
EXAMPLE 4
Production o£ human trypsin I in ToC9l3 (A. ory^ae IF04177 areA/i,)
A cDNA encoding human pancreatic trypsinogen I (TRYI) was isolated usir.c standard procedures and the sequence published by M. Smi et al, Gene (1986) 41 : 305-310(cf. Danish patent application no. 693/95). A BamHl site (GGATCC) was introduced immediately upstream of the start codcn (ATG(Met)) with che short sequence ACC between.
This EamHl site was used to fuse the cDNA to the BamHl linker in the Taka-amylase promoter in the fungal expression plasn^.id p777 described in EF application no. 87103806. The 3'end of the cDNA was fused 41 'bp downstream of the stop codon to a Nrul site in p777. This in-serts the TRYI cDNA between the A. Crysae Taka-amylase promoter and the A. niger gluccamylase transcrip¬tion terminator, The resulting plasmid was called pHW470 fcf. Danish patent applicarion no. 693/55).
pHW470 was transformed into ToCSlS by co-trans former ion v.'ith the plasmid pMT1606. 3ASTA resistant transformants were reisolated twice through conidicspores. 8 transformants were grown for four days at 30'C in YFM (YPD (Sherman, ?. et al (19S1)-Methods in Yeast Genetics. Cold Spring Harbor Labcratcry. Cold Spring Harbor, NY) ) in which the gluccse v.'as replaced with 2% maltose) , Supernacants were analysed for the content of human trypsin by SDS-FAGE followed by Western blotting and incubation with a rabbit antibody raised against porcine trypsin. The blotting membrane was then incubated with goat anti rabbit antibody coupled to peroxidase and reacted with

3-amino-9-ethyl carbazole. Supernatants from three of the transformantB contained a stained band of the expected size. The concentration of trypsin in the three positive eupernatants was 2-5 mg/I.
The presence of trypsin was further verified by incubation of samples of eupernatants with L-Benzoyl-arginoyl-paranitro anilide (L-BAPNA). Samples from the three immuno positive strains cleaved the eubstrate, which resulted in the develop¬ment of a yellow colour. Samples from ToC$13 and IF04177 did not show any activity against this substrate. The specific activity of human trypsin in this assay in not known, it is thus not possible to calculate the concentration of trypsin in the supernatants from these data.
Transformants of pHW470 in the wild type strain IF04177 were also made. More than 20 L-BAPNA positive transformants were looked at, but it was not possible to detect any immonoreactive bands in supernatancs from these transformants. The detection limir was approximately 0.5 mg/1 in this assay.

We claims:
1. A fungus, wherein the areA gene by recombinant DNA
technology has been modified in a way by which it cannot be
expressed in a way providing for a functional AreA activator.
2. The fungus of claim 1, wherein said inactivation has been
obtained by deletion of all or parts the areA gene.
3. The fungus of claim i, wherein said inactivation has been
obtained by interfering with the regulation of the expression
signals regulating the expression of the areA gene itself.
4. The fungus of claim i, wherein said inactivation has been obtained by using anti-sense technology.
5. The fungus of clairr, 1, wherain said inactivation has been obtained by inserting extra DNA internally in the areA gens.


Li) producing a DNA construct comprising the areA gene wherein an internal part has been substituted, deleted, or extra DNA has been inserted,
iii) transforming said fungus with the construct, and
iv) selecting traneformants which are areA' .
9. A method for producing a fungus according to claim l, wherein said inactivation has been obtained by using anti-sense technology, which method comprises
i) construction of an expression plasmid which gives rise to synthesis of an RNA molecule complementary to the mRNA
transcribed from the areA gene,
ii) transformation of the hcsz fungus with said expression plssmid and a suitable marker, either on separate plasmids or on ths same plasmid,
iii) selection of transforTnants using said marker, and iv) screening selected transfcrmants for strains exhibiting h reduction in the synthesis of the AreA product.
10. A process for the croduction, cf a desired gene product, whereby a fungus according tc may of the claims 1 to 7 is crltivared in a suitable growth medium at appropriate con-ait ions and the desired gene product is recovered and purified.
11. A process for the production of a desired gene product, whereby a fungus according tc any of the claims 1 to 7, which has been tran.sformed tc .integrate a ZiNA sequence coding ror the desired gene product into the genome of the fungus in a functional manner, cultivated in a suitable growth medium at appropriate ate conditions and the desired gene prcOuct is recovercd and curified.

12. A process for producing a desired polypeptide comprising
cultivating a fungus in an appropriate growth medium and
recovering said polypeptide from said culture, said fungus
carrying a recombinant DNA construct capable of causing
expression of said polypeptide or a precursor thereof in said
fungus, said fungus further being characterized by producing
lower amounts of functional AreA than the wild-type of said
fungus.
13. A method according to claim 12, wherein said fungus has
been modified to produce lower than wild-type amounts of AreA
by a process comprising transforming a parent of said fungus
with a DNA construct capable of causing reduced production of
functional AreA when integrated in the geneome of said fungus.
14. A method according to claim 12, wherein said polypeptide
is secreted to the extracellular medium by said fungus.
15. A method according to claim 12, wherein said fungus
produces higher amounts of said polypeptide than a similar
fungus where said similar fungus produces AreA in amounts
similar to those produced by the wild-type ol aaid fungus, said
similar fungus being identical to and fungus in all other
respects.


19. The process of any of the claims 10 to 116, wherein said
desired gene product is a therapeutically active peptide or
protein.
20. The process of claim 19, wherein said therapeutically active peptide or protein is selected from the group comprising insulin, growth hormone, glucagon, somatostatin, interferon, PDGF, factor VII, factor VIII, urokinase, tPA, EPO, or TPO.
21. A gene produci: produced in accordance with any of the
processes 10 to 20.
22. A DNA sequence coding for the areA gene from A. aryzae (SEQ ID No. 1) or functional alleles thereof.
23. An ArsA activator from A. oryzae (SEQ ID No. 2).

(24) A fungus substantially as hereinbefore described with
reference to the accompanying drawings.
(25) A nethod for producing a fungus substantially as
hereinbefore described with reference to the acconpanying
drawings.
(26) A process for the production of a desired gene product
substantially as hereinbefore described with reference to the
accompanying drawings.
(27) A gene product substantially as hereinbefore described
with reference to the accompanying drawings.