AU747607B2

AU747607B2 - A nucleic acid cassette & method to isolate mutants and to clone the complementing gene

Info

Publication number: AU747607B2
Application number: AU65450/99A
Authority: AU
Inventors: Leendert Hendrik De Graaff; Henriette Catharina Van Den; Jacob Visser
Original assignee: Danisco Ingredients AS
Current assignee: International N&H Denmark ApS
Priority date: 1995-06-23
Filing date: 1999-12-23
Publication date: 2002-05-16
Anticipated expiration: 2016-06-24
Also published as: AU747607C; AU6545099A

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT 9.9.

99 09 9 9 *9 o 99e 09 9.

*e fi Applicant(s): DANISCO INGREDIENTS A/S (DANISCO A/S) Invention Title: A NUCLEIC ACID CASSETTE METHOD TO ISOLATE MUTANTS AND TO CLONE THE COMPLEMENTING GENE The following statement is a full description of this invention, including the best method of performing it known to me/us: 1A A nucleic acid cassette method to isolate mutants and to clone the complementing gene Background of the Invention All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

a e *o o*o ooo.

oooo \\melbfiles\home$\WendyS\Keep\species\65450-99 Danisco.doc 12/03/02 1B The subject invention lies in the field of microorganism mutation and selection of the mutants. In particular the invention is directed at obtaining metabolic mutants in a simple. direct and specific manner. In a preferred embodiment It is also possible to obtain desired mutants not comprising recombinant DNA, thereby facilitating' incorporation thiereof in products for human consumption or application, due to shorter legislative procedures. The method according to the invention involves random mutation and specific selection of the desired metabolic mutant. The method can suitably be carried out automatically.

Such a mutant can exhibit either increased or decreased metabolic activity. The specificity of the method lies in the selection conditions applied. The mutants obtained are mutated in their regulatory function with regard to a predetermined part of metabolism. Dependent on the selection conditions derepressed mutants can be iso~lated that will thus exhibit overexpression or mutants can be found in which particular metabolic enzymic activity is eliminated. It thus becomes possible to eliminate undesirable metabolic enzymic activity or to increase desirable 20 metabolic activity.

The methods according to the invention can suitably be carried out on well characterised sources that are already widely used in :industry. The overexpressing mutants can., for example be used as major sources of enzymes producing huge amounts at low cost. The initial strain 25 to be mutated will depend on several factors known to a person skilled in the art such as: efficient secretion of proteins, the availability of large scale production processes, experience with downstream processing of fermentation broth, extensive genetic knowledge and the assurance of using safe organisms.

In another aspect of the invention it has now also become possible to ascertain and identify specific metabolic gene regulating functions.

To date a method for preparing mutants that was industrially.

applicable and could be automated was a method of mutating without selection and subsequent analysis of the mutan.ts for the aspect- which was to be amended. An alternative method with selection always required an enrichment step, followed by selection on the basis of growth or non growth. This meant a large number of undesired mutants had first to be weeded out. Also the existing method resulted in a high number of mutants with an incorrect phenotype and thus exhibits low selectivity.

Some years ago Gist-Brocades developed and introduced the pluGBug marker gene free technology for AsDergillus niger. In the GIST 94/60 p 5-7 by G. Selten a description is given of a vector for Asperzillus nizer comprising glucoamylase regulatory regions to achieve high expression levels of the gene it regulates. This was selected as regulatory region on the basis of the naturally high expression of glucoamylase by native Aspergillus niger. Using recombinant DNA techniques the regulatory region was fused appropriately to the gene of interest as was the selection marker Anegillus AmdS, allowing selection of the desired transformants after transferring the expression cassette to the A. nizer host. Multiple copies of the expression cassette then become randomly integrated into the A. niger genome. The enzyme produced as described in the article was phytase. Subsequently the generation of marker free transformants can be achieved. In the known system the generation of marker free recombinant strains is actually a two step process since the amdS gene can be used bidirectionally. First in a transformation round to select initial transformants possessing the offered expression cassette and subsequently in a second round by counterselecting for the final recombinant strain which has lost the amdS gene again. The amdS gene encodes an enzyme which is able to convert acetamide into ammonium and acetate. Acetamide is used as sole N-source in the transformation round. In the recombination round fluoro acetamide is used as selective N-source. with a second appropriate N sou ce such as e.g. urea. As the product fluoroacetate is toxic for other cells the 25 propagation will be limited to those cells which have lost the amdS gene by an internal recombination event over the DNA repeats within the expression cassette. The largest problem with the known method is the fact that the resulting strain is a recombinant strain. The desired characteristic has to be introduced by incorporation of "foreign" nucleic 30 acid, which can lengthen the time required for and sometimes even prevent legislative approval. In addition the method is not suited for developing strains with amended metabolism. Due to the presence of enzyme cascades and multiple feedback loops the mere incorporation of a particular gene cannot always lead to the desired result. Overproduction of a particular product as encoded can be compensated for by concomitant overexpression of another product or down regulated thus annulling thd effect of the incorporated gene. The incorporation of DNA will therefore often be a case of trial and error with the incorporation of the desired nucleic acid being selectable but the desired phenotype not necessarily concomitantly being achieved. Furthermore the loss of the marker gene is a spontaneous process which takes time and cannot be guaranteed to occur for all transformants comprising the nucleic acid cassette.

It is known that strain improvement in microorganisms can be achieved by modification of the organism at different levels. Improvement of gene expression at the level of transcription is mostly achieved by the use of a strong promoter. giving rise to a high level ofMRNA.

encoding the product of interest, in combination with an increase of gene dosage of the expression cassette. Although this can lead to an increase of the product formed, this strategy can have a disadvantage in principle. Due to the presence of multiple copies of the promoter the amount of transcriptional regulator driving transcription can become limited, resulting in a reduced expression of the target gene or genes of the regulator. This has been observed in the case of AsPerzillus nidulans strains carrying a large number of copies of the amdS gene (Kelly and Hynes, 1987; Andrianopoulos and Hynes, 1988) and in the case of A.nidul1s strains carrying multiple copies of a heterologous gene under the alcA promoter (Gwynne et al.. 1987). In the latter case an increase of the alcR gene. encoding the transcriptional regulator of the alcA gene, resulted in the increase of expression of the expression cassette (Gwynne et al., 1987; Davies, 1991). In analogy to the effects found in Asperzillus nidulans. in Asrerzillus nizer similar limitations were observed in using the glucoamylase (glaA) promoter. due to limitation of the transcriptional regulator driving transcription (Verdoes et al, 1993; Verdoes et al 1995; Verdoes, 1994). Cloning of the gZaA regulatory gene has thusfar been hampered by lack of selection strategy-.

In the case of the arabinase gene expression a clear competition for transcriptional regulator was found upon the increase of arabinase gene dosage (Flipphi et al, 1994), reflecting a limitation of a transcriptional regulator common to all three genes studied.

*.In addition to the abovementioned drawbacks of the state of the art isolation and determination of regulator genes has until now been extremely difficult due to the fact that most of the regulatory proteins exist in very low concentrations in the cell making it difficult to determine -hich substance is responsible -for regulation. In addition generally the regulatory product is not an enzyme and can only be screened for by-a DNA binding assay which makes it difficult to determine and isolate and is very time consuming. Thus far the strategies used to clone regulatory genes are e.g.: by complementation, which however requires a mutant to be available.

by purification of the regulatory protein, which is extremely laborious, since the protein can only be characterised by its DNA binding properties. Some of these purifications include affinity chromatography using a bound DNA fragment as a matrix. One of the drawbacks in this type of purification is that often more than one protein binds both specifically as well as non-specifically to the fragment.

based on gene clustering wherein the regulatory gene is genomically clustered with the structural genes which are regulated by its gene product, e.g. the prn cluster, the atc cluster.

Detailed description of the invention We have now achieved a system that can be used for shortening the length of time required for registration of mutant microorganisms capable of overproduction of particular desirable enzymes. The system overcomes the problem of multiple random inserts of "foreign" nucleic acid and in particular of the selection marker gene. It does not even require foreign nucleic acid to achieve the desired characteristic. The resulting mutant strain will not comprise heterologous nucleic acid. In addition the system according to the invention enables specific mutation of metabolism and prevents a large deal of experimentation leading to undesired phenotypes.

The subject invention is directed at a nucleic acid cassette comprising a nucleic acid sequence encoding a bidirecti-onal marker, said nucleic acid cassette further comprising a basic transcriptional unit operatively linked to the nucleic acid sequence encoding the bidirectional marker and said nucleic acid cassette further comprising an inducible enhancer or activator sequence linked to the basic transcription unit in such a manner that upon induction of the enhancer or activator sequence the bidirectional marker encoding nucleic acid sequence is expressed, said inducible enhancer or activator sequence being derived from a gene associated with activity of part of the metabolism, said inducible enhancer or activator sequence being derived from a gene associated with metabolism.

A basic transcription unit comprises any elements required for :transcription of the gene to which the transcription is linked. It can comprise the promoter with or without enhancer sequences. The basic transcription unit must be operative in the host organism. The basic transcription unit must be located such that it is operatively linked to the bidirectional marker gene for transcription thereof to be possible.

Suitable examples are well known for a number of host cells such as e.g. Aspergillus, Trichoderma, Penicillium, Fusarium, Saccharomvces, Kluwveromvces and Lctobacillus. In the-Examples the basic transcription unit tGOX derived from the Aspergillus niger goxC transcription unit (Whittington et at. 1990) is illustrated in an operable embodiment of the invention.

The inducible enhancer or activator sequence is preferably normally involved in regulation of an enzyme cascade or involved in a part of metabolism involved with one or more feed back loops. In a further embodiment a nucleic acid cassette according to the invention comprises an inducible enhancer or activator sequence that is normally involved in carbon metabolism. Suitable examples of inducible enhancer or activator sequence to be used in a nucleic acid cassette according to the invention are the Upstream Activating Sequence (UAS) as comprised on any of the following fragments of nucleic acid: a fragment originating from the promoters of the abfA, abfB and abnA genes encoding respectively arabinofuranosidase A. arabinofuranosidase B and endoarabinase.

a fragment originating from the glaA gene encoding glucoamylase, a fragment containing the alcR binding site such as on the alcR and alcA promoter, a fragment originating from the CUP1 gene, 25 a fragment originating from the PH05 gene a fragment originating from the GAL1, GAL7 or GAL0 genes.

a fragment originating from the xlA gene a fragment originating from the pgln gene.

By way of example these fragments can be derived from the following organisms as is described in the literature: a fragment originating from the promoters of the abfA, abfB and abnA genes encoding respectively arabinofuranosidase A, arabinofuranosidase B and endoarabinase of Aspergillus niger (Flipphi M.J.A. et at. 1994) a fragment originating from the glaA gene encoding glucoamylase of AsDergillus nizer, (Fowler T. et al 1990) a fragment containing the alcR binding site such as on the alcR promoter of Aspergillus nidulans (Felenbok B. et al. 1994), a fragment originating from the CUP1 gene of Saccharomvces cerevisiae (Hinnen A. et al. 1995) a fragment originating from the PH05 gene of Sacchromvces cerelsiae (Hinnen A. et at. 1995) a fragment originating from the GAL1, GAL7 or GALIO genes of Saccharomvces cerevisiae (Hinnen A. et at. 1995).

a fragment originating from the xlnA, xlnB, xlnC or xlnD genes of Asperillus nidulans, a fragment originating from the xlnB. xlnC or xlnD genes of Aspergillus niger (see elsewhere in this description), a fragment originating from the xlnA or xlnD genes of Asperillus tubigensis (de Graaff et al. 1994).

a fragment originating from the pgall gene of Asperzillus nier (see elsewhere in this document).

In the Examples UAS of zlnA is illustrated in an operable embodiment of the invention.

A bidirectional marker is an art recognised term. It comprises a selection marker that can be used to indicate presence or absence of expression on the basis of the selection conditions used. A preferred /bidirectional marker will confer selectability on the basis of lethality or extreme reduction of growth. Alternatively different colouring of colonies upon expression or lack of expression of the bidirectional marker gene is also a feasible embodiment. Suitable examples of known bidirectional markers are to be found in the group consisting of the facB, the NiaD, the AmdS, the Can!, the Ura3, the Ura4 and the-PyrA genes. We hereby point out that PyrA homologues are also referred to in 25 the literature as PyrG, Ura3, Ura4, Pyr4 and Pyrl. These genes can be found in i.a. the following organisms the facB gene in Aspergillus nidulans, the NiaD gene in Aspergillus niger, the NiaD gene in Aspergillus orvzae, the AmdS gene in Aspergillus nidulans, the Canl gene in Schizosaccharomvces Pombe, the Ura3 gene in Saccharomvces cerevisiae.

the Ura4 gene in Saccharomvces pombe and the PyrA genes in Asoerillus.

Trichoderma. Penicillium. Fusarium, Saccharomyces and Kluvvernaces.

Selection of facB mutants i.e. with a negative phenotype can occur on the basis of fluoro-acetate resistance. Selection for FAC B' i.e. a positive phenotype can occur on acetate as a carbon source (Katz. M.E.

and Hynes M.J. 1989). Selection of niaD mutants i.e. with a negative phenotype can occur on the basis of chlorate resistance. Selection for SNIA D' i.e. a positive phenotype can occur on nitrate as a nitrogen source (Unkles S.E. et alt.1989a and 1989b). Selection of amdS mutants i.e. with a negative phenotype can occur on the basis of fluor acetamide resistance. Selection for AMD S' i.e. a positive phenotype can occur on acetamide as a nitrogen source. As most fungi do not have a gene encoding an acetamidase function AMD S is a dominant marker for such fungi. It has been used as such in Asperzillus nizer, AsDerzillus niger var.

tubigensis, Aspergillus niger var. awamori, Asoergillus foetius.

Aspergillus orvzae, AsPerrillus svdowii, AsDerrillus Aspergillus aculeatus, Penicillium species, Trichoderma species among others (Kelly and Hynes 1985 and Bailey et at. 1991). Selection of cani mutants i.e. with a negative phenotype can occur on the basis of canavanine resistance, wherein canavanine is an arginine analogue.

Selection for CAN 1* i.e. a positive phenotype can occur on arginine (Ekwall K. 1991). The gene encoding orothidine 5'-P-decarboxylase is known as pyrA, pyrG or ura3. It has been found for various organisms e.g.

Asperilli, Trichoderma, Penicillium, Fusarium, Saccharonces and Kluweromvces. Selection of pyrA mutants i.e. with a negative phenotype, also described as pyrG or ura3 can occur on the basis of fluoro orotic acid resistance. Selection for PYR A' and homologues thereof i.e. a positive phenotype can be done on the basis of uridine or uracil prototrophy. In the Examples pyrA from Asperrillus nier (Wilson et at.1988) is illustrated in an operable embodiment of the invention. Other examples are known to a person skilled in the art and can be readily found in the literature. The selection marker to be used will depend on the host organism to be mutated and other secondary considerations such as ease of selectability, reliability and cost of substrates to be used 25 amongst others.

The nucleic acid cassette incorporated in a transformation or expression vector is also included in the scope of the invention. Also included is the application of such nucleic acid cassette or vector in transformation and selection methods. In particular in methods for producing mutants exhibiting overexpression of an enzyme involved in a predetermined part of metabolism, methods for producing mutants :"exhibiting reduced or inhibited expression of an enzyme involved in a predetermined part of metabolism and methods for determining and isolating regulatory genes involved in predetermined parts of metabolism.

35 Thus a method for preparing and selecting a mutant strain of microorganism, said mutation enhancing a predetermined part of metabolism in comparison to the non mutated strain, said method comprising introducing into a host a nucleic acid cassette according to any of the preceding claims.

said host not exhibiting the phenotype associated with expression of the bidirectional marker prior to introduction of the nucleic acid cassette, -culturing a resulting microorganism under conditions wherein the enhancer or activator sequence comprised on the nucleic acid cassette is normally active and under conditions wherein the bidirectional marker is expressed and wherein preferably expression of said bidirectional marker will lead to growth and non expression to non growth, -selecting a transformant that exhibits the phenotype corresponding to the expression of the bidirectional marker gene under the aforementioned culturing conditions, -subjecting the selected transformant to mutagenesis in a manner kn~own per se, -culturing the resulting strain under conditions acceptable for a strain with a phenotype corresponding to the expression of the bidirectional marker and under conditions that in the non mutated parent comprising the nucleic acid cassette result in non-expression of the bidirectional marker and in the presence of a metabolisable substrate for the predetermined part of metabolism, -selecting a strain resulting from the cultivation step following mutagenesis that exhibits a phenotype corresponding to the expression of the bidirectional marker gene under selection conditions that for the non mutated parent comprising the nucleic acid cassette result in ~:25 non-expression of the bidirectional marker falls within the scope of the invention.

:In a suitable embodiment of this method -the inducible enhancer or activator sequence is the Upstream Activating Sequence (UAS) derived from the gene x~nA, 30 -the predetermined part of the metabolism is the xylanolytic part of carbon metabolism.

-the culturing step wherein the resulting microorganisms are cultivated under conditions wherein the enhancer or activator sequence is normally active and wherein the bidir-atioflal marker is expressed comprises cultivation in the presence of inducer of UAS and absence of repressor of UAS and a metabolisable source of carbon.

-the selecting of a transformant that exhibits the. phenotype corresponding to the expression of the bidirectional marker-gene occurs under the aforementioned culturing conditions.

the culturing step after mutagenesis of the selected transformant occurs under conditions acceptable for a strain with a phenotype corresponding to the expression of the bidirectional marker and under conditions that in the non mutated parent comprising the nucleic 46id cassette result in non-expression of the bidirectional marker i.e. in the presence of repressor of UAS and in the presence of a metabolisable source of carbon and optionally also in the presence of inducer of UAS, the selection of a strain resulting from the cultivation step following mutagenesis of a strain that exhibits a phenotype corresponding to the expression of the bidirectional marker gene occurs under selection conditions that for the non mutated parent comprising the nucleic acid cassette result in non-expression of the bidirectional marker.

In a further embodiment of this method the nucleic acid cassette comprises a nucleic acid sequence encoding the bidirectional marker pyrA.

the host does not exhibit the pyrA* phenotype associated with expression of the bidirectional marker prior to introduction of the nucleic acid cassette, -the culturing step wherein the resulting microorganisms are cultivated under conditions wherein the enhancer or activator sequence is normally active and wherein the bidirectional marker is expressed comprises cultivation under conditions wherein the enhancer or activator is normally active i.e. in the presence of inducer of the enhancer or activator and in the absence of repressor of the enhancer or activator and under conditions wherein the bidirectional marker is expressed.

-the selecting of a transformant that exhibits the phenotype corresponding to the expression of the bidirectional marker gene occurs under the aforementioned culturing conditions, -the culturing step after mutagenesis of the selected transformant occurs under conditions acceptable for a strain with a phenotype :corresponding to the expression of the bidirectional marker and under conditions that in the non mutated parent comprising the nucleic acid cassette result in non-expression of the bidirectional marker i.e. in the absence of inducer of the enhancer or activator or in the presence *of repressor of the enhancer or activator and in the presence of a metabolisable substrate for the predetermined part of metabolism.

-the selection of a strain resulting from the cultivation step following mutagenesis of a strain that exhibits a phenotype corresponding to the expression of the bidirectional marker gene occurs under selection conditions that for the non mutated parent comprising the nucleic acid cassette result in non-expression of the bidirectional marker i.e. in the absence of inducer of the enhancer or activator or the presence of repressor of the enhancer or activator.

Suitably the embodiments just mentioned can further be characterised by the nucleic acid cassette comprising a nucleic acid sequence encoding the bidirectional marker pyrA, the host not exhibiting the pyrA+ phenotype associated with expression of the bidirectional marker prior to introduction of the nucleic acid cassette, the inducible enhancer or activator sequence being the UAS derived from the gene x mA, the culturing step wherein the resulting microorganisms are cultivated *under conditions wherein the enhancer or activator sequence is normally active and wherein the bidirectional marker is expressed comprising cultivation under conditions wherein UAS is normally active i.e. in the presence of inducer of UAS such as xylose or xylan and in the absence of repressor of UAS i.e. absence of glucose and under conditions wherein the bidirectional marker is expressed.

-the selecting of a transformant that exhibits the phenotype corresponding to the expression of the bidirectional-marker gene occurs under the aforementioned culturing conditions, -the culturing step after mutagenesis of the selected transformant occurring under conditions acceptable for a strain with a phenotype *corresponding to the expression of the bidirectional marker and under conditions that in the non mutated parent comprising the nucleic acid cassette result in non-expression of the bidirectional marker i.e. in the absence of inducer of UAS such as xylose or xylan or in the :presence of repressor of UAS i.e. in the presence of glucose and in the presence of a metabolisable source of cirbon, -the selection of a strain resulting from the cultivation step following mutagenesis of a strain that exhibits a phenotype corresponding to the expression of the bidirectional marker gene occurring under selection conditions that for the non mutated parent comprising the nucleic acid cassette result in non-expression of the bidirectional marker i.e. in the absence of inducer of UAS such as xylose or xylan or the presence of repressor of UAS i.e. in the presence of glucose.

:In addition a method for preparing and selecting a non reconminant mutant strain of microorganism,-said mutation enhancing a predetermined part of metabolism in comparison to the non mutated strain falls within the preferred scope of the invention. This method comprising carrying out the steps of the method according to the invention as described in the preceding paragraphs followed by crossing out in a manner known per se the nucleic acid of the introduced nucleic acid cassette.

As indicated previously a method for preparing and -selecting a mutant strain of microorganism, said mutation inhibiting a predetermined part of the carbon metabolism in comparison to the non mutated strain, said method comprising introducing into a host a nucleic acid cassette according to the invention as described above, said host not exhibiting the phenotype associated with expression of the bidirectional marker prior to introduction of the nucleic acid cassette and said host exhibiting activity of the type characterising the predetermined part of metabolism to be reduced or inhibited, culturing a resulting microorganism under conditions wherein the enhancer or activator sequence of the nucleic acid cassette is normally active and wherein non expression of the bidirectional marker 25 of the nucleic acid cassette will result in growth and detection of the resulting microorganism and wherein expression of said bidirectional marker will preferably be lethal or strongly inhibit growth, selecting a transformant that exhibits the phenotype corresponding to the expression of the bidirectional marker gene under the aforementioned culturing conditions subjecting the selected transformant to mutagenesis in a manner known per se, culturing the strain resulting from the mutagenesis under conditions 35 acceptable for growth of a strain with a phenotype corresponding to the non expression of the bidirectional marker and in the presence of a metabolisable substrate and under conditions that illustrate the reduced or inhibited activity of the predetermined part of metabolism

S

in comparison to the non mutated host either with or without the nucleic acid.cassette.

selecting a strain resulting from the cultivation step following mutagenesis that exhibits a phenotype corresponding to the reduced or inhibited activity of the predetermined part of the metabolism under selection conditions that illustrate the reduced or inhibited activity of the predetermined part of metabolism in comparison to the non mutated host with or without nucleic acid cassette such as a reduced zone of clearing upon growth on a substrate which serves as a substrate for the part of metabolism for which the activity is to be reduced or inhibited.

In a further embodiment of such a method the inducible enhancer or activator sequence is the Upstream Activating Sequence (UAS) derived from the gene xlnA the predetermined part of the metabolism is the xylanolytic part of carbon metabolism, the culturing step wherein the resulting microorganisms are cultivated under conditions wherein the enhancer or activator sequence is normally active and wherein the bidirectional marker is expressed comprises cultivation in the absence of repressor of the UAS of the nucleic acid cassette, in the presence of a metabolisable source of carbon and preferably also in the presence of inducer of the UAS, the selecting of a transformant that exhibits the phenotype_ corresponding to the expression of the bidirectional marker gene occurs under the aforementioned culturing conditions, the culturing step after mutagenesis of the selected-transformant occurs under conditions acceptable for growth and detection of a strain with a phenotype corresponding to the non expression of the bidirectional marker, under conditions that are unacceptable for growth and detection of a strain with a phenotype corresponding to the 30 expression of the bidirectional marker i.e. in the presence of uridine and fluoro-orotic acid and under conditions that in the non mutated parent comprising the nucleic acid cassette result in activity of the predetermined part of the carbon metabolism i.e. in the presence of inducer of the UAS and a metabolisable carbon source and the absence 35 of repressor of the UAS for example the presence of sorbitol or an alternative non repressing source of carbon in combination with an inducer like xylan or D-xylose.

the selection of a strain resulting from the cultivation step following mutagenesis that exhibits a phenotype corresponding to the

°O°

13 reduced or inhibited activity of' the predetermined Part of' the carbon metabolism occurs under selection conditions that illustrate the reduced or inhibited activity of the predetermined part of carbon metabolism in comparison to the non mutated host either with or *without the nucleic acid casette such as a reduced zone of clearing upon growth on xylan.

An example of' the method according to the preceding paragraph is provided, wherein the nucleic acid cassette comprises a nucleic acid sequence encoding the bidirectional marker pyrA.

the host does not exhibit the PYRA+ phenotype associated with expression of' the bidirectional marker prior to introduction of the nucleic acid cassette.

the culturing step wherein the resulting microorganisms are cultivated under conditions wherein the enhancer or activator sequence is normnally active and wherein the bidirectional marker pyrA is expressed i.e. comprises cultivation in the presence of inducer of the enhancer or activator and in the absence of' repressor of' the enhancer or activator and under conditions wherein the bidirectional marker py2'A is expressed.

-the selecting of' a transformant that exhibits the phenotype corresponding to the expression of' the bidirectional marker gene pyrA occurs under the aforementioned culturing conditions, -the culturing step after mutagenesis of the selected transformant occurs under conditions acceptable for growth and detection of a strain with a phenotype corresponding to the non expression of the bidirectional marker i.e. PYR-phenotype, under conditions that are unacceptable for growth and detection of a strain with a PYRA phenotype, such a phenotype corresponding to the expression of the bidirectional marker i.e. such conditions comprising the presence of uridine and fluoro-orotic acid and under conditions that in the non mutated parent comprising the nucleic acid cassette result in activity of the predetermined part of the metabolism i.e. in the presence of *inducer of' the enhancer or activatcr anrd a metabolis able- substrate and the absence of repressor of the activator or enhancer or an alternative non repressing substrate in combination with an inducer.

1n a preferred embodiment the method according to the preceding 0: 2 paragraphs is a method, wherein furthermore the nucleic acid cassette comprises a nucleic acid sequence encoding the bidirectional marker pyrA, the host does not exhibit the PYRA+ phenotype associated with expression of the bidirectional marker prior to introduction of the nucleic acid cassette, the inducible enhancer or activator sequence is the UAS derived from the gene xlnA the culturing step wherein the resulting microorganisms are cultivated under conditions wherein the enhancer or activator sequence is normally active and wherein the bidirectional marker 2rA is expressed comprises cultivation under conditions wherein the UAS is normally active i.e. in the presence of inducer of the UAS such as xylose or xylan and in the absence of repressor of the UAS i.e. absence of glucose and under conditions wherein the bidirectional marker PYrA is expressed, the selecting of a transformant that exhibits the phenotype corresponding to the expression of the bidirectional marker gene pm occurs under the aforementioned culturing conditions, the culturing step after mutagenesis of the selected transformant occurs under conditions acceptable for growth and detection of a strain with a phenotype corresponding to the non expression of the bidirectional marker i.e. pyrA-phenotype, under conditions that are unacceptable for growth and detection of a strain with a PYRA" phenotype, such a phenotype corresponding to the expression-oV the bidirectional marker i.e. such conditions comprising the presence of uridine and fluoro-orotic acid and under conditions that in the non mutated parent comprising the nucleic acid cassette result in activity of the predetermined part of the carbon metabolism i.e. in the presence of inducer of the UAS and a metabolisable carbon source and the absence of repressor of the UAS for example the presence of sorbitol or an alternative non repressing source of carbon in combination with an inducer.,like xylan, or D-xylose, *oo.

the selection of a strain resulting from the cultivation step following mutagenesis that exhibits a phenotype corresponding to the reduced or inhibited activity of the predetermined part of the carbon 35 metabolism occurs under selection conditions that illustrate the reduced or inhibited activity of the predetermined part of carbon metabolism in comparison to the non mutated host either with or without the nucleic acid cassette such as a reduced zone of clearing upon growth on xylan.

*o oo oe* The methods described above can advantageously be carried out with a host characterised in that prior to introduction of the nucleic acid cassette it comprises nucleic acid corresponding at least in part to the nucleic acid sequence encoding the bidirectional marker, said correspondence being to a degree sufficient to allow homologous recombination in the chromosome of the bidirectional marker encoding nucleic acid comprised on the nucleic acid cassette. This aspect ensures the integration of the nucleic acid cassette at a predefined location.

In a further preferred embodiment the nucleic acid cassette will be incorporated in multiple copies to ensure that the mutagenesis step does not inactivate the bidirectional marker as this would result in incorrect results when detecting marker negative phenotypes and a decrease in the number of marker positive phenotypes.

In preferred embodiments of the invention a nucleic acid cassette has been constructed which can be used in a method for producing mutants exhibiting overexpression of an enzyme involved in a predetermined part of metabolism, a method for producing mutants exhibiting reduced or inhibited expression of an enzyme involved in a predetermined part of Smetabolism and a method for determining and isolating regulatory genes involved in predetermined parts of metabolism. In particular we illustrate the system as used for mutants in carbon metabolism. In the examples mutants with altered xylanolytic characteristics are described as well as arabinase and polygalacturonase mutants leading to mutants in the arabinolytic and pectinolytic pathways. In the examples Astergillus is used as the strain to be mutated, however any other industrially acceptable microorganism will suffice. Examples of such organisms are Saccharomvces e.g. Saccharomvces cerevisiae, Saccharomvces nombe, Aspergillus e.g. Asoergillus nidulans. Trichoderma, Penicillium. Fusarium Kluweromvces and Lactobacillus. Other examples will be obvious to a 30 -person skilled in the art and a number are also mentioned elsewhere in the description. An overexpressing or nulexpressing strain for a predetermined part of the metabolism can now be produced. We can also determine the identity and nucleic acid sequence of the activating regulator of an inducible enhancer or activator sequence. In particular 35 when such activating regulator is involved in metabolism, more specifically when such activating regulator is involved in a part of metabolism with an enzyme cascade or feed back loop or multiple feed back loops. The nucleic acid sequence of such a regulatory gene can subsequently be used to enhance expression of target genes. Said target gene being a gene that is regulated by the regulatory gene. In a preferred embodiment such a target gene will have a binding site for the expression product of the regulator gene. Combination of a promoter normally :associated with a target gene of the regulator with the regulatory gene in an expression cassette said promoter being. operably linked to a homologous or heterologous sequence encoding a homologous or heterologous protein or peptide to be expressed can lead to an expression cassette extremely useful for expression of homologous and even heterologous proteins or peptides. The regulator encoding gene can be under control of its native promoter or any other promoter that can be expressed in the host cell of choice. The promoter can be constitutive or inducible, whatever is most desirable for the particular production process. Such a combination expression cassette falls within the scope of the invention as does a vector or a plasmid comprising such a cassette.

The degree of expression is no longer restricted by the presence of too small an amount of regulator and thus the degree of expression of the gene to be expressed is much higher than in a corresponding host cell -where the gene is expressed under control of the same promoter but without the additional presence of the regulator gene. Such increased expression is preferably achieved in cells of organisms normally comprising components of the part of. the metabolic pathway to be influenced. Suitable host cells are filamentous fungi cells. The incorporation of a combined expression cassette of the type just.

described above in a host cell comprising a target gene of the regulator can lead to increased expression of the target gene or to increased expression of the target genes if multiple target genes are present.

Preferably the target gene will be endogenous to the host cell. A host cell comprising the combination expression cassette falls within the **:scope of the invention.

In the examples the nucleic acid sequence xlnR of the regulator of the xylanolytic pathway xylR is provided. The target genes for this regulator ::have been found to comprise the genes xlnA., xlnB, xlnC and xlnD as well as axeA. The increase in xylanase A expression is illustrated and can serve to indicate the general applicability of the xylR action on a target gene of the xylR regulator. A number of sequences are kniown in the state of the art comprising the xlnA, B, C and D genes mentioned and the axeA gene and such information is readily available to a person skilled *in the art and is to be considered incorporated herein. The pro moters of preferred interest to be used in combination with xlnR nucleic acid can 17 be selected from xlnA, xlnB. xlnC and xlnD. The use of the axeA promoter also forms a suitable embodiment of the invention. The promoters are known in the state of the art to the person skilled in the art and are considered to be incorporated herein. The xlnA promoter is described in de Graaff et al 1994. The xlnB promoter has been described by Kinoshita et al 1995). The xlnD promoter has been described in EP 95201707.7 and is included in the Sequence Listing in the sequence of sequence id no 8 of this document. The promoter sequences can either be readily synthesized on the basis of known sequences or be derived from organisms or vectors comprising them in standard manners known per se. Where the term promoter, enhancer or regulator is used naturally a fragment comprising the promoter, enhancer or regulator can be employed as long as the operability of such is not impaired. It is not necessary in the constructs according to the invention to merely incorporate the relevant sequence, any flanking non interfering sequences can be present.

Not only is the nucleic acid sequence xlnR encoding the expression product xylR covered by the subject invention but also sequences encoding equivalent expression products and mutant expression products as well as the expression products themselves of the nucleic acid sequences according to the invention. Any application of xylR or xylR encoding sequences (=xlnR) disclosed herein is also applicable to the mutants and the nucleic acid sequences encoding such mutants and is to be considered incorporated mutatis mutandi.

Examples of suitable fungal cells to be used for expression of nucleic acid sequences and cassettes according to the invention-are Asperzilli such as Aserzillus nicer. Aspergillus tubizensis. Aspergillus aculeatus.

Asperzillus awamori. Aspergillus orvzae. Asergillus nidulans.

Aspergillus carbonarius. Aspergillus foetidus. Aspergillus terreus.

Aspergillus svdowii. Aspergillus kawachii. Asperzillus iaponicus and 30 species of the genus Trichoderma. Penicillium and Fusarium. Other cells such as plant cells are also feasible host cells and elsewhere in the description alternative host cells are also described.

Genes of particular interest for expressing using the expression cassette according to the invention or in combination with a nucleic acid sequence according to the invention are those encoding enzymes. Suitable genes for expressing are genes encoding xylanases, glucanases.

oxidoreductases such as hexose oxidase, a-glucuronidase, lipase.

"esterase, ferulic acid esterase and proteases. These are non limiting examples of desirable expression products. A number of sequences are o.

known in the state of the art comprising the genes mentioned and such information is readily available to the person skilled in the art and is to be considered incorporated herein. The genes can either be readily synthesized on the basis of' known sequences in the literature or databases or be derived from organisms or vectors comprising them in a standard manner known per se and are considered to be knowledge readily available to the person skilled in the art not requiring further elucidation.

An expression product exhibiting 801'-100% identity with the amino acid sequence of xylR according to sequence id no. 9 or as encoded by the nucleic acid sequence of xlnR of' sequence id. no. 9 (from nucleotide 948) is considered to be an equivalent expression product of the xylanase regulator (xylR) according to the invention and thus falls within the scope of' the invention. The equivalent expression product should possess DNA biLnding activity. Preferably such DNA binding activity should be such that the expression product binds to the nucleic acid of the target gene to the same degree or better than the expression product binds with the amino acid sequence provided in sequence id no 9. The preferred target genes for determining binding activity are those encoding xylA, xylB, xylC and xylD i.e. the xlnA, xlnB, xlnC and xlnD genes.

Mutants of' the xlnR gene expression product xylR and the encoding nucleic acid sequence xlnR according to sequence id no 9 at least maintaining the same degree of target binding activity are also claimed. Mutants considered to fall within the definition of equivalent expression products comprise in particular mutants with amino acid changes in comparison to the amino acid sequence of sequence id no 9. Such equivalents are considered suitable embodiments of the invention as are the nucleic acid sequences encoding them. Mutants with 1-15 amino acid substitutions are suitable and substitutions of for example 1-5 amino acids are also considered to form particularly suitable embodiments of the invention that form equivalent expression products. It is common knowledge to the person skilled in the art that substitutions of particular amino acids with other amino acids of' the same type will not ~:.seriously influence the peptide activity. On the basis of hydropathY profiles a person skilled in the art will realise which substitutions can :9 be carried out. Replacement of hydrophobic amino acids by other hydrophobic amino acids and replacement of hydrophilic amino acids by other hydrophilic amino acids e.g. will result in an expression product ~:that is a suitable embodiment of the invention; Such substitutions are considered to be covered by the scope of the invention under the term equivalents. Point mutations in the encoding nucleic acid sequence according to sequence id no 9 are considered to result in nucleic acid sequences that fall within the scope of the invention. Such point mutations can result in silent mutations at amino acid level or in substitution mutants as described above. Substitution mutants wherein the substitutions can be of any type are also covered by the invention.

Preferably the identity of the mutant will be 85-100%, more preferably 90-100%, most preferably 95-100% in comparison to the amino acid sequence of sequence id no 9. As already claimed above amino acid sequences exhibiting 80-100% identity with the amino acid sequence of sequence id no 9 are covered by the term equivalent so that deletions and/or substitutions of up to 20% of the amino acid sequence, preferably of less than 15% and more preferably less than 10% most preferably of less than 5% of the amino acid sequence according to the invention are covered.

Such a mutant may comprise the deletion and/or substitution in one or more parts of the amino acid sequence. Such a deletion and/or -rsubstitution mutant will however comprise an amino acid sequence corresponding to a Zn finger binding region and an amino acid sequence corresponding to the RRRLWW motif. Deletion mutants of 1-5 amino acids are considered to fall within the scope of the invention. Deletion mutants with larger deletions than 5 amino acids an d/or with more than substitutions and/or point mutations can also maintain the DNA binding activity. Such larger number of deletions and/or substitutions and/or point mutations will preferably occur in the N terminal--half of the amino acid sequence and the corresponding part of the encoding nucleic acid sequence. The most important regions considered to be involved in regulation and activation are present in the C terminal half of the amino acid sequence from the zinc finger binding region and as such the deletion mutants will preferably comprise at least this portion of the :amino acid sequence. Preferably no mutation will be present in the zinc finger binding region corresponding to that encoded in sequence id no 9 from nucleotides at position 1110 to 1260. If a mutation is present preferably it should not involve the spanning between the 6 cysteines coordinating the zinc and most preferably any mutation should not involve any of the 6 cysteines themselves. In addition preferably no mutation is *.:present in the RRRLWW motif present in the amino acid sequence of sequence id no 9. A deletion may occur in one or more fragments of the amino acid sequence. Such a deletion mutant will however comprise an amino acid sequence corresponding to a Zn finger binding region and an amino acid sequence corresponding to the RRRLWW motif. Deletions of 1-15 amino acids, preferably of 1-10 amino acids and most preferably 1-5 amino acids are suitable embodiments to ensure equivalence.

Embodiments of equivalent nucleic acid sequences are a nucleic acid sequence which encodes an expression product having the same amino acid sequence as xylR of sequence id no. 9 or as encoded by the nucleic acid sequence of xlnR of sequence id. no. 9. A nucleic acid sequence encoding an expression product exhibiting 80%-100% identity with the amino acid sequence of xylR according to sequence id no 9 or as encoded by the nucleic acid sequence encoding xylR of sequence id no 9 is also considered to be an equivalent nucleic acid sequence of xlnR and falls within the scope of the invention. Another embodiment of an equivalent nucleic acid sequence according to the invention is a nucleic acid sequence capable of hybridising under specific minimum stringency conditions as defined in the Examples to primers or probes derived from nucleic acid sequence id no 9. said primers or probes being derived from the non zinc finger binding region and said primers or probes being at least 20 nucleotides in length. Generally suitable lengths for probes and primers are between 20-60 nucleotides, preferably 25-60. Preferably a probe or primer will be derived from the C-terminal encoding half of the sequence of id no 9 from the zinc finger binding region. A preferred embodiment of a nucleic acid sequence according to the invention will be capable of hybridising under specific conditions of at least the stringency illustrated in the examples to the nucleic acid sequence of id no 9. An equivalent nucleic acid sequence will be derivable from other organisms by e.g. PCR using primers based on the nucleic acid sequence id Sno 9 as defined above. An expression product of an equivalent nucleic acid sequence as just defined is also considered to fall within the scope 30 of the invention. Vice versa a nucleic acid sequence encoding an equivalent amino acid sequence according to the invention is also eoo considered to fall within the scope of the term equivalent nucleic acid sequence. In particular equivalent nucleic acid sequences and the expression products of such sequences derivable from filamentous fungi 35 and plants are preferred embodiments of the invention. Preferably an equivalent nucleic acid sequence will comprise a nucleic acid sequence :-encoding a zinc finger binding region corresponding to that encoded in sequence id no 9 from nucleotides from position 1110 to 1260. In a preferred embodiment the equivalent nucleic acid sequence should encode

O•OQ

the 6 cysteines coordinating the zinc. In a further embodiment thu spacing between the cysteines should correspond to that of sequence id no 9.

Embodiments comprising combinations of the characteristics of the various embodiments of the equivalent nucleic acid sequences and expression products described above also fall within the scope of the invention. Mutants with mutation in the zinc finger binding domain are also claimed as these could exhibit increased DNA binding. Mutants exhibiting dereased DNA binding are also considered to fall within the scope of the invention. Such mutants may possess a mutation in the zinc finger binding domain. In particular mutants of the amino acid sequence provided in sequence id no 9 are claimed.

Fragments of the nucleic acid sequence according to sequence id no 9 of at least 15 nucleotides also fall within the scope of the subject invention. Such fragments can be used as probes or primers for detecting and isolating equivalent sequences. In particular a combination of two or more such fragments can be useful in a kit for detecting and/or isolating /'.such equivalent sequences. Preferably a fragment will be derived from the C terminal half of the amino acid sequence of sequence id no 9. In a suitable embodiment of the kit one fragment will not comprise a part of the nucleic acid sequence forming the zinc finger domain. In the examples a suitable combination of fragments to be used as primers is illustrated.

Any sequence obtainable through PCR as illustrated in the examples with these primers is considered to fall within the scope of the invention.

The hybridisation conditions used in the examples provide the minimum stringency required for selectivity. More stringent conditions can be applied such as the stringent hybridisation conditions described by Sambrook et al for increased homology of the obtained sequences with the sequence id no 9. A lower salt concentration generally means more 30 stringent conditions. Any fragment of the sequence according to sequence id no 9 being or encoding a polypeptide exhibiting the target gene binding activity of the complete sequence is also included within the scope of the invention as are equivalent nucleic acid sequences or amino acid sequences thereof, with equ i valent being defined as defined above 35 with regard to hybridisation and/or mutation and/or degeneracy of the genetic code for the complete sequence.

A vector or plasmid comprising the nucleic acid sequence encoding xylR or an equivalent sequence thereof as defined above also falls within the scope of the invention, as do a vector or plasmid comprising such a sequence and a host cell comprising such an additional sequence.A transformed host cell such as a microorganism or a plant cell carrying at least one additional copy of' an encoding nucleic acid sequence according to sequence id no 9 or an equivalent thereof falls within the scope of the invention. Preferably the various embodiments are organised such that the sequence can be expressed in the vector. plasmid or host cell. The regulatory gene can comprise the complete sequence of id no 9 or merely the encoding sequence therof in combination with an alternative promoter that is operable in the host cell. Suitable examples of host cells have been provided elsewhere in the description. Suitable operable promoters for the various host cells that can be incorporated with the encoding sequence of sequence id no 9 will be clear to a person skilled in the art. In particular for the host cells explicitly mentioned in the description constitutive promoters or inducible promoters are knxown and available to work the invention without undue burden.

A priocess for production of homologous or heterologous proteins or peptides is provided, said process comprising expression of a nucleic ,'Acid sequence encoding the homologous protein or peptide in a host cell.

said host cell further comprising an additional copy of a nucleic acid sequence encoding a regulatory gene such as xlnF{ or an equivalent thereof which is also expressed. A process as just described is preferably carried out with a combination nucleic acid expression cassette comprising the regulatory gene operably linked to a first promoter and said cassette further comprising a second promoter, said second promoter normally being associated with a target gene of the regulator. said target gene promoter being operably linked to the nucleic acid sequence encodi ng the homologous or even heterologous protein or peptide to be 000* 0expressed. The first promoter can be the promoter natively associated with the regulator gene but may also be a promoter of choice that is operable in the'host cell. The deg-ree of expression is no longer restricted by the presence of too small an amount of' regulator and thus the degree of expression of the gene to be expressed is much higher than In a corresponding host cell where the gene is expressed without the additional presence of the regulator gene. Such increased expression is preferably achieved in cells of organisms normally comprising components of the part of' the metabolic pathway to be influenced. Suitable host a so cells are a plant cell or a microorganism. suitably the microorganism can be a fung-us in particular it can be a filamentous fungus. Examples of suitable host cells have been given elsewhere in the descriportion and may be considered to be incorporated here. The incorporation of a combined expression cassette of the type just described above in a host cell comprising a target gene of the regulator can lead to increased expression of the target gene or to increased expression of the target genes if multiple target genes are present. Preferably the target gene will be native to the regulator. Preferably such a target gene will be endogenous to the host cell. In the examples the nucleic acid sequence of the regulator of the xylanolytic pathway xlnR is provided. The native target genes for this regulator have been found to comprise the genes xlnA, xlnB, xlnC and xlnD as well as axeA and as such these genes are preferred target genes. Other targets exist and are considered to be included in the term target gene. Various embodiments of the host cells according to the invention are covered in the claims. If both regulator sequence and target gene are natively present in the host cell the regulator sequence will be present in multiple copies. Such a micro6rganism will over express the gene regulated by the target gene promoter in comparison to the native microorganism.

Because now the sequence for the xylanase regulator is known it has become possible to knock out the xylanase regulator. The creation of a knockout host cell once the nucleic acid sequence of the gene to be knocked out is known is standard technology. This method can be carried out analogously to that described in Berka et al (1990) and example 11 of EP 95201707.7, which is a copending European patent applicationof which a copy has been included upon filing the subject document and the example itself has also been copied into this document in the examples. Such a knockout renders a host cell which can be free of xylanolytic activities.

A host cell free of xylanolytic activity due to knocking out the xlnR gene can be used to produce homologous or heterologous expression S. products of choice free of xylanolytic activity. A host cell with a knocked out xlnR gene falls within the scope of the invention. Such a host cell is preferably a plant cell or a filamentous fungus. Such a filamentous fungus is preferably an Aserzillus. Examples have been provided elsewhere in the description of numerous suitable host cells.

A host cell with a mutation in the regu4lator gene which ran be arrived at using the selection and mutation method of the invention can be "subjected to complementation with a regular active copy of the regulator gene. Such a complemented strain will subsequently express the products e of any target genes that are regulated by the regulator. These target S""gene products will be absent in the case of the non complemented 24 Regulator negative mutant. Upon comparison of protein bands obtained in a manner known per se from both of the strains it will become apparent what target products are regulated by the regulator and subsequently the corresponding novel target genes can be determined in a manner known per se once its expression product has been determined. In this manner other target genes than those already known can be found for the xylanese regulator xylR in the instant examples.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

a.

a 24A Example 1: Construction of the plasmids Example 1.1: Construction of the selection plasmid pIM130 The selection plasmid pIM130 was constructed as depicted in Fig. 1. In PCR1 a fragment was generated from the plasmid pIM120 (de Graaff et al.. 1994) using oligonucleotide 1 (SEQ ID NO: 1) C TCCCGT-3' (Formula 1) and oligonucleotide A (SEQ ID NO: 2) 5'-CAATTGCGACTTGGAGGACATGATGGCAGATGAGGG-3' (Formula 2) Oligonucleotide 1 was derived from the Asoerrillus tubizensis JnA promoter (de Graaff et al., 1994) positions 600-619 (SEQ ID NO: 5) to which 10 nucleotides containing a Nsil site were added. The 3' end of oligonucleotide A was derived from the Asoergillus nizer go:C transcription unit (Whittington et al., 1990) ending just before the translation initiation site (positions 708-723)(SEQ ID NO: while the 5' end was derived from the coding region of the A,niger pyrA gene (Wilson et al., 1988) (starting at the translation initiation site (positions 341 to 359, SEQ ID NO: 7).

Fragment A was generated by a PCR containing 10 pl 10*reaction buffer (100 mM Tris-HC1, pH 8.3, 500 mM KC1. 15 mM MgCl 2 0.01% gelatin), 16 pi 1.25 mM of each of the four deoxynucleotide triphosphates, 1 ng of the plasmid pIM120 DNA and 1 ug of each of the oligonucleotides 1 and A in a final volume of 100 pl. This reaction mixture was mixed and 1 pl TAQ polymerase (5 U/pl) (Life Technologies) was added. The DNA was denatured by incubation for 3 min at 92 0 C followed by 25 cycli of 1 min 92°C, 1 min 48 0 C and 1 min 72°C. After these 25 cycli the mixture was incubated for min at 72 0 C. Analysis of the reaction products by agarose electrophoresis revealed a fragment of about 250 bp, which corresponds to the size expected based on the sequence of the genes.

In PCR2 a fragment was generated from the plasmid pGW635 (Goosen et o: 30 aZ.. 1987) using oligonucleotide 2 (SEQ ID NO: 3) *r 5'-AGAGAGGATATCGATGTGGG-3' (Formula 3) and oligonucleotide B (SEQ ID NO: 4) -CCCTCATCTGCCCATCATGTCCTCCAAGTCGCAATTG-3' (Formula 4) The 5' end of oligonucleotide B was derived from the A.nizer goxC basic transcription unit (positions 708-723, SEQ ID N0:6)(Whittington et al., 1990), while the 3' end was derived from the coding region of the A.nizer pyrA gene starting at the translation initiation site. Oligonucleotide 2 was derived from the pyrA coding region (positions 339-359. SEQ ID N0:7) and is spanning an EcoRV restriction site at position 602 (SEQ ID NO:7).

Fragment B was generated in an identical manner as fragment A except that in this case the reaction mixture contained 1 pg each of oligonucleotide 2 and B and 1 ng of plasmid pGW635 DNA. Analysis of the reaction products by agarose electrophoresis revealed a fragment of about 250 bp, which corresponds to the size expected based on the sequence of the pyrA gene.

Fragments A and B were isolated from agarose gel after electrophoresis. The fragments were cut from the agarose gel, after which they were recovered from the piece of agarose by electro-elution using ISCO cups. Both on the large and the small container of this cup a dialysis membrane was mounted, the cup was filled with 0.005 x TAE buffer per 1000 ml: 242.0 g Trizma base (Sigma). 7.1 ml glacial acetic acid, 100 ml 0.5 M EDTA pH 8.0) and the piece of agarose was placed in the large container of the cup. Subsequently the cup was placed in the electro-elution apparatus, with the large container in the cathode chamber containing 1*TAE and the small container at the anode chamber containing 1°TAE/3 M NaAc. The fragments were electro-eluted at 100 V during 1 h. After this period the cup was taken from the electro-elution apparatus and the buffer was removed from the large container, while from the small container the buffer was only removed from the upper part. The 30 remaining buffer (200 pl) containing the DNA fragment was dialyzed in the cup against distilled water during 30 min. Finally the DNA was precipitated by the addition of 0.1 vol. 3 M NaAc. pH 5.6 and 2 vol. cold ethanol. The DNA was collected by centrifugation (Eppendorf centrifuge) for 30 min. at 14.000 x g. at 4"C. After removal of the S: 35 supernatant the DNA pellet was dried using a Savant Speedvac vacuum o:i centrifuge. The DNA was dissolved in 10 ul TE buffer (TE: 10 mM Tris/HCl pH7.2. 1 mM EDTA pH 8.0) and the concentration was determined by agarose gel electrophoresis. using lambda DNA with a known concentration as a reference and ethidiumbromide staining to detect the DNA.

Fragments A and B were fused in PCR3 which was identical to PCR1 except that in this case the reaction mixture contained 1 pg of each of the oligonucleotides 1 and 2 and approximately 50 ng of each of the fragments A and B. Analysis of the reaction products by agarose gel electrophoresis revealed a fragment of about 500 bp, which corresponds to the size expected based on the sequences of the genes.

The resulting fragment C was isolated from agarose gel as described and subsequently digested using the restriction enzymes Nsil and EcoRV.

The DNA was digested for 3 h. at 37*C in a reaction mixture composed of the following solutions; 5 pu 0.5 pg) DNA solution; 2 pl of the appropriate 10 x React buffer (Life Technologies); 10 U restriction enzyme (Life Technologies) and sterile distilled water to give a final volume of 20 pl. After digestion the DNA fragment was analysed by agarose gel electrophoresis and subsequently isolated from the gel as described.

For the final construction of pIM130 5 pg of the plasmid pGW635 was digested as described using 50 U of the restriction enzymes EcoRV and Xbal in a final volume of 500 pl. After separation of the products a 2.2 kb EcoRV/Xbal fragment (fragment D) was isolated from the agarose gel by electro-election. Analogously 1 pg vector pGEM-7Zf(+) (Promega) was prepared by digestion with the restriction enzymes NsiI/XbaI. which was after digestion electrophoresed and isolated from the agarose gel by electro-elution.

The plasmid pIM 130 was constructed by the following ligation reaction: 100 ng pGEM-7Zf(+) Nsfl/Xbal fragment was mixed with 50 ng fragment C and 50 ng fragment D and 4 pl 5 ligation buffer (composition; 500 mM Tris-HCl, pH 7.6; 100 mM MgC 2 1; 10 mM ATP; 10 mM dithiotreitol; 25% PEG-6000) and 1 pl (1.2 U/pl) T4 DNA ligase (Life Technologies) was added to this mixture in a final volume of 20 ul. After incubation for 16 h at 14*C the mixture was diluted to 100 pl with sterile water. 30 pl of the diluted mixture was used to transform E. coli DH5a competent cells, prepared as described by Sambrook et at., 1989.

Two of the resulting colonies were grown overnight in LB medium (LB medium per 1000 ml: 10 g trypticase peptone (BBL), 5 g yeast extract (BBL), 10 g NaCl. 0.5 mM Tris-HCl pH 7,5) containing 100 pg/ml ampicillin. From the cultures plasmid DNA was isolated by the alkaline lysis method as described by Maniatis et at. (1982), which was used in restriction analysis to select a clone harbouring the desired plasmid pIM130. Plasmid DNA was isolated on a large scale from 500 ml cultures E.coli DH5a containing pIM130 grown in LB medium containing 100 pg/ml 27 ampicillin (Maniatis et al., 1982) The plasmid was purified by Csl centrifugation, phenolyzed. ethanol precipitated and dissolved in 400 ul TE. The yield was approximately 500 pg.

Example 1.2: Construction of the plasmid plM135 From the plasmid pIM120 a second plasmid was constructed which contains the goxC basic transcription unit fused to the pyrA coding region and termination region. In PCR4 a fragment was generated from the plasmid pIM120 using oligonucleotide 3, 5'-CACAATGCATCGTATAACTAACCTCGTTCG-3' (Formula which was derived from the gozC basic transcription unit (positions 640- 660, SEQ ID NO:6) to which 10 nucleotides containing a Nsfl site were added, and oligonucleotide 2 (Formula The fragment generated was isolated from gel, digested with Nsil and EcoRV and cloned together with the 2.2 kb EcoRV/Xbal fragment of pGW 6 35 in the plasmid pGEM-7Zf(+).

which-was digested with XbaI/NsiI, as described in Example 1.1., resulting in the plasmid pIM135.

The plasmid pIM135 can be used as construction vehicle for preparing vectors according to the invention with any desirable inducible enhancer or activator sequence, a UAS of a gene involved in metabolism. pIM135 comprises a basic transcription unit (tGOX) operatively linked to a bidirectional marker gene (pyrA).

Example 2: Transformation of A.nirer using the plasmid pIM130 250 ml of culture medium, which consists of Aspergillus minimal medium (MM) (contains per liter: 6.0 g NaN0 3 1.5 g KH 2 POhA 0.5 g MgSO,.7H 2 0. 0.5 g KC1, Carbon source as indicated. pH 6.0 and 1 ml Vishniac solution *o (contains per liter 10 g EDTA, 4.4 g ZnS0h.7H 2 0, 1.0 g MnC 2 1.4HO0. 0.32 g CoC1 2 .6H 2 0. 0.32 g CuS0h.5H 2 0. 0.22 g (NHa) 6 Mo0 2 ,.4H 2 0. 1.4 7 g CaCl 2 .2H 2 0.

30 1.0 g FeSO0.7H 2 0. pH 4.0) supplemented with 2 glucose. 0.5 Yeast Extract. 0.2 Casamino acids (Vitamin free). 10 mM L-arginin. 10 uM nicotinamide, 10 mM uridine, was inoculated with 1 106 spores per ml of strain NW205 (cspAl, pyrA6, nicAl, argB1J) and mycelium was grown for 16 18 hours at 30 *C and 250 rpm in a orbital New Brunswick shaker. The mycelium was harvested on Myracloth (nylon gauze) using a BUchner funnel and mild suction and was washed several times with SP6 SP6: 0.8 NaCl.

mM Na-phosphate buffer pH 150 mg Novozyme 234 was dissolved in ml SMC (SMC: 1.33 M Sorbitol. 50 mM CaC1 2 20 mM MES buffer, pH 5.8) to which 1 g (wet weight) mycelium was added and which was carefully resuspended. This suspension was incubated gently shaking for 1 2 hours at 30 every 30 minutes the mycelium was carefully resuspended and a sample was taken to monitor protoplast formation using a haemocytometer to count the protoplasts. When sufficient protoplasts were present (more then 1 108) these were carefully resuspended and the mycelial debris was removed by filtration over a sterile glasswool plug. The protoplasts were collected by 10 minutes centrifugation at 3000 rpm and 4 *C in a bench centrifuge and were carefully resuspended in 5 ml STC (STC: 1.33 M Sorbitol, 50 mM CaCl 2 10 mM Tris/HC1, pH This wash step was repeated twice and the protoplasts were finally resuspended in STC at a density of 1 108 per ml.

The transformation was performed by adding 1 pg of pIM130 DNA (dissolved in a 10 20 pl TE to 200 pl of protoplast suspension together with pl of PEG buffer (PEG Buffer: 25 PEG-6000, 50 mM CaCI 2 10 mM Tris/HCl pH mixed gently by pipetting up and down a few times, and incubated at room temperature for 20 minutes. After this period 2 ml PEG buffer was added, the solution was mixed gently and incubated at room temperature for another 5 minutes and subsequently 4 ml of STC was added and mixed gently on a vortex mixer. This was also done using 5pg pIM130 and 1 and 5 pg of plasmid pGW635 DNA. As a negative control 20 pi of TE was added to the protoplasts.

One ml portions of this suspension were then added to 4 ml of osmotically stabilised top agar and poured on plates (swirl gently to cover plate with top agar) containing MMS having either 100 mM D-glucose or 100 mM D-xylose as a carbon source. These media (MMS) were osmotically stabilised using 0.8 M KCL or by using 1.33 M Sorbitol.

To determine the percentage regeneration serial dilutions of the protoplasts were prepared before transformation (untreated protoplasts.

kept on ice) and after transformation (obtained from the negative 30 control). 100 pl of the 10 3 10 10- 5 and 10- 6 dilutions were plated in duplicate on 10 mM uridine supplemented MMS plates.

For the positive control, in which the fungus was transformed using the plasmid pGW635, colonies were found on all plates. However, the transformation frequency was much lower (1-10 transformants per pg 35 plasmid DNA) on KC1 stabilised medium than on sorbitol stabilised medium (100-1000 transformants per pg plasmid DNA). This was due to the much higher regeneration frequency on the latter medium, which was about in comparison to 2-5% on the KCl stabilised medium.

In case of the transformations using the plasmid pIM130 transformants were found on the medium containing D-xylose as a carbon source but not on medium containing D-glucose. The frequency on sorbitol stabilised medium was about 100 per pg of plasmid DNA while the frequency on the KC1 stabilised medium was less than one per pg of DNA.

Example 3: Analysis of transformants The transformants from pIM130 obtained in Example 2 were analysed phenotypically by plating on MM containing 100 mM D-glucose. 100 mM Dglucose/l% Oat spelt xylan (Sigma #X0627) and 1% Oat spelt xylan. A selection of the transformants were replica plated to these media and incubated at 30 0 C. About 75% of the transformants were growing on xylan containing medium, while no growth was found on media containing Dglucose. The remaining 25% of the colonies grew on all three media tested.

A selection of five transformants having the expected phenotype (growth on xylan containing medium, non-growth on D-glucose containing media) was analysed by Southern analysis. Fungal DNA was isolated by a /modified procedure used to isolate plant RNA essentially as described by de Graaff et ac., 1988). Mycelium, which was grown overnight in culture medium, was harvested, washed with cold saline, frozen in liquid nitrogen and stored at -80*C. Nucleic acids were isolated by disrupting 0.5 g frozen mycelium using a microdismembrator (Braun). The mycelial powder obtained was extracted with freshly prepared extraction buffer.The extraction buffer was prepared as follows: 1 ml tri-isopropylnaphtalene 25 sulfonic acid (TNS) (20 mg/ml) was thoroughly mixed with 1 ml p-aminosalicylic acid (PAS) (120 mg/ml) and 0.5 ml 5 x RNB buffer was added (5 x RNB contains 121.10 g Tris, 73.04 g NaCl and 95.10 g EGTA in 1 1, pH After the addition of 1.5 ml phenol, the extraction buffer was equilibrated for 10 min. at 55'C. The warm buffer was then added to the mycelial powder, and the suspension was thoroughly mixed for 1 min.

using a vortex mixer. After addition of 1 ml chloroform the suspension was remixed for 1 min. After centrifugation at 104 x g for 10 min.. using a Sorvall high speed centrifuge, the aqueous phase was extracted once more with an equal volume of phenol/chloroform and was then 35 extracted twice with chloroform. DNA was isolated from the aqueous phase using the following procedure; the DNA was immediately precipitated from the aqueous phase with 2 vol. ethanol at room temperature and subsequently collected by centrifugation using a Sorvall high speed centrifuge at 10" x g for 10 min. and washed twice by redissolving the DNA in distilled, sterile water and precipitating it again with ethanol.

RNA was removed by adding RNase A (20 g pg/ml) to the final solution.

High molecular weight DNA (1-2 Ig) isolated from A.niger N402 (cspAl) as a wild-type and five pIM130 transformants as described was digested with Hpal (Life Technologies) according to the manufactors intructions.

The resulting fragments were separated by agarose gel electrophoresis, and transferred to High-bond N membrane as described by Maniatis et al.

(1982). Hybridisation using a 3 P-labelled 3.8 kb XbaI fragment, prepared as described by Sambrook et al., 1989, containing the A. nirer pyrA gene as a probe was done according to the following procedure (Sambrook et prehybridization in 6 x SSC (20xSSC per 1000 ml 175.3 g NaC,.

107.1 g sodiumcitrate.5.5

H

2 0, pH 0.1% SDS, 0.05% sodium pyrophosphate, 5* Denhardt's solution (100xDenhardts solution per 500 ml 10 g Ficoll-400. 10 g polyvinylpyrrolidone. 10 g Bovine Serum Albumin (Pentax Fraction V) and 20 pg/ml denatured herring sperm DNA at 68'C for hrs and hybridization in an identical buffer which contained the Sdenatured radiolabelled probe at 68'C for 15-18 hrs, followed by two washes in 3 x SSC, 0.1 SDS at 68*C and two washes in 0.2 x SSC, 0.1% SDS at 68*C. The membrane was covered with Saran wrap and autoradiographed overnight at -70'C using Konica X-ray films and Kodak X-Omatic cassettes with regular intensifying screens.

As a result a 10 kb hybridising band is found in the N402 lane, while this band is missing in the transformants NW205::130#1. NW205::130#2 and NW205::130#3. In the transformants NW205::130#1 and NW205::130#3 a 25 hybridising fragment is found, while in NW205::130#2 a 20 kb band is found. These results correspond respectively to a single and a double copy integration at the homologous pyrA locus. In the transformants NW205::130# 4 and NW205::130#5 the plasmid was integrated at a nonhomologous locus. Transformant NW205::130#2 was selected for mutagenesis.

The UAS fragment of pIM130 comprises the binding site required for the positive regulator to exhibit the stimulatory activity of the UAS. Thus inhibition of xlnR in the host cell comprising pIM130 will result in negative expression of the bidirectional marker present on pIM130. Expression of xlnR is induced by xylan or xylose. Thus the 35 presence of such substrates should result in expression of the bidirectional marker of pIM130 if the host possesses xlnR.

The UAS fragment of pIM130 does not comprise the site required for inhibitory activity of creA that is present on the native UAS of the Asperrillus niger xlnA gene. Thus the presence of glucose which renders A. nizer CRE A' and subsequently inhibits the UAS of xlnA and the other xylanolytic enzyme encoding genes such as xlnD and axeA and also inhibits the xlnR gene encoding the activator of the UAS of the aforementioned xylanolytic enzyme encoding genes i.e. represses xylanolytic enzyme expression which results in negative expression of pIM130.

Example 4: Selection of mutants Example 4.1: Selection of derepressed mutants Spores of NW205::130#2 were harvested in 5 ml ST (ST: 0.8 NaCl 0.05 Tween 20), shaken at high frequency and mycelial debris was removed by filtration over a sterile glasswool plug. The spores were collected by centrifugation for 10 minutes at 3000 rpm at room temperature in a bench centrifuge and were resuspended in 5 ml saline.

This wash step was repeated twice and the spores were finally resuspended in saline at a density of 1 107 per ml. 10 ml of the spore suspension /'was dispensed in a glass petridish and was irradiated using UV at a dosage of 18 erg/mm 2 /min for 2 min. After mutagenesis 105 and 106 spores were plated (10 plates each) on MM+ 10 mM L-arginine 10 uM nicotinamide containing 3% D-glucose and on plates containing 3% Dglucose/3% oat spelts xylan (Sigma #X0627).

After 4-7 days 5-10 mutant colonies per plate (106 spores inoculated) were found which on basis of their morphology could be divided into three 25 classes; large, well sporulating colonies, intermediate-sized, well 'sporulating colonies and small poorly sporulating colonies. A random selection of 20 of these mutants was made and the selected mutants were tested on media containing different carbon sources or substrates. The mutant colonies were found to be able to grow on media containing D- 30 glucose, D-glucose/xylan and xylan, while the parental strain NW205::130#2 only was able to grow on medium containing xylan as a carbon source. In addition these mutants were tested on different chromogenic substrates; 4-methylumbelliferyl-p-D-xyloside (-xylosidases, endo- Sp. xylanases)(Sigma #M7008), 4-methylumbelliferyl acetate (acetyl-xylan esterases) (Sigma #M0883), 4-methylumbelliferyl-a-L-arabinofuranoside (arabinofuranosidases) (Sigma #M9519) and on Remazol Brilliant blue--..

modified xylan (endo-xylanases) (Sigma #M5019). The methylumbelliferyl derivatives were added in a 1 mM final concentration to media containing 6 D-glucose, D-glucose/xylan and xylan, while the RBB-xylan was added in a 32 concentration of 1 mg/ml to media containing D-glucose/xylan andjxylan.

For all these substrates tested enzyme activity was found in the mutants after growth on D-glucose containing media, while no expression was found in the parental strain NW205::pIM130#2. On media containing xylan an increased expression of these enzymes was found in -comparison to NW205::pIM130. Of the mutants tested mutant 5 B had the highest expression levels. In the instant case selection occurred on a substrate normally active as inhibitor of xylanolysis i.e. glucose. To be certain that expression could occur in the absence of repression an inducer of xylanolytic genes xylan was also included. Such a control test is preferably included in a method according to the invention. The mutant clearly exhibited derepression.

For the comparison of the activity levels produced, both A.niger N402 and mutant 5B were cultured on MM containing 1.5% crude Wheat arabinoxylan as a carbon source. Samples were taken at 24, 42. 72 and 96 hrs and the activities of a-L-arabinofuranosidase, endo-xylanase and p-xylosidase were measured. The results (Fig. 2 A,B,C) indicated an increase in activity for the mutant strain for all three enzymes. a-Larabinofuranosidase and endo-xylanase activity was most strongly increased.

Example 4.2: Selection of non-expressing mutants Spores of strain NW205::130#2 were harvested and mutated as described Sin Example 4.1 and subsequently plated on MM containing 100 mM D-xylose -25 supplemented with 10 mM uridine, 10 mM L-arginin and 0.8 mg/ml fluoroorotic acid (Sigma #F5013). These plates were incubated for 4-7 days at 30 0 C. 64 of the growing colonies, having a PYR' phenotype, were analysed for xylanase expression by plating on MM containing 1% xylan mM uridine 10 mM L-arginin 10 uM nicotinamide. Of these 64 mutants tested 10 gave a reduced zone of clearing on these xylan containing plates and had potential reduced xylanase levels. The phenotype of these Smutants was verified on D-glucose. D-glucose/xylan and xylan containing media in the presence and absence of uridine. All 10 mutants did not grow on media without uridine.

.35 For further analysis these mutants were precultured 18 hrs at 30°C on MM containing 50 mM fructose 10 mM uridine 10 mM L-arginin 10 4M nicotinamide after which the mycelium was harvested and 1 g wet mycelium was transferred to MM containing 1% xylan and to MM containing 10 mM Dxylose 10 mM uridine 10 mM L-arginine 10 PM nicotinamide. After hrs incubation at 30 0 °C both the mycelium and the culture filtrate were harvested. The culture filtrate was dialysed against 1 mM NaP 1 pH 5.6 after which the xylanase (Bailey et aZ. (1991)) was determined and 8xylosidase activities were determined (Table Both the xylanase as well as the -xylosidase expression levels were found to be strongly reduced in these selected mutants.

ooooo oo° *5*

S

S. S S S S S* Activity endo-xylanase D-xylosidase a-L-arabinofuralosidase FStrain xylan xylose xyian xylose xylan xylase (nkat m1- 1 (nkat mwE') (nkat ml- (nkat ml- (nkat mE'1) (nkat mlE NW4205 5 102 5 *lO0 0.35 0.110 0.33 0.37 NW205::130 5 102 1 120.36 0.51 0.110 0.29 NW205::130 2Ac2-15 5 02 1 o20.26 0.30 0.30 0.23 NW205::130 Acl-I 2 1 0.01 0.01 0.36 NW205::130 Ac t

I-

1 2 0.3 0.01 0.01 0.24I fW2o5::130O Ac t I-6 2 0.3 0.01 0.01 0.31 .W05:30 Ac3-14 1 0.14 0.01 0.01 0.29 r NW205:: 13 c l 820140 0 10 3 14W 0 5 0 A 2 010 1 0 .0 1 0 .0 1 0 1 9 NW205::1'30 Ac2-8 2 0.34.10.102 tNf205::1'30 Ac2-50 1 0.2 0.01 0.01 0.28 NW205::1'30 Ac2-i 2 0.2 0.01 0.01 0.23 NW205::130 Ac 1 I-1 1 5 0.2 0.01 L 0.01 0.28 Example 5. Complementation of non-expressing mutants 5.1 Construction of an A.niger genomic plasmid library SFor the construction of a plasmid library 10 pg of genomic DNA of Anige NW128 (cspAl, nicAl, pyrAB, goxC17), isolated as described in Example 3, was partially digested for 30 min at 37 0 C according to the manufactors instructions using 3.5 U Sau3A (Life-Technologies) in a final volume of 100 pl. After separation of the fragments by agarose electrophoresis. fragments ranging in size from 6.7 kb to 9.4 kb were cut from the low melting point agarose gel and were isolated as described in Sambrook et al., (1989). From totally 6 digestions 4 pg of fragments were isolated in a final concentration of 100 ng/pl. 600 ng of the resulting fragments were ligated in 100 ng BamHI digested pUCl8 (Pharmacia #27526201) according to the manufacturors instructions. After ligation 4pl of the resulting ligation mixture was used to transform 100 pl E.cof Efficiency competent cells (Life Technologies #18258-012) according to the manufactors instructions. Six subsequent transformation experiments resulted in about 5 1 0 A colonies. After resuspension of these colonies and growth for 3 hrs at 37°C in TY medium (medium per 100 ml: 16 g Select Peptone 140 (Life Technologies), 10 g NaCI and 10 g Yeast extract)containing 100 pg/ml Ampicillin, plasmid DNA was isolated and purified by CsCl centrifugation.

5.2 Complementation of non-expressing mutants 25 For the complementation in non-expressing mutants a selection of three mutants, NW205::130 Acl-4, NW205::130 Acl-15 and NW205::130 Ac4- 4. protoplasts were prepared of these strains and transformed as described in Example 2. In these transformation experiments 108 protoplasts were used and combined with; 20 pg DNA of the Aniger plasmid 30 library as described in Example 5.1. and with 10 pg DNA of the plasmid library combined with 10 pg DNA of the autonomouly replicating plasmid pHELP1 (Gems and Clutterbuck, 1993) and with 20 pg DNA of the plasmid library combined with 10 pg DNA of the autonomously replicating plasmid pHELP1. As a positive control 2 pg pGW635 was used. After the transformation procedure the mixtures were plated on MM stabilised with 1.33 M Sorbitol containing 50 mM D-xylose as a carbon source supplemented with 10 pM nicotinamide and 10 mM L-arginin. After about 4 days colonies appeared on the positive control plates and were counted, while after 6 days colonies could be picked from the complementation plates.

The resulting transformant colonies were analysed by plating on medium containing 1% Oat spelt xylan, 1 mM 4-methylumbellyferyl-B-Dxyloside. 10 M nicotinamide and 10 mM L-arginin. After 6-7 hrs incubation at 30°C xx fluorescent colonies were detected. After 2-3 days a clearing of the xylan around these colonies-appeared.

Example 6: Cloning of the A.nizer xtnR gene Example 6.1: Isolation of the A.nizer xrnR gene The A.nizer ztnR gene was isolated from transformants obtained and selected as described in Example 5.2. From 13 transformants obtained from NW205::130 Ac 1-4. NW205::130 Ac 1-15 and NW205::130 Ac 4-4 total DNA was isolated, as described by de Graaff et at., 1988. After mycelium was cultured under selective inducing) conditions for pyrA and xylanolytic expression on 1.5 crude wheat arabino-xylan as C source from which free replicating plasmids were isolated using Nucleobond AX100 columns (Macherey Nagel). 200 pg of total DNA dissolved in 400 Il of sterile water was mixed with 2 ml of S1 buffer (50 mM Tris-HCl pH 8.0, mM EDTA, 100 pg RNase 2 ml of S2 (200 mM NaOH. 1 SDS), followed by an incubation at room temperature for 5 minutes, and 2 ml of S3 (2.60 M KAc, pH followed by an incubation on ice for 5 minutes. After clearing of the suspension at 15.000 g for 30 minutes adsorption, washing. elution and precipitation of the plasmids was done all according to the manufacturers' instructions for "working procedure for the 25 purification of plasmids and cosmids" (5.3 modified alkaline/SDS lysis).

20 pl of the resulting plasmid DNA, dissolved in 150 ul sterile water.

was used in E.coli DH5a transformation. (Sambrook et at., 1989). 12 E.coli colonies resulting from each of these plasmid preparations were grown for 9-12 hrs at 37 0 C in 250 ml LB medium containing 50 pg/ml 30 Ampicillin. after which a miniprep plasmid DNA isolation was performed on 1.5 ml of the culture as described for the boiling lysis protocol by Sambrook et al., 1989 and the cells were pelleted by centrifugation and stored at -20*C. Analysis of these DNA preparations, after HinDIII digestion, by agarose electrophoresis revealed three classes of plasmids; pHELPI type plasmids, genomic library type plasmids and large complex type plasmids. From colonies containing the latter type of plasmids, a large scale plasmid isolation, using Nucleobond AX100 columns according to the manufacturers' instructions for the modified alkaline/SDS lysis (Macherey-Nagel) was performed on the frozen pellet of the 250 ml culture. This resulted in the isolation of two plasmid types. A and B, complementant A and B respectively. Both these plasmids were digested.

using SaZI. Pstl, EcoRl, HinDIII and after agarose electrophoresis, the fragments were analysed in triplicate by Southern analysis using denatured radiolabelled pHELP1. plasmid A and plasmid B. This showed that both plasmids A and B, besides the pHELP part, shared the same genomic region. Based upon the differences between the hybridisation signals found using the pHELP plasmid, showing the vector and AMA1 sequences, and the plasmid A and B signals, fragments hybridising with both plasmid A and B, but not with pHELP1, were identified and subcloned. A 4 kb EcoRI fragment and a 6.5 kb HinDIII fragment of plasmid B, and a 3 kb PstI fragment of plasmid A were found to hybridise with both plasmids A and B and were subcloned in pGEM7/EcoRI, pGEM7/HfnDIII and pBluescript/PstI, resulting in plasmid pNP1. 2 and 3 respectively. After propagation and purification these plasmids were used in complementation experiments using the mutant NW205::130 Ac 4-4 as described in Example 5.2. In these experiments the mutant was directly transformed without using pHELP1. In these experiments the plasmid pNP2, containing the 6.5 kb HinDIII fragment gave rise to complementation of the mutation. Further subcloning and transformation of pNP2-derived plasmids revealed a 5 kb BamHI-Xbol fragment, subcloned in pBluescript and resulting in plasmid pNP8, giving rise to complementation of strain NW205::130 Ac 4-4 Example 6.2: 'Subcloning of the A.niger zLnR gene For the subcloning of the zlnR gene, the A.nizer genomic library, constructed as described by Harmsen et at., 1990, was screened for phages containing the xlnR region by using the 4 kb EcoRI fragment of pNPl as probe. 3 x 103 pfu per plate were plated in NZYCM top-agarose containing 0.7% agarose on five 85-mm-diameter NZYCM agar) plates Sas described (Maniatis et al., 1982) using E. coli LE392 as plating bacteria. After overnight incubation of the plates at 37*C two replicas Sof each plate were made on HybondN* filters (Amersham) as described in oo: 35 Maniatis et at. (1982). After wetting the filters in 3xSSC the filters were washed for 60 min. at room temperature in 3xSSC. Hybridisation using the 32 P-labelled 4 kb EcoRI fragment, prepared as described by Sambrook et Sa., 1989, was done according the following procedure (Sambrook et al., 1989); prehybridisation in 6 x SSC (20xSSC per 1000 ml 175.3-g NaCl, 107.1 g sodium citrate.5.5 H20 pH 0.1% SDS, 0.05% sodium pyrophosphate. 5 Denhardt's solution (100xDenhardts solution per 500 ml 10 g Ficoll-400. 10 g polyvinylpyrrolidone, 10 g Bovine Serum Albumin (Pentax Fraction V) and 20 pg/ml denatured herring sperm DNA at 68*C for 3-5 hrs and hybridisation in an identical buffer which contained the denatured radiolabelled probe at 68*C for 15-18 hrs, followed by two washes in 2 x SSC, 0.1 SDS at 68'C and two washes in 0.2 x SSC, 0.1 SDS at 68C. The membrane was covered with Saran wrap and autoradiographed overnight at -70*C using Konica X-ray films and Kodak X-Omatic cassettes with regular intensifying screens.

This screening resulted in about 12 positive phages, of which eight were purified. Each positive plaque was picked from the plate using a Pasteur pipette and the phages were eluted from the agar plug in 1 ml of SM buffer containing 20 pi chloroform, as described in Maniatis et al.

(1982). The phages obtained were purified by repeating the procedure described above using filter replicas from plates containing 50-100 plaques of the isolated phages.

After purification the phages were propagated by plating 5x10 3 phages on NZYCM medium. After overnight incubation at 37'C confluent plates were obtained, from which the phages were eluted by adding 5 ml SM buffer and storing the plate for 2 h. at 4*C with intermittent shaking.

After collection of the supernatant using a pipette, the bacteria were removed from the solution by centrifugation at 4,000 x g for 10 min. at S4C. To the supernatant 0.3% chloroform was added and the number of pfu is determined. These phage stocks contain approximately 109 pfu/ml.

DNA of five selected phages. A-R.B-RC-R.E-R.F-R. isolated as described in Sambrook et al. (1989). was analysed by Southern analysis.

The DNA was digested for 5 h. at 37'C in a reaction mixture composed of 30 the following solutions; 5 pl 1 pg) DNA solution; 2 ul of the appropriate 10 x React buffer (Life Technologies); 10 U Restriction enzyme (Life Technologies) and sterile distilled water to give a final volume of 20 pl. The samples were incubated for 10 min. at 65'C and rapidly cooled on ice, before loading on a 0.6% agarose gel in 1*TAE buffer. The DNA fragments were separated by electrophoresis at 25 V for 15-18 h.

.i After electrophoresis the DNA was transferred and denatured by alkaline vacuum blotting (VacuGene XL. Pharmacia LKB) to nylon membrane (Hybond N. Amersham) as described in the VacuGene XL instruction manual 39 (pp. 25-26) and subsequently prehybridised and hybridised using the denatured radiolabelled 5 kb BamHI-Xbal fragment of plasmid pNP8 with hybridisation conditions as described. The hybridisation pattern was obtained by exposure of Kodak XAR-5 X-ray film for 18 h. at -70'C using a regular intensifying screen. In all 5 clones, fragments originating from the same genomic region were found, for which a restriction pattern was constructed.

Based on the restriction map a 5 kb BamHI-XbaI fragment was selected for subcloning. 100 ng pBluescript BamHI-XbaI digested vector was mixed with 250 ng 5 kb BamHI-XbaI DNA of phage B-R and 4 pl 5 ligation buffer (composition; 500 mM Tris-HCl, pH 7.6; 100 mM MgCl 2 mM ATP; 10 mM dithiotreitol; 25% PEG-6000), and 1 pl (1.2 U/Pl) Th DNA ligase (Life Technologies) was added to this mixture in a final volume of pl. After incubation for 16 h at 14'C the mixture was diluted to 100 pl with sterile water. 10 ul of the diluted mixture was used to transform E. coli DH5a competent cells, prepared as described by Sambrook et al.

(1989). Six of the resulting colonies were grown overnight in LB medium (LB medium per 1000 ml: 10 g trypticase peptone (BBL). 5 g yeast extract (BBL), 10 g NaCl, 0.5 mM Tris-HCl pH 7.5) containing 100 pg/ml ampicillin. From the cultures plasmid DNA was isolated by the boiling lysis method as described by Maniatis et at. (1982). which was used in restriction analysis to select a clone harbouring the desired plasmid pIM23.0. Plasmid DNA was isolated on a large scale from 500 ml cultures E, i DH5a containing pIM230 grown in LB medium containing 100 pg/ml 25 ampicillin (Maniatis et al., 1982 The plasmid was purified by CsCl centrifugation, ethanol precipitated and dissolved in 400 pl TE. The yield was approximately 500 ug. E. li containing pIM230 was deposited at the CBS under the conditions of the Treaty of Budapest on..June 1996 under the accession number CBS 678.96 30 Example 6.3: Subcloning of the A. niger xlnR cDNA To obtain a cDNA clone of part of the zinc finger regio of the sZnR gene.

a reverse transcriptase and second strand synthesis reaction were carried out on 1 pg of polyA' RNA from an A.niger N402 wild-type strain grown on 35 xylan for 30 hrs with an oligonucleotide starting at position 1476-1496 (Seq id no in analogy to the method as described in the ZAP'"-cDNA synthesis kit (Stratagene). An aliquot (1/50) of the second strand reaction was used as template in PCR with primer R026 and R025 (derived from positions 9 46-970 of Seq id no 9) in 35 cycles of 60 seconds of subsequent 95C, 58'C and 72*C, followed by an incubation of 5 minutes at 72C. The resulting fragment of 500 bp was subcloned in the pGEM-T vector (Promega): and sequenced.

Example 7: The primary structure of the zlnR gene Example 7.1: Sequence analysis of the ztnR gene The sequence of the A. nizer ZnR gene, its promoter/regulation region, the structural part of the gene and the termination region, was determined by subcloning fragments from both pNP8 as pIM230, in combination with the use of specific oligonucleotides as primers in the sequencing reactions.

For nucleotide sequence analysis restriction fragments were isolated and were then cloned in pEMBL, pUC, pBluescript, pGEM DNA ,vectors, digested with the appropriate restriction enzymes. The nucleotide sequences were determined by the dideoxynucleotide chain-termination procedure (Sanger et al., 1977) using the Pharmacia ALF express automated sequencer and the Thermosequenase sequencing kit (Amersham). In the case of gene specific oligonucleotides the Pharmacia Cy5 internal labelling kit was used in combination with the T7 DNA polymerase sequencing kit (Pharmacia). The reactions and the electrophoresis was performed according to the manufacturers' instructions. Computer analysis was done 25 using the PC/GENE programme (Intelligenetics). The sequence determined is given in SEQ ID NO:9.

Example 7.2: 30 Description of the zlnR gene The sequence as given in SEQ ID N0:9. comprising the ztnR structural gene, is preceded by a 947 nucleotide long upstream region. In the upstream non-coding region CT-enriched sequences are found but no TATAA box. The structural part of the xZnR gene ranges from position 948 35 till position 3690. interrupted by two introns. The intron at position 1174 till 1237 was certified by sequencing the genomic fragment in pIM230. described in example 6.2 and part of the cDNA, as described in example 6.3. A second intron is indicated from position 3 496 till position 35 49. The second intron sequences follow the conserved intron sequences, normally found in fungi, for splice junctions and lariat sequence.

The xlnR gene encodes a protein of 875 amino acids in length. The derived polypeptide contains a typical N-terminal zinc binuclear cluster domain encoded by nucleotides from position 1110 till position 1260, with typical six cysteines coordinating the zinc. In this region furthermore a number of similarities with other fungal regulatory proteins are shown as listed in e.g. Fig. 1 of Kraulis et al. (1992) which is herein incorporated by reference.

A typical RRRLWW motif is found from position 2623 till position 2649. this motif is found, with slight variations, in a number of binuclear zinc cluster regulatory proteins as noted by Suirez et al.

(1995).

Example 7.3: Sequence analysis of xZnR in A.niger mutants To determine whether the mutation, in the case of the A. nier Smutants which do not express the xylanolytic system, as described in example 4.2, is located in zLnR, the sequence of the zxnR gene of these mutants was determined. For this a library enriched for 5.5 kb BamHI xznR containing fragments was made for each of the NW205::130 Ac mutants. For the construction of this xlnR enriched library. 5.5 kb fragments of BomHI, HinDIII, Xhol, SstI and KpnI digested genomic DNAs were isolated for each strain. These fragments were mixed with BamHI digested, 25 dephosphorylated pUC18 vector (Ready-To-Gon pUC18 BamHI/BAP Ligase, Pharmacia) and ligated. The ligation mixture was used to transform E.colt DH5a (MAX Efficiency DH5a o n Competent Cells, Gibco-BRL) for Amp resistance according to the manufacturers' protocol, which resulted in a primary library of 5*102-103 colonies. These A.nizer zmnR enriched libraries, 30 after replating the primary library on master plates, were screened by colony filter hybridisation according to standard protocol (Sambrook et al., 1989), with the use of the denatured radiolabelled BamHI-XbaI insert of pIM230 as a probe.

For each mutant strain, positive colonies were picked from the master plate with a toothpick and were grown for 15-18 hrs at 37'C in 5 ml LB medium containing 100 pg/ml Ampicillin, after which a miniprep plasmid DNA isolation was performed as described for boiling lysis by Sambrook et al. (1989). Analysis of these DNA preparations by agarose electrophoresis and comparing digestion patterns with zlnR specific patterns revealed colonies containing the correct 5.5 kb BamHI xlnR fragment.

The sequence of the A. niger mutants was determined with the use of specific zlnR oligonucleotides as primers in the sequencing reactions as described in example 7.2. For mutant NW205::130 Ac 1-15 a single basepair substitution was determined at position 3479, resulting in the change of the Leucine at position 823 of SEQ ID N0:9 into a Serine. For mutant NW205::130 Ac 2-5 a single basepair substitution was determined at position 3655, resulting in the change of the Tyrosine at position 864 of SEQ ID NO:9 into an Aspartic acid. These mutations identify both mutants as xlnR mutants.

Example 8: Expression of zZnR in A.niger Example 8.1: Complementation of A.niger mutants non-expressing the xylanolytic system For the complementation in all non-expressing mutants all ten NW205::130 Ac mutants, as described in Example 4, were transformed as described in example 2 of this document by combining protoplasts and pGW635 as a control for transformation frequency and pIM230 DNA for testing the complementation ability of pIM230.

The resulting transformant colonies were analysed for xylanolytic .activity and compared with their parental mutant strain, by plating them on appropriate medium containing 1 oat spelts xylan'as C source, and after 2-3 days a clearing of the xylan around the transformant colonies.

but not the parental mutant strain, appeared for all ten, thereby showing the restoration of xylanolytic activity.

Example 9: Effect of xlnR gene dosage on the expression of the A.niger xylanolytic system To study the potential use of the zLnR gene for strain improvement for an increased xylanolytic expression, the strain N902::200-18. harbouring multiple copies (about 6) of the A.nigr ZnD gene encoding B-xylosidase, was transformed to arginine prototrophy in a co-transformation experiment, as described in Example 2 of this document using 19 pg of the xZnR harbouring plasmid pIM230 and 2pg of the plasmid pIM650 harbouring the A.nidulans arzB gene (Johnstone et al., 1985). The transformants obtained were screened for increased endo-xylanase 43 expression, on MM plates containing 1% Oat spelts xylan. Four colonies, having the fastest and largest halo formation, were selected to determine zlnR copy numbers. For this DNA of these transformants and the recipient strain, was isolated and serial dilutions were spotted onto Hybond N membrane. The copy number was estimated from the signals found after hybridisation, using a radiolabelled 4.5 kb SmaI/Xbal fragment spanning the coding sequence of the slnR gene. Based on comparison to the recipient strain the ztnR copy number was determined to be 8 in N902::200-18-R14 and 32 in N902::200-18-R16. For both these transformants the effect of the increased gene dosage of zlnR was analysed by Northern analysis after strains were grown in liquid culture. This was done in a transfer experiment into 2% oat spelts xylan as a carbon source, after a preculture in 1 fructose for 18 h. Mycelial samples were taken 8 and 24 hrs after transfer, from which total RNA was isolated using TriZol (Life technologies) according to the manufacturers instructions and analysed by Northern blot analysis (Sambrook et al., 1989). Xylanase B expression levels were strongly increased in these transformants in comparison to the recipient strain, as detected after hybridisation using the radiolabelled 1 kb EcoRI/XhoI fragment of A.niger ztnB (Kinoshita et al., 1995).

To further study the potential use of the rlnR gene for strain improvement for an increased endo-xylanase expression. A.niger was transformed, as described in Example 2 of this document according to the following scheme; 1. pGW635 (pyrA) (positive control), 2. pDB-K(XA). 3 25 pDB-K(XA) pIM230) and 4 pGW635 pIM230. The plasmid pDB-K(XA) contains both the A.nizer pyrA gene and the A.niger xylanase A-gene from A.tubigensis in the vector pEMBL18. Transformants were obtained for all conditions used, strains overexpressing endo-xylanases were selected by halo size in a plate screening on Oat spelts xylan (20 transformants from each group were tested).

From each group one transformant was selected and grown for activity C assays. The strains were pregrown for 18 hrs on medium containing fructose, after which the mycelium (2.5 g wet weight in 50 ml) was transferred to medium containing 1.4% crude arabino xylan. All cultures 35 were performed in shake flasks. Incubations were done for 40 hrs at and the xylanase A levels were determined by HPLC analysis, while Bxylosidase and endo-xylanase activities were determined for both.

Chromatography was carried out on standard Pharmacia-LKB HPLC equipment (Uppsala, Sweden) running a SOURCE 15 Q. HR 5/5-column at 44 Buffer A 20 mM TRIS-buffer. pH 7.5 and buffer B 20 mM TRIS-buffer. pH with 1M NaCl. Gradient from 0-50% buffer B over 30 min. Detection at 280 nm. Pharmacia-LKB UV-MII. absorbance range at 0.1-0.2, see figure 3.

100 pl culture media was diluted with 1000 pl 20 mM TRIS-buffer, pH and 1000 pl diluted sample was applied to the column. The xylanase A activity is then seen as a peak eluting at approx. 30% B-buffer. From each group of transformants the one showing the highest xylanase A activity/peak in the HPLC analysis is shown in figure 3.

The endo-xylanase activity was determined by measuring the release of dyed fragments from azurine-dyed cross-linked wheat arabinoxylan (Xylazyme tablets) from Megazyme, Warriewood, Australia. The xylanase activity was calculated by comparing with an internal standard enzyme of 100 XU/gram (Xylanase-Unit) at 40'C in 0.1 M acetate buffer, pH 3.4. The same four transformants were assayed on water insoluble arabinoxylan at a pH were only xylanase A contributes to the endoxylanase activity, the results of this analysis is given in table 2 Table 2 Transformant XU/gram pyr+ (control) 4 DB-K(XA)-15 52 DB-K(XA)/pIM230-21 111 pIM230-26 24 From the results shown in figure 3 and table 2 it is clear that 25 transformation with the xylanase A encoding gene as expected gives a large increase in the xylanase A enzyme activity. More surprisingly, also after transformation using the activator gene xlnR. also a large increase in the level of xylanase A is found. This indicates a limitation in the level of activator XYL R xlnR gene product) in the untransformed 30 parent. Therefore, it is expected that in a pDB-K(XA) multicopy transformant the amount of transacting regulatory factor will be even more limiting. This is confirmed by the result for the pDB-K(XA)/pIM230 transformant, which has a xylanase A level twice as high as the pDB-K(XA) multicopy transformant.

Example 10: Screening filamentous fungi for the zlnR gene 415 To analyse whether it is possible to isolate the xlnR counterpart from other fungi by heterologous hybridisation, using the 4.5 kb SmaI/XbaI fragment of the xLnR gene as a probe. DNA was isolated from the follow'ing strains; A.nie N902 (argB15, cspAl. fwnAl metBlO pyrA5), L, tuibngenzsis NW184 (cspAl. fwnAl. pyrA22). A. nidulans WG096. (paAl.

yA2) of FGSC 187, Lai1.l1a NW240 (pyrA3) of CBS 101.43, A clau NW217 (fwnAl. cs'pAl. pyrA4. lysAl) of CBS 115.80, A.foetidu (waoro) NW183 (cspAl. fwnAl. pyrAl3, lysAl) of CBS 115.52., A. japonicus CBS114.51 and Trichoderma reesei QM9fl4. 1-2 pag DNA was digested with BamHI or with XhoI and subsequently analysed by Southern analysis as described in Example 3. The hybridisation conditions used were; hybridisation in 6 x SSC (2OxSSC per 1000 ml :175.3 g NaCi. 107.1 g sodium citrate.5.5H 2 0, pH 0.1% SDS. 0.05% sodium pyrophosphate, Denhardt's solution (100 x Denhardt's solution per 500 ml 10 g Ficoll- 400. 10 g po lyvinylpyrroli done. 10 g Bovine Serum Albumin (Pentax Fraction V) and 20 Vg/ml denatured herring sperm DNA at 56*C for 18-24 -".hrs followed by two 30 min. washes in 5 x SSC. 0.1 SDS at 68*C and two min. washes in 2 x SSC. 0.1% SSC at 56*C. After hybridisation the membrane was covered with Saran wrap and autoradiographed overnight at -70*C using Konica X-ray films and Kodak X-Omatic cassettes with regular intensifying screens.

As a result hybridising fragments were found for all. fungi analysed, very strong hybridisation signals were found in A. niger, A 1 nizenis, .j foetidus, while in the other strains investigated clear hybridisation signals were found.

Example 11: Application of the selection system using other promoter fragments Plasmids were constructed containing promoter fragments from the ALnizer abfA gene (Flipphi et at.. 1994) and the A~ie abfB gene (Flipphi et al., 1993). For the construction containing the abfA promoter fragment. a 1.4 kb XhoI/PstI fragment from pIM900 (Flipphi et at., 1994) was ligated in SalI/PstI digested pAlter (Promega), as described in Example 1. Using the Altered Sites 11 in vitro mutagenesis system (Promega) a Xhol restriction site was created at positions -83 till -88 relative to the translation initiation site (Flipphi et at. 1994). From the resulting plasmid a 953 bp SstI/XhoI fragment was isolated. This fragment and a 1.5 kb XhoI fragment from PIM130 were ligated into pBluescript (Stratagene) digested with SstI/Xhol. Plasmids containing the 46 correct orientation of the 1.5 Xhol fragment were identified by a digestion using BglII. The resulting plasmid is pAP8.

Analogously a 910 bp PstI fragment from the abfB promoter was isolated from plM991 (Flipphi et aZ.. 1993) and ligated in PstI digested pEMBL19. The resulting plasmid was digested using SatI and was ligated with the 1.5 kb XhoI fragment from plasmid pIM130. Plasmids containing both fragments in the correct orientation were identified by a digestion using BamHI. The resulting plasmid is pIM132.

A third plasmid containing a fragment from the A.nizer pgall was constructed by cloning a 1450 bp XbaI/XhoI fragment into the vector pAlter. Using the Altered Sites II in vitro mutagenesis system (Promega) a Xhol restriction site was created at positions -107 till -112 relative to the translation initiation site (Bussink et al., 1991). From the resulting plasmid a 1.2 kb XbaI/XhoI fragment was isolated. This fragment and a 1.5 kb XhoI fragment from pIM130 were ligated into pBluescript (Stratagene) digested with XbaI/XhoI. Plasmids containing the correct orientation of the 1.5 Xhol fragment were identified by a digestion using /HfnDIII. The resulting plasmid is pIIP7. From the same pgall gene a 223 bp HinDIII/PstI fragment (Bussink et at., 1992) was isolated and ligated in HinDIII/PstI digested pEMBL19. The resulting plasmid was digested using Sail and was ligated with the 1.5 kb Xhol fragment from plasmid pIM130. Plasmids containing both fragments in the correct orientation were identified by a digestion using HinDIII. The resulting S plasmid is pHPII.

S: 25 All four plasmids were introduced in A.niger NW219 by transformation as described in Example 2, using 10 mM L-arabitol as an inducer in transfomation experiments using the constructs having the abf promoter fragments (plasmids AP8 and pIM132) and 1% polygalacturonic acid (USB chemicals) in the case of the plasmids harbouring the pgall fragments. While for the plasmids AP8 and pIM132 transformants were found at high frequency, for the transformation experiment using the pgall promoter fragment containing plasmids pIIP7 and pHPII only 5 and 7 transformants were found respectively.

S"The transformants resulting from the transformation experiment using the plasmids pAP8 and pIM132 (abf promoter fragments were analysed phenotypically as described in Example 3 using MM containing 10 mM Larabitol/50 mM sorbitol, 10 mM L-arabitol/ 50 mM D-glucose and 50 mM Dglucose. The expected phenotype; growth on 10 mM L-arabitol/50 mM sorbitol. non-growth on 10 mM L-arabitol/ 50 mM D-glucose and 50 mM D- 47 glucose, was found for 10 out of 29 transformants resulting from plasmid pAP8 and 13 out of 30 transformants resulting from plasmid pIM132. The transformants resulting the plasmids pIIP7 and pHPII were tested on MM containing 1% polygalacturonic acid. 1% polygalacturonic acid/50 mM Dglucose and 50 mM D-glucose. Both classes of transformants however, did not show the expected phenotype, all transformants were able to grow on all three media. This suggests that these transformants result from a double cross-over at the pyrA locus.

Based on the results for the pgall promoter fragment containing plasmids, we have tested whether we could improve transformation frequency by improving induction. We assumed that transformation more or less failed due to lack of inducer, since no monomeric inducer for polygalacturonases was available and thus the polymer needs to be degraded to release inducer to give expression. We tested whether supplementation of the medium using a small amount of uridin could overcome this problem. For this the NW219 and a transformant NW219::pIM132#30 were tested on media containing 1% Oat spelts xylan, giving induction of abfB. in combination with an increasing amount of uridin; respectively 0. 0.001, 0.005, 0.01. 0.1. 1. 5 and 10 mM. In this experiment the recipient strain NW219 did not grow on media containing less then 0.01 mM uridin, while the NW219::pIM132#30 transformant strain grew under all conditions used. However, the degree of sporulation and colony morphology varied, the lowest uridin concentration giving wildtype-like sporulation being around 0.01 mM uridin.

Based on these results A.nizer NW219 transformation using the plasmids pIIP7 and pHPII was repeated using MMS as described in Example 2 containing 1% lemon pectin (degree of esterification Pectin factory) and two conditions for uridin supplementation 0.01 mM and 0.005 mM respectively. This resulted in an increased number of transformants found. These transformants were tested on media containing 1% lemon pectin as described above, 1% lemon pectin/ 50 mM D-glucose and 50 mM D-glucose, for which the expected phenotype is respectively growth, non-growth and non-growth. For the pIIP7 resulting transformants 10 out of 30 transformants and for the pHPII 9 out 30 transformants the expected 35 phenotype was found.

S"A selection of the transformants showing the expected phenotype were analysed by Southern analysis. DNA was isolated and analysed as described in Example 3.For the transformants resulting from the abfA promoter fragment containing plasmid pAP8 the DNA was digested using 48 CZaI, for pIM132 (abfB) resulting transformants using Clal and for the transformants resulting from pgall plasmids pIIP7 and pHPII using CaI.

Based on:-the autoradiograph obtained after hybridisation using the following radiolabelled fragments; the 3.8 kb Xbal and the 1.2 kb CalI fragment of the pyrA gene, transformants were selected based on estimated copy number. The following transformants were selected; AP8/16 (abfA), NW219::132#8 (2 copies) and NW219::132#30 (3-4 copies)(abfB), NW219::pIIP7#3 (2 copies) and NW219::pHPIIP#9 (2 copies) (pgall) The selected transformants were subjected to mutagenesis as described in Example 4. Arabinofuranosidase derepressed mutants were selected on MM 50 mM D-glucose and on MM 10 mM L-arabitol/50 mM Dglucose, while polygalacturonase derepressed mutants were selected on MM 50 mM D-glucose and on MM 1% lemon pectin/50 mM D-glucose. After 4-7 days 5-10 mutant colonies per plate were found, which on basis of their morphology could be divided into three classes: large, well sporulating colonies, intermediate sized, well sporulating colonies and small poorly sporulating colonies. A random selection of 20 of these mutants was made and the selected mutants were tested on media containing different carbon sources or substrates. The arabinofuranosidase mutant colonies were found to be able to grow on media containing D-glucose, D-glucose/L-arabitol and L-arabitol, while the parental strains only were able to grow on medium containing L-arabitol as a carbon source. Analogously the polygalacturonase mutants also were able to grow on MM 50 mM D-glucose and on MM 1% lemon pectin/50 mM D-glucose and MM 1% lemon pectin. The 25 parental strains could only grow on MM 1% lemon pectin. In addition the arabinofuranosidase mutants (20 of each) were tested on the chromogenic substrate methylumbelliferyl-a-L-arabinofuranoside as described in Example 4. While the parental strains did not show any arabinofuranosidase expression of D-glucose/L-arabitol containing media.

30 as detected by fluorescence of the substrate, the mutants showed variable levels of expression, the highest levels were found in mutants selected on the D-glucose medium.

The polygalacturonase mutants were tested on MM 1% lemon pectin/50 mM glucose, two and three days after inoculation of the plates polygalacturonase activity was visualized by staining as described by Ried and Collmer (1985). In this case for the parental strain a small halo was found, while in the mutants various degrees on increased halo formation was detected.

According to Example 4 also non-expressing mutants were 49 selected on media containing FOA. For this strains NW219::132#30(abfB) were mutated and plated on MM 10 mM L-arabitol 1 mg/ml FOA 10 mM uridin. After 7-10 days mutants were selected and plated to MM containing mM L-arabitol/50 mM sorbitol, 10 mM uridin and having a top agar containing 0.5% AZCL-arabinan (Megazyme. Sydney, Australia) for the detection of endo-arabinan expression. Two of the 30 transfomrants tested. 132/30 F12 and F26, were not able to release the dye from the substrate, an indication for the absence of endo-arabinan activity. Upon cultivation of these transformants in liquid MM containing 10 mM Larabitol and 10 mM uridin and subsequent measurement of arabinofuranosidase activity in the culture filtrates, an at least 4-fold decrease in activity was found.

The strain NW219::pIIP7#3 was mutated and plated on MM containing 1% lemon pectin/50 mM sorbitol. 10 mM uridin and 1 mg/ml FOA. After incubation of the plates for 6-10 days mutants were picked and screened for polygalacturonase activity as described above. Of 65 mutants selected three mutants were found to have a decreased haloformation after polygalacturonase activity staining, indicating a decrease in polygalacturonase expression.

Examples 7, 8 and 11 of EP 95201707.7, which is a copending European patent application of which a copy has been included upon filing the subject document and which examples have also been copied into this document.

Example 7 of EP 95201707.7: Transformation of A. niger using the plasmid pIM200 250 ml of culture medium, which consists of MM supplemented with 2 glucose, 0.5 Yeast Extract, 0.2 Casamino acids (Vitamin 30 free). 2 mM leucine. 10 PM nicotinamide, 10 mM uridine, was inoculated with 1 106 spores per ml of strain NW155 (cspAl, argB13, pyrA6, nicAl, leuAl, prtF28) (derived from NW228, Van den Hombergh et al, 1995) and Smycelium was grown for 16 18 hours at 30 'C and 250 rpm in a orbital New Brunswick shaker. The mycelium was harvested on Myracloth (nylon 35 gauze) using a Bchner funnel and mild suction and was washed several times with SP6 (SP6: 0.8 NaCI. 10 mM Na-phosphate buffer pH 150 mg Novozyme 234 was dissolved in 20 ml SMC (SMC: 1.33 M sorbitol, 50 mM CaC 2 20 mM MES buffer, pH 5.8) to which 1 g (wet weight) mycelium was added and which was carefully resuspended. This suspension was incubated gently shaking for 1 2 hours at 30 every 30 minutes the mycelium was carefully resuspended and a sample was taken to monitor protoplast formation using a haemocytometer to count the protoplasts. When sufficient protoplasts were present (more then 1 108) these were carefully resuspended and the mycelial debris was removed by filtration over a sterile glasswool plug. The protoplasts were collected by minutes centrifugation at 3000 rpm and 4 *C in a bench centrifuge, the supernatant was removed and the pellet was carefully resuspended in 5 ml STC (STC: 1.33 M Sorbitol, 50 mM CaCl2, 10 mM Tris/HCl. pH This wash step was repeated twice and the protoplasts were finally resuspended in STC at a density of 1 108 per ml.

The transformation was performed by adding 20 ,g of pIM200

DNA

and 5 pg pGW635, containing the A. nijer ovA gene (dissolved in a 10 pl TE), to 200 pl of protoplast suspension together with 50 l of PEG buffer (PEG Buffer: 25 PEG-6000, 50 mM CaCl 2 10 mM Tris/HCl pH 7.2), mixed-gently by pipetting up and down a few times, and incubated at room temperature for 20 minutes. After this period 2 ml PEG buffer was added.

the solution was mixed gently and incubated at room temperature for another 5 minutes and subsequently 4 ml of STC was added and mixed gently on a vortex mixer. One ml portions of this suspension were then added to 4 ml of 0.95 M sucrose osmotically stabilised top agar and poured on osmotically stabilised plates. As a control A. niger was also transformed using pGW635.

Example 8 of EP 95201707.7: Analysis of transformants The transformants from pIM200 obtained in Example 7 were analysed phenotypically by plating on MM containing 1% Oat spelt xylan and 1 mM 4-methylumbelliferyl-5-D-xyloside. Of the 26 transformants tested, five had an increased fluorescence. These transformants, together with a PYR" transformant as a reference, were grown on MM containing 1% Oat spelt xylan for 20, 27 and 42 hrs, after which the activity towards PNP-X was measured. The results are summarised in Table

C.

An increased level of i-xylosidase activity was found in all five transformants selected, the highest level being more then 30 times the wild-type activity. These results were confirmed by Western blot analysis. using the anti -xylosidase antibody, prepared as described in Example 3 of the EP 95201707.7. and the Bio-Rad Immun-blot GAR-AP assay kit following the suppliers instructions.

Table C B-xylosidase activities in A,..igf transformants activity (mU/ml culture filtrate) after: 20 hr 27 hr 42 hr pGW 635 15 16 17 XlsAl 82 86 51 XlsA4 90 112 78 XlsA8 211 239 384 X1sA9 63 110 74 XlsA12 96 295 527 Example 11 of EP 95201707.7: Disruption of the A niger nD gene Example 11.1: Construction of the disruption plasmids pIM203 and pIM204 The gene disruption plasmids pIM203 and pIM204 were constructed by generating an internal fragment of the xinD gene by PCR. The fragment was generated using the oligonucleotides derived from the xZnD sequence

(SEQ

ID NO: Xylos001 was derived from positions 1157 till 1176 and xylos00 4 was derived from positions 3147 till 3164. The fragment was generated by PCR containing 10 pi 10*reaction buffer (100 mM Tris-HCl, pH 8.3. 500 mM KC1, 15 mM MgCl2, 0.01% gelatine), 16 ul 1.25 mM of each of the four deoxynucleotide triphosphates. 1 ng of the plasmid pIM200

DNA

and 1 pg of each of the oligonucleotides in a final volume of 100 pl.

This reaction mixture was mixed and 1 ul TAQ polymerase (5 U1/l) (Life Technologies) was added. The DNA was denatured by incubation for 3 in at 92°C followed by 25 cycli of 1 min 92C, 1,5 min 52 0 C -and 1.5 min 720C.

30 After these 25 cycli the mixture was incubated for 5 min at 72*C.

Analysis of the reaction products by agarose electrophoresis revealed a fragment of about 2000 bp, which corresponds to the size expected, based Son the sequence of the gene. The resulting fragment was subcloned in the vector pGEM-T (Promega) resulting in the plasmid pIM202. Plasmid pIM203 was constructed by ligation of a Smal/Pstl fragment of pILJ16 (Johnstone et aL.. 1985). containing the A. nidulans argB gene (Upshall et at..

1986). in the EcoRV/Pstl digested pIM202 vector. Plasmid pIM204 was constructed by ligation of the Nsil/Xbal fragment of pIMl30 (this document. EP 95202346.3). containing the pyrA gene under the control of 40 the UAS of the zlnA promoter of A. tubiZensis, in the Spel/Nsil digested pIM202 vector.

Example 11.2: Disruption of the zlnD gene in A. niger The plasmids containing the 2lnD internal fragment as well as the argB gene (pIM203) or the pyrA gene (plM204), as described in Example 11.1 of the copending E? application, as a selection marker in transformation, were used to disrupt the A. nizer xinD gene. For this A niger N902 (argB15, cspAl, fwnAl metBlO pyrA5) was trans&ozmed, as described in Example 2 of this document, using the plasmids pIM203 and pIM204 selecting for arginine or uridine prototrophy respectively. The resulting transformants were screened for activity on methylumbelliferyl- B-D-xyloside on a 1 xylan plate as described in Example 8 of the copending EP application. For both groups of transformants twenty were screened. Of these transformants one of each group had a severe decreased level of MUX activity after 24 h of growth. Southern analysis of the selected transformants, as described in Example 3 of the copending application, demonstrated for the pIM203 transformant a multicopy integration at the homologous xlnD locus. In case of the plM204 transformant a single homologous integration at the xznD locus had /occurred. Analysis for PNP-X activity, as described in Example 8 of the copending EP application, of these transformants revealed an at least 100-fold decrease in O-xylosidase activity.

Example 11.3: Effect of overexpression and inactivation of zlnD gene on the expression of xylanolytic system of A. niger.

To determine the effect of zZnD expression on the expression of the xylanolytic spectrum, A. nizer N902, two ztnD multicopy-transformants 25 in N902 and the xrD gene disruption strains were grown in liquid ~culture. This was done in a transfer experiment into 2% oat spelts xylan or 3 D-xylose as a carbon source, after a preculture in 1 fructose for 18 h. Beta-xylosidase activity was determined as PNP-X activity in the culture filtrate. With both C sources a clear overexpression could be seen for the pIM200 transformants against an almost absence of PNP-X .activity for both (pIM203 and pIM20 4 inactivation transformants. The xLnD gene disruption transformants showed an initial decreased level of endo-xylanase expression, which however increased in time finally after 16 hrs resulting in increased activity levels in comparison to the A.

35 niger wild-type. Thus resulting in xylanase preparations free of Bxylosidase.

The culture filtrates were subsequently analysed by HPLC analysis.

using a Dionex system and Pulsed Amperometric Detection. For this 1 ml of culture filtrate was boiled immediately after harvesting, to inactivate the xy18flolytic enzymes. after which the sample was centrifuged fdi' min. (14I.000 rpm .at 4*C. Eppendorf' centrifuge) The resulting supernatant was diluted 5-fold in bidest and 20 iii was analysed by HPLC using a Dionex CarboPac 100 column. The analysis indicated that, while in the wild-type and in the over-expression transfornants only in the initial stage xylose oligomers could be detected in the culture filtrate. in the disruption mutant xylobiose and to a lesser extent xylotriose accumulated in the culture filtrate, thus resulting in a source for xylooligomers, in particular xylobiose and xylotriose.

The entire disclosure in the complete specification of our Australian Patent Application No. 62443/96 is by this cross-reference incorporated into the present specification.

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06 58 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Agricultural University Wageningen STREET: Costerweg CITY: Wageningen COUNTRY: The Netherlands POSTAL CODE (ZIP): 6701 BH (ii) TITLE OF INVENTION: A novel method to isolate mutants and to clone the complementing gene (iii) NUMBER OF SEQUENCES: 9 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (EPO) INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) o* (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CACAATGCAT CCCCTTTATC CGCCTGCCGT INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 37 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 04 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CAATTGCGAC TTGGAGGACA TGATGGGCAG ATGAGGG 37 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: AGAGAGGATA TCGATGTGGG INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 37 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: CCCTCATCTG CCCATCATGT CCTCCAAGTC GCAATTG 37 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 20 5 4 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Aspergillus tubigensis (ix) FEATURE: NAME/KEY: TATAsignal LOCATION: 848..854 (ix) FEATURE: NAME/KEY: exon LOCATION: 950..1179 (ix) FEATURE: NAME/KEY: intron LOCATION: 1179..1228 (ix) FEATURE: S: NAME/KEY: exon LOCATION: 1229..1631 (ix) FEATURE: NAME/KEY:

CDS

LOCATION: join(950..1179, 1229..1631) OTHER INFORMATION: /EC_number= 3.2.1.8.

/product= "1,j4-b eta-xylanxylanohydrolase" /gene= "xlnA" /standard-name= 'endo-xylanase" (ix) FE-ATURE: NANE/KEY: mat-peptide LOCATION: 1031. .1631 (ix) FEATURE: NANE/KEY: sig_peptide LOCATION: 950. .1031 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AACGTCTGCA GTCCCGTACT GTIACOAAA GTAATACG=r

CTACTGGCGTA

GTT1'ATGCT

AMCCAGCT

GGTTGACCT

GAGCTCCAAA

AGGCTTGCTA

GWACTG;TGT

AAAACTCT

CCI=ATCC

717TAGCCAAG

CAACTATAGA

GCAAAATGCA

CGCAGAATAT

MTCCAGTIG

GCCGGAGTCA

GTAGGCTCCT

GGCTAGGCAG

ATAAGCGAGA

GCCTFCATCA

GTACC TTCGC

CMATAAATAA

TCAGTAGTAG

GCAGGGAACC

GCCTGCCGTC

TCCAGTG=2

ACTOTCCCTA

TATGACTGAG

AAATAGAGGT

CATACATCCA

GCCCCTACTC

AGAGTOGGGT

GACCTGGTA

=TGCCATAC

CCTACACAAA

GACCTTTGGC

AGAGACATAA

ATCAGTGGGT

GGTGAAGAAA

CATTTAGCCA

AGGTTGGTGG

GAAATAGGCT

TTG=ICAAC

AGAGCGGGCT

TTCACAGCAT

ATGCCAGGCC

CCTGATGGGT

AAAGTIGCC

AGITGATGGC

TCTTCAGCGA

GAACTCCTCG

CAGTGITCT

CCTI'GCAGTA

ACATAATCAT

CCCCAC7TCC

AATGTAGTCC

CTACACAGGA

CGAGGTrGIT

GTGCAGGGGA

CGCAGCAATA.

TCAGC=1CT U a AOTGGTGGAT ATACAACT= TCCCACTCCC TAGTTACTTC AAGGGTAG CCCCAGTCTI' TCCTGCATTC CTACCTGAGT GTCCGGATGG TCCGCGCCGA GCCAACTCCC GGTGGCCTrC CGCAGCG'I ACTGAGCCTA CATACGTCT GTATGAGCGA GAACATGACT TCTOAGCCAG CCGCCTCCAC TAACTGCAGC X1TTAGCCAA GTGCGG;TCCA AACGGCCATG--AATarAGACA AGAGC=1A AGGTGATGCG AAGGGATAAA TAGTC~1IT ITGACCAGGA CAGGCCT TCAATCATC ATG AAG Met Lys -27 GCA TC GCC GOT COT Ala Phe Ala Ala Pro GOT ATC AAO TAO GOG Gly le Asn Tyr Val 120 180 240 300 360 420 480 5140 600 660 720 780 840 900 955 1003 1051 a a a.

p. GTO ACT GCG GOT TTT GCA GOT 0TT ITG GTO ACG Val Thr Ala Ala Phe Ala Gly Leu Leu Val Thr -25 -20 -15 GCC CCA GAA COT CAT CTG OTO TCG CGA AGT CC Ala Pro Glu Pro Asp Leu Val Ser Arg Ser Ala 1 CMA MC TAC MAC GGC MAC CTI' GOT CAT TTC ACC TAC GAC GAG ACT CC Gin Asn Tyr Asn Gly Asn Leu Gly Asp Phe Thr Tyr Asp Giu Ser Ala 15 GGA ACA TTi' TCC ATG TAC TGG GMA CAT GOA G70 AGC TCC GAC 'rrr G'rC Gly Thr Phe Ser Met Tyr Trp Ciu Asp Gly Val Ser Ser Asp Phe Val) 30 OTT OT CTG GGC TOG ACC ACT OT TCT TCT AA GTGAGTGAOT Val Gly Leu Gly Trp Thr Thr Gly Set' Ser Asn 4045 OTA'ITCTr1'A ACCMAGGTCT AGGATCTAAC 07C'I-rrCAG C OCT ATO ACC TAC TCT 1099 .1147 1189 1244 1292 CTC GCT 070 TAC .GGC Leu Ala Val Tyr Gly 0CC GMA TAC ACC OCT TCT 0CC TCC OCT TCC TAC Ala Glu Tyr Ser Ala Ser Gly Ser Ala Ser Tyr

TOG

Trp

GAT

Asp

GAT

Asp

TC

Ser 120

ACC

Ser

TOO

Trp

OTC

Val

TAT

Tyr

GGA

Gly 105

ATC

Ile

ACO

ml'

GCG

Ala

MAC

Asn

MAC

Asn

AGC

Ser

ACG

ml'

COC

Arg

CAC

His

TAT

Tyr

CT

Pro

ACC

Thr.

OA

Gly

ACA

Thr

CAT

~CT CM OCT GAO TAC TAC ATO 07C GAO OAT TAC GOT ~iu Asp Tyr Gly Pro

TGC

Cys

TAC

Tyr

ACA

ml'

TCT

Ser 140 000 Gin

ACT

Set'

CMA

Gin

AGC

Ser

GGA

Giy Ala 7CC Ser

OTC

Val.

110

ACG

mhr

ACG

mhr

GOC

Glu 0CC Ala 95

TOC

Cys Trc Phe 070 Val

MAT

Tyr Tyr Ile 80 ACA AGC CTT mhr Set' Leu ACC GAC ACT Thr Asp Thr ACO CAG TAC mhr Gin Tyr 130 ACT =T CC Thr Val. Ala 145 AGC GAC TTC Set' Asp Phe 160 GGC AGC OCT

GOT

Gly

CGA

Arg 115 Trc Phe

AAC

Asn

MAT

Asn

ACT

ACC

Thr 100

ACA

mhr

TCC

Ser

CAT

His

TAT

Tyr 07C Val

MAC

Asn

GUT

Va).

ITC

CAC

Gin 165

ACA

Thr

TAC

Tyr

GAA

Giu

CGA

Arg

MAC

Asn 150 G TC Val.

ATC

Ile

TOT

Ser cCc Pro

GAG

Giu 13 Phe Val

TOT

Ser 13140 1388 1436 His Gly Phe Gly Asn GC 070 Ala Val GMA OCA TCG AGC Giu Ala Trp Ser GOT OCT Cly Ala Gly Ser Ala Set' Va).

170 175 180 1532 1580 1628 1681 1741 1801 1861 p. TCT TGAGAGATTA GTGCCCTAGT AGTCGGAAGA TATCAACGCG

GCAGTTTGCT

Ser CTCAGGT00T 07TGATCATCC GATCCCGTCT CTGGCO1TAC ATTOAGOCTO

TATAAGT

TGT00GGCCG AGOTGTCAGC GCCTGCCTIT TCAOC'ITGCA CAGATMATCA

ACTCTC=I

TCTATCTCTT GCGT=CCTC OCTGCTTATC CTATCCATAG ATMAITAT TCCCCAOTAC 62 CACAACTGT TCGGTCGCAG TAGTCACTCC GAGCAAGGCA TTGGGAAATG GGGGATGCGG 1921 GGTGCTGCGT ACCCTCTAAC CTAGGGCATT ITAAAGGATA 7rrACCCTCC AGATATTCTA 1981 TAGATACAGA CTTC=AGGA CTGCGGGTAA TATAGAGAGC GAP.A'TFCTA CATTCGATG 20141 CAGTI'CAATG CGA 20514 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 3026 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Aspergillus niger STRAIN: Am40 (CBS 120.49) (ix) FEATURE: NAME/KEY: TATAsignal LOCATION: 6'43. .6148 (ix) FEATURE: NAME/KEY: CDS LOCATION: 7214. .2538 OTHER INFORMATION: /EC number= 1.1.3.14.

:/product= "glucose oxidase" gene= "goxC"

FEATURE:

NAME/KEY: mat_peptide LOCATION: 790. .2538 (ix) FEATURE: NAME/KEY: sigpeptide LOCATION: 7214. .790 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CTGCAGGAC CTGAAGCCTG CCTAGTTTGA TCACCCTGAA ACCAGCACTG CCTGTC'ITGA *CCTTGGTGGT GAGTTTGCAC GTGGGCTGG;C TG'TTCAAATA AACTCTCCAA TTGACCCTCT 120 CCCCG;TGGAG AACACAGCAA ACACTATAGO C717CCATTG AGGGCATGAC GAGGACCCTA 180 TGGTTTTGC ACTTGGCGAG GGCTGACCGG AGCACGAATC GGGAAGGGCA GAACTCAGAA 240 'rCGGTGOTC GGA1TCGTAT

TATGGGOCCA

GATCAOGACG

ATTATGGCT

TCAGCTCACG

TGGATTATCG

GTCCTCCTGT

ATC ATO CA Met G2.

-22 GCC CTG CC Ala Leu Pr ACT OAT CC Thr Asp Pi

TCGGCATGCC

AGTTCCTCOT

ATAAGCCACT

CCAATCCTGC

CGGATTA'ITG

GCCCCTATCA

AACAAGTTG

CACC17CTGA 63 GAAAOTCOOT

ATCCCTIGGC

CCACGAGGCT

GCCTACCGTC

ACGAGGATGA

CATGGCCTCT

GCTCCACCTGA" TCTAAGGAfl TATTCGGGAT

ACCGACGGCT

GTATOCATTA

TOAGGATOOC

'rTCTOGACCA T1'GACTCG.AG TCAGCAACCA

GCCITCCTC

GCCACGATGA TTTGCGTCCA- AGCG TGAGGC AO7TGAGCTAA ACAGAACGAG AGACOCAGAG CGCT 7TTGGA CTATCCAGGG.

GAGCACACGG AGGATEIAGT TTCT TGGAAA GCAGAGGAAT CGTATAAGTA ACCTCGITCG TCTCAI7CCC

TCATCTGCCC

GTC TCC CTC OCT GCO Val. Ser Leu Ala Ala GAA 0CC AGC CTC CTO Giu Ala Ser Leu Leu G ACT CTC CT1' OTO AGC TCG C77 OTO n Thr Leu Leu Val Ser Ser Leu Val

A

0 *0 CAC TAC ATC AGO AOC AAT GOC ATT His Tyr Ile Arg Ser Asn Gly Ile 1 TCC GOC COC ACG Ser Oly Arg Thr

GT

Gly AAG OAT OTC Lys Asp Val 15 CTG ACT GGA Leu Thr Gly OTC GAO TAC Val Asp Tyr ATC ATC Ile Ile 300 360 420 48o 540 600 660 720 768 816 864 912 960 1008 1056 104 1152 1200 OGA GOT Gly Gly CTC ACC ACC Leu Thr Thr

OCT

Ala

GC

Gly CCC AAC ATC Pro Asn Ile AGA GOT CCT Arg Gly Pro 60 AGO ACT OTA Ser Ser Va].

CTC OTC ATC Leu Val Ile

OAA

Giu

MAC

Asri CGT CTO ACO GAG AAC Arg Leu Thr Giu Asn TCC TAG GAO TCG GAG Ser Tyr Giu Ser Asp.

GOC GAG ATC TTl' 000 Glv Asp I1:'Phe Gly AiT GAO GAC le Giu Asp

CTO

Leu 65

GAO

ciu 0CC TAC Ala Tyr

OCT

Al a OAC CAC 0CC Asp His Ala

TAC

Tyr ACC GTO GAG Thr Val Glu ACC GC MAT Thr Asn Asn

CMA

Gin Val

TO

Trp ACC OCO 070 ATO Thr Ala Leu le MAT GOT 000 ACC Asn Gly Oly Thr 110 GAG ACT GTC TTT Oiu Thr Val Phe COC TCC Arg Ser TOG ACT Trp Thr OGA MAT OCT CTC Oly Asn Oly Leu 100 COO CCC CAC MAG Arg Pro His Lys GGC TCT ACT Gly Ser Thr

CTA

Leu 105 OCA CAG CTT.

Ala Gin Vai GAG 707 Asp Ser 120 070 GCC Val Ala OGA AAT GAG Oly Asn Oiu ~cC Gly 130 MAC TO GAC Asn Trp Asp GCC TAC TCC CTC CAG GCT GAG COT OCT Ala Tyr Ser Leu Gln Ala Glu

ATC

lie

ACT

Thr 170

OTC

Val

AAA

Lys

ACC

Thr .orr Leu

OTT

Val 250 0CC Gly

AAG

Lys

CTC

Leu

ATC

lie

ACC

Thr 330

OA

Gly

OCT

Ala 155

GTC

Val

AAG

Lys

GAO

Asp 77G Leu

CCC

Pro 235

GT

Gly

GTG

Val

CAC

His

GAA

Glu

GAC

Asp 315

ACC

Thr

CAG

Gin 140

OCT

Ala

CAT

His

OCT

Ala

TTC

Phe

CAC

His 220

AAC

Asn

AAG

Lys

GAA

Glu

GAG

Glu

TAT

Tyr 300

ACC

Thr

OCT

Ala

GCC

Ala 000 Gly

GCC

Ala

CTC

Leu

GGA

Gly 205

GAA

Glu

TAC

Tyr

GTG

Va rrc Phe

OTC

Val 285

TCC

Ser arc Val

ACC

Thr

OCT

Ala

CAC

His

GGA

Gly

ATG

Met 190

TGC

Cys

GAO

Asp

CAA

Gin

CTC

Leu

GGC

Gly 270

CTC

Leu

GOT

Gly rr Va aTC Val

TOO

Trp 350

TAC

Tyr

CCC

Pro 175

AGC

Ser GOa Gly

CAA

Gln car Arg 0.T Leu 255

ACC

Thr

CTG

Leu

ATC

Ile

GAC

Asp

CCC

Arg 335

'TT

Phe

TTC

Phe 160 CoC Arg

OCT

Ala

GAC

Asp 0TC Val

CCC

Pro 240

ACC

Ser

CAC

His 0CC Ala

OGA

Gly

CTO

Leu 320

TCC

Ser 0CC Ala Arg 145

AAC

Asn

GAC

Asp aTC Val

CCC

Pro

CCC

Arg 225

AAC

Asn

CAC

Gin

AAG

Lys

GCG

Ala

ATG

Met 305

CCC

Pro

CGC

Arg

ACC

Thr Ala

GCA

Ala

ACC

Thr

GAA

Olu

CAT

His 210 7CC Ser

CTG

Leu

AAC

Asn 0CC Gly 0CC Gly 290

AAG

Lys

CTC

Val

ATC

Ile 77C Phe CoC Arg 7CC Ser

GGC

Gly

GAG

Asp 195

GGT

dly

OAT

Asp

CAA

Gin

GGC

Gly

AAC

Asn 275 7CC Ser 7CC Ser 0CC Gly

ACC

Thr

AAC

Asn 355

OCA

Ala

TGC

Cys

CAT

Asp 180

CGG

Arg 070 Val

GCC

Ala 0TC Val

ACC

Thr 260

ACC

Thr

OCT

Ala

ATC

lie ToG Leu

TCT

Ser 340

GAG

Glu

CCA

Pro

CAT

His 165

GAO

Asp

GGC

Gly

TCC

Ser

OCT

Ala CT0 Leu 245

ACC

Thr

CAC

His aTC Val

CT

Leu

AAC

Asn 325

OCT

Ala

ACC

Thr

AAT

Asn 150 car Oly

TAT

Tyr

GTT

Val

ATG

Met CoC Arg 230

ACC

Thr

OCT

Pro

AAC

Asn

TCT

Ser

GAG

Glu 310

CTG

Leu

GOT

Oly Phe Phe CCC AAA CAG Ala Lys Gin OTT AAT 007 Val Asn 0ly TCT CCC ATC Ser Pro Ile 185 CCC ACC AAG Pro Thr Lys 200 TTC CCC AAC Phe Pro Asn 215 GAA TOG OTA Glu Trp Leu OGA CAG TAT Gly Gin Tyr 0T GCC OTT Arg Ala Val 265 OTT TAC GCT' Val Tyr Ala 280 CCC ACA ATC Pro Thr lie 295 CCC OTT 007 Pro Leu Gly CAG GAO CAG Gin Asp Gin OCA OA CAG Ala Gly Gin 345 G0T GAC TAT Gly Asp Tyr 360 1248 1296 1344 1392 1440 1488 1536 1584 1632 1680 1728 1776 1824 1872 efl.

0* I 0* TCC GAA AAG GCA CAC GAG CTG GTC AAC ACC AAG CTG GAG CAG TOG 0CC Ser Giu Lys GAA4 lu

ATC

lie

TAC

Tyr 410

TOG

Trp

GAC

Asp

GAG

Olu

ATC

lie

CCC

Pro 490

TAC

Tyr

TCC

Ser CGrG Val

ACG

Thr

AAA

GAG A Glu

CAG

Gin 395

TCG

Ser

GAG

Asp Ccc Pro

CTG

Leu

TCC

Ser 475

GOT

Gly

ATC

Ile

ATG

Met

TAT

Tyr

CAA

Gin 555

ATT

3CC Ala 380

TAC

Tyr

GAA

Olu

CTT

Leu

TAG

Tyr

GAG

Asp 460

AAC

Asn

GAT

Asp

CCC

Pro

ATC

Mel

G

G13 54C

AT(

Mel

TC

Ala 1 365

GTC

Val

GAG

Glu

GTC

Leu

CTG

Leu Cr- Leu 445

CTG

Leu

TCC

Ser

AAC

Asn

TAC

Tyr

CCG

Pro 525

GTG

Vai

TCG

Ser 3 GAT

GCC

Ala

AAC

Asn 77C Phe

CCG

Pro 430

CAC

His

CTC

Leu

GOT

Gly

CTC

Leu

CAC

His 510

AAG

Lys

GAG

Gin

TCC

Ser GC1

CGT

Arg

TAC

Tyr

GTC

Leu 415

TTC

Phe

CAC

His G T Oly 0CC Ala

GCO

Ala 495 ric Phe

GAG

Glu

OA

Gly

CAT

His

ATC

Ile 575 GGG GGA Gly Gly I 385 CGC GAC Arg Asp 400 GAG ACT Asp Thr ACC CGA Thr Arg TiC OCC Phe Ala GAG GCT Gin Ala 465 ATG CAG Met Gin 480 TAT GAT Tyr Asp COT CCT Arg Pro ATG GGC Met Gly CTG CGT Leu Arg 545 GTC ATG Val Met 560 TTG GAA Leu Glu ETC CAC ?he His rO AT rrp lie GCC OGA Ala Gly OGA TAC Gly Tyr 435 TAC GAC Tyr Asp 450 GCC OCT Ala Ala ACC TAG Thr Tyr 0CC CAT Ala Asp AAC TAG Asn Tyr 515 GOT or- Gly Val 530 GTC ATT Val Ile ACG OTG Thr Val GAT TAT Asp Tyr Us Giu Leu Leu Asr Thr Lys Leu Giu Gin Trp Ala

AC

ksn

OTC

Jal'

GTA

Val 420

OTT

Vai

CCT

Pro

ACT

Thr rrc Phe

ITG

Leu 500

CAT

His Vai

GAT

Asp Ti-c Phe

GCT

Ala 580

ACC

Thr

AAC

Asn I 405

CCC

Ala

CAC

His

CAG

Gln

CAA

Gin

OCT

Ala 485

AGC

Ser

GGC

Gly

GAT

Asp

GOT

Gly

TAT

Tyr 565

TCC

kCC [hr 390

CAC

His

AGC

Ser

ATC

lie

TAG

Tyr

CTO

Leu 470 000 Gly 0CC Ala

OTG

Val

AAT

Asn

TCT

Ser 550 0CC Ale

ATC

375 0CC TTG CTC Ala Leu Leu AAC GTC GCC Asn Val Ale TTC OAT OTC Phe Asp Val 42! CTC GAC AAC Leu Asp LyE 440 rTC CTC AAC Phe Leu Ast 455 0CC CGC AA Ala Arg Asr GAG ACT AT( Olu Thr Iii TOG ACT GA( Trp Thr G1 50 GOr ACT TG Gly Thr Cy 520 OCT 0CC CG Ala Ala Ar 535 AT CCT CC lie Pro Pr ATG GCG CT Met Ala Le

GAG

C

I

-I

u

C

s

T

0

A

u 1920 -1968 2016 2064 2112 2160 2208 2256 2304 2352 2400 2448 2496 2538

S

Lys le Ser Asp Ale 570 Ser Met Gin TGACTGTAT GATGGOATA TOACTGAGGA TA7TAGGGGA TGGTACTTAG ATOCTGOGGA 2598 66 GGTATAATCA TAGATTGGAT AGAA'ITGGTA GGTTACATAG ACAGGITACA TGAATAGACG 2658 'rrCGTrATAT GTGAGCAGAC ATTACTACCA AACAAGGGCA TTTCAGTr AGTCGAACGA 2718 TAGTCATATG =TIACGG GAAGAAAGVI' TCACTAKITA TTAAGCAAAC GGATCAGGGG 2778 TTGCCAGCTA AAATACAATC ATCCGATGTT CTA1TrCT T CAAA'ITGATC GACCAGTCAG 2838 TTAATGAATG CATGAGAGCA ACTCTGCGCA TCCTCTAGCT ATCTAGTCAA TAATAAGCAT 2898 GGTTAAG ATGAAACACC GCCATAGACA TA2TCTGTI'G CTGGTGAAGC AAGCCCTCGC 2958 TAAATATGCT GATAACTTCC TATGCCAGTA GAATA=I1C CCACTCTGCT GCGCGCTCTC 3018 AAAAGC TT 3026 INFORMATION FOR SEQ ID NO.: 7: SEQUENCE CHARACTERISTICS: LENGTH: 1517 base pairs TYlPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: ORGANISM: Aspergillus niger STRAIN: L112 (ix) FEATURE: NAME/KEY: exon LOCATION: 339. .495

FEATURE:

NAME/KEY: intron LOCATION: 496. .563 (ix) FEATURE: NAME/KEY: exon LOCATION: 5 64i..1237 (ix) FEATURE: NAME/KEY: CDS LOCATION: join(339. .495, 564..1237) OTHER INFORMATION: /product= decarboxylase" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GCAGGGAAAA ATACGAGCTC CAATGAACCT GGGTG;TGGCA ACTTCAATGG AAAGGAACTG CCTTTGCAGG TGTGGCTGAA CCCCACGGTT CCGGTrCGGAG GCGGCGAAAT CACCCGATGT 120 GGCTGGTGCG TGGAGGGTCG CGATGATTTA CTGAGCTCCT C7ITGCTCG ACATTGAATG 180 67 TGCATTGT TC ACCTCATATA AGCGCCACTC CCTCCTAAAT TATTCGGTAC TA'II'CGCA- 2'4o TCTCTGCATC TACCAAI7AG CGCCTATCAG TCGAAACTCC AAGCTACTCA TKITrCACAA 300 GCCTCT1TCA TCCCCCCATT AACCCCTCCA CCGACACC ATC TCC TCC AAG TCG 353 Met Ser Ser Lys Set 1 CAA TO ACC TAC ACT CCC COT GCC AGC MAG CAC CCC AAT GCT CTO CCC ±101 Gln Leu Thr Tyr Thi- Ala Arg Ala Ser Lys His Pro Asn Ala Leu Ala 15 AAG CCC CTG TTC GAA ATI' OCT GAG GCC AAG AAO ACC AAT CTG ACC OTC 449 Lys Arg Leu Phe Glu Ile Ala Giu Ala Lys Lys Thr Asn Val Thr Val.

30 TCT CCC CAC OTT ACC ACC ACT AAG GAG CTA CTA CAT CTT OCT GAC- C 495 Ser Ala Asp Val Thr Thr Thr Lys Glu Leu Leu Asp Leu Ala Asp ~45 GTAGGCCGAC CCGCCATTCT CCCTC'VITAT CCTCCATACA AACTTATTAA CGGTGATACC 555 GGACTSGAG CT CTC COT CCC TAC ATC CCC GTG ATC AMA ACC CAC ATC CAT 604 Arg Leu Gly Pro Tyr le Ala Val Ile Lys Thr His le Asp 60 ATC CTC TCT CAC TTC AGC GAC GAG ACC ATT GAG CCC CTC MO GOCT cTr 652 Ile Leu Ser Asp Phe Ser Asp Glu Thr Ile Oiu Oly Leu Lys Ala Leu 75 CC CAG MCG CAC MAC 17C CTC ATC TTC GAG GAC CCC MAA TTC ATC CAC 700 Ala Gin Lys His Asn Phe Leu Ile Phe Giu Asp Arg Lys Phe Ile Asp 90 :ATI' CCC AAC ACT OTC CAG MCG CAA TAC CAC COT GOT ACC CTC CCC ATC 7148 Ile Giy Asn Thr Val Gin Lys Gin Tyr His Arg Cly Thi- Leu Arg Ile 100 105 110 :::TCA CMA TCC CCC CAT ATC ATC MAC TOC ACC ATC CTO CCT CCC GAG COT 796 Ser Ciu Ti-p Ala His Ile Ile Asn Cys Ser Ile Leu Pro Cly Glu Cly 115 120 125 130 ATC OTC GAG OCT CTC GCT CAC ACO GC TCT GCA CCC CAC TiC TCC TAC 8&44 Ile Val Ciu Ala Leu Ala Gin Thr Ala Ser Ala Pro Asp Phe Ser Tyr 135 140 145 :*:CGC CCC GAA COT COT CTC TTC ATC TiC C GMA ATG ACC TCT MAG COT 892 GlyProGluArgGlyLeuLeuIleLeuAla Clu Met Thr Ser Lys Gly 150 155 160 7CC TTC CCC ACC GOC CAC TAC ACT ACT TCT TCCOi C AT TA T CCC CCG 94o0 Ser Leu Ala Thi- Cly Gin Tyr Thr Thi- Ser Ser Val Asp Tyr Ala Arg 165 170 175 AAA TAC MOG MC TTC GTC AT GOA TIT OTO TCC ACC CCC 7CC TTC GOT 988 Lys Tyr Lys Asn Phe Val Met Oly Phe Val Ser Thi- Arg Ser Leu Cly 180 185 190 GAG GTG CAG TOG GMA 0TC AGO TCT COT TOG CAT Glu Val Gin Ser Glu Val Ser Ser Pro 200

OTG

Val 205 7CC AAG Ser Lys GAG GAG GAO TTT G070 Giu Glu Asp Phe Val 210 GGA OAT MAG CTC GOT Gly Asp Lys Leu Gly, 225 TTC ACC ACT Phe Thr Thr

GGT

Gly 215

ACT

Thr A.AC ATT TO Asn Ile Ser 220

ATC

Ile CAG CAG TAO Gin Gin Tyr ATT ATO GOG le Ile Ala 245 GOG CAA CAG Ala Gin Gin 260

CAG

Gin 230

GOT

Gly COO GCA TOG Pro Ala Ser

OT

Al a 235 000 Aila Gly Arg Gly COO OT ATO Arg Giy Ile

TAO

Tyr 250

GOT

Gly 000 COG GAO CO Ala Pro Asp Pro 255 GAG 000 TAO 070 Glu Ala Tyr Leu 270 Aia Asp Phe 240 GTG CAG GOT Val Gin Ala 000 COT 070C Ala Arg Val 1036 1084 1132 1180 1228 TAO CAG AAG Tyr Gin Lys

GAA

Glu 265

TOG

Trp r, Arfr-rrATrft 1277 p a.

p. 000 GGA MOC TAATACTATA AANIAiAA jv G111U Gly Gly Asn 275 ATGATATAGA AATGCAACIT GOOGCTAOGA. TACGOATAOA

AACTAATGTO

AOTCAGACTG OGOCATOGGA TGTCAAAAOcG OTTGATCC

TGCAGGOTAT

CACGGGATA ATOGOGTACG ACGAT1' GAT GCAGATAAGC

AGOCTGCGAA

CTGTAACTOT TGCOTAGAGC AAATGGCGAC GGOTGGCTGA

TAAGGGACGG

INFORMATION FOR SEQ ID, NO: 8: SEQUENCE

CHARACTERISTICS:

LENOT H: 4108 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iii) ANTI-SENSE:

NO

(vi) ORIGINAL

SOURCE:

ORGANISM: Aspergillus niger (CBS 120.49) STRAIN: NVw147 (ix) FEATURE: NAME/KEY: TATA -signal LOCATION: 787. .794 (ix) FEATURE: NAME/KEY:

ODS

LOCATION: 855. .3266 OTHER INFORMATION: /EO number= 3.2.1.37

GAGOACGGT

TATAGGOTOG

GTACTTAGTC

TGATMkGCTT 1337 1397 1457 1517 69 /product= "1 ,4-beta-D-xylan xylanbhydrolase" /gene= "xlnD" /standard-name= "beta-xylosidase" (ix) FEATURE: NAME/KEY: sig_peptide LOCATION: 855. .932 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 933.-.3266 (ix) FEATURE: NAME/KEY: polyA_site LOCATION: 3383 (ix) FEATURE: NAME/KEY: polyA_site LOCATION: 34o~4 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: a a. I a a a

CTGCAGGCCA

GAGATGGCGA

ACGGCGGCGC

GTGCCGGGGC

CTGGTGGAAT

ACC'IIrGACG

GGTG.ACGA

GAAGAGAGGC

GCTGGTG=r

ACCCCATTTG

ACATI'A CC C 'ITGI1ITAAT

GGCTAAACCC

AACCGCTATA

AGACTGGCTC

GGGCCGCGAT

AGATGAGTAT

AGTIGCCC

GrCGGGAGGC

ATGGGGAGGT

ATGAGGAGGT

AGAAGAAGAT

TGGGCGAGTI'

GACGGGGATG

GGTGCCTGAG

TGCTAAATGC

CCGCGATGAA

GAAGAAGGGT

ACCGTATGCO

OGGCATGTTG

CAAGGAGCTG

GGTGGAGAAG

GITGCAGAAG

CGAGGAGATT

GTTGAGGGAA

GCTTCATATI'

CGAAACTCTA

AAACATTCCA

GTCAATCGGT

GGCTGGGCAC

GTGGCGFI'GC

AATCGOGAGC

GATAATACGT

'ITGTTCAAGG

TGCGGGCTG

OTGTTGAAT

GAGACGOTGA

TCCCAAGCGA

CTTCGAAGAC

G=TCITCCT

CA7TCTCCGG TGTATCCTGC GAAGATGGOT GAOTGGAAGA AAATCCTCAA

CGGAGAAGCC

AGGTI'CTGGA

AGTTATCGGA

GGCGCAGAG

CTCCGAACGGG

TGACGAAGCG

TGCAAGAGOT

ATGTGCTGCA

TGTCACGCCA

AATGCCAATO

AATGCCGGCT

CGCATCTCCG

C'ICTATCC

GATTGAGCCG

AGCCACCCC

TGGGGAGA7T

TGTGATTAGG

GCAGATC

AC7CCATCCT

GGTAKTCG

GGAGGATGTG

GTAGACOGCIT

TAGAAAGGGC

TTTAACTATC

AAATCAITCA

CATCTCCGCA

CCCCCCCCAC

120 180 24I0 300 360 4 480 540 600 660 720 780 84 0 890 AAATCTACCC CAGATI'CAGT CCCCGGCCAC AACC ATG GCG CAC TCA ATG TCT COT Met Ala His Ser Met Ser Arg -26 -25 -20 CCC GTG GCT CCC ACT Pro Val Ala Ala Thr GCT CTT CCC CAG CC Ala Leu Ala Gin Ala CCC GCT GCT CTG CTG GCT OTO OCT CTI' CCT CAA Ala Ala Ala Leu. Leu Ala Leu Ala Leu Pro Gin MAC ACC AGO TAO. GTC GAO TAO MAC ATC GMA GCC Asn Thr Ser Tyr Val Asp Tyr Asn Ile Giu Ala OCT 'TOG TGC Pro Leu Cys GGT CCC 070 Gly Pro Leu GAO CGA GCA Asp Arg Ala CO MAC ACC Ala Asn Thr 9*

GCA

Ala Phe

ATC

le 115

TOO

Ser

GGO

Gly

TG

Trp Val

TCA

TAO

Tyr

AGO

Ser 100

OTO

Leu

ATO

Ile 070 Leu

GGT

Gly

TAO

Tyr 180

MOC

CAA

Gin

GAO

Asp

ACC

Thr

ATC

le

GAC

Asp

CCC

Arh 1 61

GO(

Al~

CTC

ATA C Ile C

OGO

Arg

OCA

Ala

GC

Gly

GTA

Val

TOA

Ser

ACC

Thr

TOT

Ser 070 Val 150

GGA

Giy

TAO

i Tyr

A

AA ACO ATO liu Thr Ile 25 kGC CAT OTO 3er His Leu 40 rOG OTO ATO Ser Leu Ile AO ACC GO ALsf Thr Giy OCA OTG AGO TI'O Pro Leu Ser Phe AAC COG Asn Pro CCC GAO Pro Asp ACA GC Thr Ala

TOO

Cys

ACC

Thr

CAG

Gin

COO

Pro ATO TGT Ile Cys

C

TGG

Trp

GGA

Giy

CG

Aila

ACC

Thr 135

TAO

Tyr

CMA

Gin

GMA

Giu

OTC

AGT GMA Ser Giu CO TAO Ala Tyr 105 GOC 070 Ala Leu 120 CAA GC Gin Cly GOC 000 Ala Pro GMA ACC Giu Thr TAO ATO Tyr Ile 185 cc GC Ala Ala 200 O C Ser I

CTCC

LeuC

GCT

Ala 90

MAT

Asn

MOC

Asn

CGO

Arg

MOC

Asn

OCA

Pro 170

ACC

Thr

AOG

Thr

~TC

.eu 75 Leu

TGG

Trp

OGO

Arg

OO

Al a

ATO

Ile 155

OGA

Gly

GGC

Gly

OC

Al a ~AT GMA sp Clu rTC ACC he Thr 60 G70 TC Val Ser

COG

Leu

CA

Arg

CAC

His

GCC

Ala

ACC

Thr .1-Ic Phe 14o

MAC

Asn

GAG

Ciu

ATC

Ile

MAG

Lys GOC 070 Cly Leu ACC TCA T1hr Ser *1 070 ATO Leu le 1.25 MOC AAO Asn Asn ACC 'I7O Thr Phe GAO arc Asp Val CAG GGT Gin Gly 190 CAC TAO His TIyr 205

ITO

GAC

Asp rro Phe

CAC

His

CO

Ala

CC

Arg

TOT

Ser 175 000 Pro

C

Ala GAO GAG CTG ATO Giu Leu GGO CTC Gly Leu CT CO Arg Ala COO CAG Pro Gin CMA ATO Gin Ile CCIC coc Gly Arg 14~ CAC COO His Pro 160 OTO CO Leu Ala GAO OCA Asp Pro GO TAT *Gly Tyr

CT

Pro

MAT

Asn

COO

Pro

COO

Ala 130

TAO

Tyr ar0 Val

COO

Al a

GMA

Ciu

GAO

Asp 210

ATO

GAO 'ITG TAT Asp Leu Tyr

MAT

Asn

TAT

Tyr 986 1130 1178 1226 1274 1322 1370 1418 1466 15114 1562 1610 1034 1082 Ser Asn Leu Lys Leu ATO GAG MOC TGG CAC MAC CAC TOO CO CTG GGO MOC GAO ATG MAC Ile Giu Asn Trp His 215 Asn His Ser Arg Cly Asn Asp Met 225 71 ACC CAC CAA GAC 'CTC TCC GAA TAC TAC ACO CCC CAA TTC CAC OTC GCC 1658 Thr Gin Gin Asp Leu Ser Giu Tyr Tyr Thr Pro Gin Phe His Val Ala 230 235 240 GCC CCC GAC GCC AAA GTC CAC ACT OTC ATG; TGC CCC TAC MAC CCC GTC 1706 Ala Arg Asp Ala Lys Val Gin Ser Val Met Cys Ala Tyr Asn Ala Val.

245 250 255 MAC GCC CTC CCT CCC TCC CCC GAC TCC TAC TI'C CTC CAG ACC CTC CTC 1754 Asn Gly Val Pro Ala Cys Ala Asp Ser Tyr Phe Leu Gin Thr Leu Leu 260 265 270 CCC GAC ACC TI'C CCA MIT CTC CAC CAC OGA TAC CTC TCC ACC GAC TGC 1802 Arg Asp Thr Phe Cly Phe Val Asp His Gly Tyr Vai Ser Ser Asp Cys 275 280 285 290 CAT CCC CCC TAT MAC ATC TAC AAC CCC CAC CCC TAT CCC TCC TCC CAC 1850 Asp Ala Ala Tyr Asn Ile Tyr Asn Pro His Gly Tyr Ala Ser Ser Gin 295 300 305 GCT GCC OCT CCC OCT GAG CCC ATC CTC CCC CCC ACC CAC ATC CAC TGC 1898 Ala Ala Ala Ala Ala Ciu Ala Ile Leu Ala Gly Thr Asp Ile Asp Cys 310 315 320 GOT ACC ACC TAC CMA TCC CAC CTG AAC GAG TCC ATC OCT CC CCA CAT 1946 Gly Thr Thr Tyr Gin Trp His Leu Asri Giu Ser le Ala Ala Gly Asp 325 330 335 CTC TCT CCC CAT CAT ATT CAG CAC CCT GTC ATCGT CTC TAC ACC ACC 1994 Leu Ser Arg Asp Asp Ile Glu Gin Gly Val Ile Arg Leu Tyr Thr Thr 340 345 350 CTC CTC CAC CCC CCA TAC TI'C CAC TCC AAC ACC ACA MAG C MAC AC 20-42 Leu Val Gin Ala Gly Tyr Phe Asp Ser Asn Thr Thr Lys Ala Asn Asn 35360 365 370 CCC TAC CCC GAC CTC TCC TOC TCC GAC CTC C'IT GAG ACC GAC-'GCA TOG 2090 Tyr Arg Asp Leu Ser Trp Ser Asp Val Leu Glu Thr Asp Ala Trp 375:: 380 385 MAC ATC TCC TAC CMA CCC CC ACC CAC CCC ATT GTC CTT CTC MCG MC 2138 A Asn Ile Ser Tyr Gin Ala Ala Thr Gin Gly le Val Leu Leu Lys Asn *390 395 400 TCC MAC MAC GTC CTC CCC CTC ACC GAG AAA OCT TAC CCA CCA TCC MAC 2186 Ser Asn Asn ValJ Leu Pro Leu Thr Ciu Lys Ala Tyr Pro Pro Ser Asn .:405 410 415 ACC ACC OTC CCC CTC ATC OCT CCC TOG CCC MAC CCC ACC ACC CMA CTC 2234L Thr Thr Val Ala Leu Ile Gly Pro Trp Ala Asn Ala Thr Thr Gin Leu 420 425 430 *:CTC GCC MAC TAC TAC CCC AAC CCT CCC TAC ATO ATC AGC CCC CCC OCC 2282 Leu Gly Asn Tyr Tyr Cly Asn Ala Pro Tyr Met Ile Ser Pro Arg Ala 435 440 445 450 72

AAC

Asn GCC r Ala P ATC T lie S CAA I Gin S GAA C Glu 1

CTGC

Leu 515

ATC

lie

AAC

Asn

TCT

Ser

CCC

Ala 77C Phe 595

GAG

Gin

GGC

Gly

AAG

Lys

TC

he .cc er

'CC

;er 3

CG

la 0O 3AC ksp 3TC Ja-i-

AAC

Asn

CGC

Gly

COT

Gly 580

CCC

Pro

ACG

Thr TrA Let

GAJ

Ci GAA G Glu C TCC I Ser 'J CCC C Ala 485

GAG

Glu TT1 Leu CTC I Leu

ACC

Thr

CGC

Gly 565

AGA

Arg

CC

Ala

TAT

Tyr Trc Phe Val 645

;AA

alu

~CA

7hr 3AC ksp 3CA kla

ATC

Ile

CAA

Gin

AAT

Asn 550

TIC

Phe

CTA

Leu

ACA

Thr

AAA

Lys

TAC

Tyr 630

AAC

Lys c 4 CC GGA TAG lia Gly Tyr GC AGC TCG jer Thr Ser AAA GTC Lys Val 070 G Val I CTC C Leu

CAG

Gin I

ATG

Met 535 Vai

GCT

Ala

GTC

Val

CAT

Asp

TOO

Trp 615

ACG

Thr

CTC

Leu

TA

le

!AT

ksp kAG Lys 520 3GC Gly

TCT

Ser

TO

Leu

ACO

Thr

ATC

Met 600

TAC

Tyx

AC(

Th

AA

Asr

ATC

lie

CGA

Arg 505

CTC

Leu

CGC

Cly

OGCA

Ala

CCC

Arg

ACG

Thr 585

AAC

Asn

ACC

Thr Phe

AT

*1 Il~ 460 GGC TTC OCT Gly Phe Ala 475 TAG CCC OCT Tyr Ala Gly 490 GAG ACT ATC Glu Ser lie CCC TCC CCC Ala Set Ala GGA CAG OTC Cly Gin Va 540 CMr CTC TGG Leu Leu Trp 555 CAT ATC ATC Asp lie lie 570 CAG TAG CCT Gin Tyr Pro GIT CGT CCI Leu Arg Pro CGC GAA GCC Gly Ciu Alf 62C SGCG GAA TC( Ala Giu Sez 635 CAG GAG A7 Gin Asp IlE 650 TTC CCC GAG GGC ACC COT Phe Ala Giu Gly Thr Gly 465 CCC CCC TTA 7CC CCC (;CA Ala Ala Leu Ser Ala Ala 480 GOT ATO GAG AAT ACC CIT Gly lie Asp Asn Thr Leu 495 GCO TOG CCG GGT AAC CAA Ala Trp Pro Gly Asn Gin 510 CCC GCA AAG AAG CCC CTG Ala Gly Lys Lys Pro Leu 525 530 GAT TGC TCT TCG CTC AAC Asp Ser Ser Ser Leu Lys 545 GGC GGA TAG CCC CC CAA Cly Gly Tyr Pro Cly Gin 560 AG CCC AAG AAG AAC CCC Thr Cly Lys Lys Asn Pro 575 CCC ACC TAC GCG GAG GAG- Ala Ser Tyr Ala Giu Clu 590 GAG GOT CAT AAC CCT GGT Glu Gly Asp Asn Pro Gly 605 610 CTC TAC GAG TC GCC CAC Val Tyr Ciu Phe Gly His 625 TCC ACC AAT ACC ACT ACA Ser Ser Asn Thr Thr Thr 640 r CIT TCC CAG ACA CAC GAA e Leu Ser Gin Thr His Glu 655 2330 2378 2426 2474 2522 2570 2618 2666 2714 2762 2810 2858 2906 2954 *r 0 0.

0 0*

S

GAC CTG GCG TG ATT ACC CAG CTC CCT OTC CTG AA ITC ACC OCC AAT Asp Leu 660 Ala Ser lie Thr Gin 665 Leu Pro Val Leu Phe Thr Ala Asn ATC AGG MAC le Arg Asn 675 73 ACT GGA MAG CTG GMA TCG GAT TAC ACC GCT ATO GTA TiC Thr Gly Lys Leu Glu Set' Asp Tyr Thr Ala Met Val Phe 68o 685 690 3002 CCC MAT ACC Ala Asn Thr G Tc CGG TG Val Cly Trp '170 AGOGCTC Leu Arg Val 725 CCC GAT TG Cly Asp Trp 740 TCT CAT Ser Asp 695 CCC CCC CCC Ala Gly Pro

CAT

Asp 710

CCC

Pro CCG CTT GGG Arg Leu Cly COTT GAG aG Val Clu Val

CAG

Glu Cly 730

GCA

Gly GCC CCC TAT CCC MAG MC TOG C Ala Pro Tyr Pro Lys Lys Trp, Leu 700 705 arc MCG CTC CCC GAG ACG AGO GAC Val Lys Val. Cly Clu Thr Arg Glu 715 720 ACC M~ CC AGO arc MAT GAG CAT Ser Phe Ala Arg VaJ. Asn Clu Asp 735 ACG '171 GAG 7irc CC 'TTO MT TIC Thr Phe Clu Leu Ala Leu Asn Leu 750 .3050 3098 3146 3194 CTC GTC =17 CCC Val Val. Phe Pro 745 GAG AGC Clu Arg 755- arc CTG Yal Leu MAG =I CCC ac MAG OTTI= CTT r GAG OT GAG GAG CMA CTC Lys Val. Arg Val Lys Val Val. Leu Clu Cly Clu Glu Clu Val.

760 765 770 MAG TGG CCC CCC MCG GAG TAGAAAATAC TA'ITCTCF1G

ATGGCTCTAG

Lys Trp Pro Cly Lys Clu 775 GGGATGAGAG TCAGCCTA'IT ACTGCATATG CATAGTGGTG ATACCATGTA TATAGCTCTA at..

*f

S

a..

lava 6* a a a *8*4 TCMAGTM'IT AG'ITCM.GTG AATAGGGATC AFTCTGAT T CCCATTGTAC CGGMCGTMC TAAGAAAGGG AM'ITGATCA.

TCTGCMATAC AGCTACAGMA TACTAAACCA ACACMATG ATCCTACCCA CMACATOCAT GATCACGGAG MATIACCMAC ATCCGTCCGA

CTCAAAAGCA

CTCCGACACC TCCTCMACTG ATCGGATACT

COCTGCCCGC

AACCGTI='C CCCMATCCAG G'1TTiCCTIG AGGGTCGGGA

GMATACCCC

MTAGTAGCG

M'ITCCACTG

AAAAATAAOG

ATMAACTG

ACMATACCCC

AAACCACTGC

TACTCTCGC

ACAAATCCCT

GCTCTTCAAA

TTAGACATAT

TGTCAAAGTC

GTGCGACTC

=ICACACAT ATAG TATGCT CTATTCCCA CTAGCGATGG TCACACACCA C'ITAATGIC AccTC'ITAGA AGACAACAG

CMAGAAAAAC

CCATCTACAG CCTATTCACA _7AGCCGCA 'ITAGCCTGC TOCTAGCATA

GCTCCTACTA

AAITAACCAG CCCTCACTCA

ACACMCTGA

TI'CCCCACCC AGCACCCTTC 'ITCACGATCA ATAAAACGTA AACMACCCCC

TCGOGCCAGG

CG7ICGCATA CTAGCCACAT

CAACCTC'IG

TGCCCAGAAG ACCC TTCTF CTCGATATCC GMACGATGAG TCT1CGTCTGC

CAMAGGAMAC

GTAGCTCTOC MTIGMAA. aTCGOC

AC

3242 3296 3356 3416 3476 3536 3596 3656 3716 3776 3836 3896 3956 4016 4076 4108 *1 a I *5

S

a.

5

S

S. a a a S 55 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 4173 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Aspergillus niger STRAIN: CBS 120.49 INDIVIDUAL ISOLATE: N400 (ix) FEATURE: NAME/KEY: CDS LOCATION: join(948..1173, 1238..3495, 3550..3690) IDENTIFICATION METHOD: experimental OTHER INFORMATION: /function= "Transcriptional activator of xylanolytic genes" /product= "Binuclear Zn finger DNA binding protein" /gene= "xlnR" /standard_name= "XYL R" (ix) FEATURE: NAME/KEY: exon LOCATION: 948..1173 (ix) FEATURE: NAME/KEY: intron LOCATION: 1174..1237 (ix) FEATURE: NAME/KEY: exon LOCATION: 1238..3495 (ix) FEATURE: NAME/KEY: intron LOCATION: 3496..3549 (ix) FEATURE: NAME/KEY: exon LOCATION: 3550..3690 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CCCGGGCTTG GTTGGTCTCC GTCTGGCTTC CCCGCCTTT TCCCCTGCAA TTCTGCATCC CCAATCCTTC TTTTTTCTTT GCCTCGCCAG GCTGTGTCTT TTITCCCCCT CCCCCTCCTC 120 CCTCGTCAGC TrCTCTTCGA CAGCATGCGT GAGGGTCTGC TACCAACTAC AATCCTTGTT 180

CTCACTG'TCT

GAAAGCTAOT

TCCGACACTA

TGGTCGGCCT

GIIIAOT

CGTTCCGTGG

TOGTCTTGAA

TTTCCCGCTA

GOTCTGAATA

TGTTCCGCCC

GATGGTCTOA

CAOTCCAGTC

AAAAOCCACT

TGTCGCTTCT

CCCTO1IA

'TTCCTTGCG

TCGCCTGGCC

GCGTAGTGCC

CCTCACTTTC

GGTGAGGATG

~CCGACCGTG

ACTC71TCTC rCCCCCCAAC

CCOGCTGACA

GITOCCCCOC

ACCGCTCCTC

CTCOTCTAGO

Crrl'CACGCT

CCCCCCAACO

TCAACCCCCT

TCGACTOOCA

7rOTCTGTGG TGTGTOTGTG AGAGAGAAAG )TOOGTTCTT CACCTTCCCC

GGACCTOCCC

rcOTI'AM-rG C TGCTAOTCT

CCTTAGITCA

ITCTCCTC~r

AGACTGAATC

TGC TICATCA

ATCTGTOCG

TGGGGCCTI'A

ACCGGOTCT

CGA77CCTCA

TGGCGCCGTC

CTOCTGCCT

GOcAATOCCO

TCTITICCT

CCAGTGTCGC

CGGCCCTTCC

TCATGACCCG

ATI'CACCAGT

TCAGACTGTC

CTAGG'rCCT TGGAG77GAT

CCTGCCCTCC

CTTAATCTCC

ATTCGCCAGC

CTGGTGAT

CCTTTCTCTC

GGITGGATA

GACGCTGCGG

CCTTCTCfl'C

CGGATCOCAC

CGCTCGCCGA GGOCTCGCAG TACOTCCTGG AACAATTOCA

GCTGTCGCGA

0* GAACCOGTGC COOCOATGOC GCGACCTCCA CrTCC1TGCG AAA 1TCC ATG TCG CAT Met Ser His ACG AAO GAT CMA CCA CCC Tfl GAT MAT GAG AAG MAC CAG AGC ACT GCC Thr Lys Asp Gin Pro Pro Phe Asp Asn Glu Lys Asn Gin Ser Thr Gly 5 10 TCG GOT Tfl' AGO GAC OCT OTO CMA AGA GAT CCC CTC OTG GAG OCT CGC Ser Gly Phe Arg Asp Ala Leu Gin Arg Asp Pro Leu Val Giu Ala Arg 20 25 30 TCT GCC G'rC CGC MAA ACC TCC TCT TCA OCT CCG OTT CGC CCC CGA ATC Ser Ala Val Arg Lys Thr Ser Ser Ser Ala Pro Val. Arg Arg-Arg le 40 45 AOC COT GCG TOT GAC CAC TOT AAC CAA CTC CGA ACG AMA TOC GAC COG Ser Arg Aia Cys Asp Gin Cys Asn GIn Leu Arg Thr Lys Cys Asp Cly 55 60 CAG CAT CCG TGC OCT CAT 7CC ATT G GTAOOCTTCC

OCTCTI'CTC

Gin His Pro Cys Ala His Cys Ile COATCGGC GATGACOCGG ACCCrTOACT GACCTCICT CTAG MA TTC GGA CTO Ciu Phe Gly Leu ACC TGC GAG TAT GCG CGA GMA COC AG AAG CO!T GGA MAA GCC TCG MOG Thr Cys Giu Tyr Ala Arg Clu Arg Lys Lys Arg Cly Lys Ala Ser Lys 85 90 240 300 420 480 5140 600 660 720 780 8140 900 956 1004 1052 1100 1148 1193 12148 1296 AAG CAT CTG GCG GCG GCA Lys Asp Leu Ala Ala Ala 100 OCT GCG GCG GCT ACC CAA GGG TOG AAT GOT Ala Ala Ala Ala Thr Gin Gly Ser Asn Gly 1344 105 110 CAT TCC GGG CAG GCC AAC GCG TOG OTA ATO GC GAG His Ser Gly Gln Ala Asn Ala Ser Leu Met Gly Glu CGA ACG TOG GAA Arg Thr Ser -Glu 125 1392 115 120 *4 GAC Asp E GAG I Glu

ACT

Thr 160 TCC I Ser

TTC

Phe

CTG

Leu

CC

Gly

TC'

Se.

24u

GCA

Ala

CCT

Pro CCc Pro

~GC

er

~GC

3er

GCA

Ala

CAA

Gin

AAC

Asn

CGT

Arg

TAO

Tyr 225

CCG

Pro

CTI

Leu PTh PhE COG C Arg P 130 CAC C His I

GGG

Gly

TCA

Ser

ACG

Thr

TCG

Ser 210

AAC

Asn

STTT

Phe

CCC

Pro i TCC Ser 290

CA

~ro

:AT

Iis kTA Ile rcG Ser

ATG

Met 195

CTA

Leu

GCG

Ala

%AC

C-Il Let 7CC Sei 271

AG(

Se GA C Cly C OTT A Leu S TCC C Ser C

I

OTA

Leti 180

AAO

Asn COO I Pro

AGO

Ser

CAC

Cln G GTn

CT

1Gly 260

OCA

Pro

ACT

:AA

In

GC

;er

;GC

fly 65

.GA

3ly

GAT

Asp

CCG

Pro 0CC Ala

'I

PhE 24

OT(

Le

TC-

Se -rr

GAC

Asp

TCOG

Ser 150

CTG

Leu I

ACG

Thr

TCC

Ser

TCC

p Ser 770 Phe 230

COO

Arg 5

TCG

1 Ser r OCT r Pro A OGA ;rG tal .35

:AC

%In

CAC

His

ACT

rhr

GOT

Cly

OTC

Val 215

GOCT

Ala roc Let

CCI

Prc

GC(

Al

TA

AAC C Asn C OCA I) Pro I

GAG

Olu

ATO

lie

CGC

Arg 200 7TA Leu Leu

CGA

1 Cly

SCCA

Pro A AAC a Asn 280 C CCT cc ;ly

?CG

jer

'CT

Ser

CAT

Asp 185

CCG

Pro

OCA

Pro

GTG

Val

AGC

Ser

GJ

G13 261 PhE ACA T Thr T CAT i His

CAC

Gin 170

GCC

Ala I

OCA

Ala

CCC

Pro

AAC

Asn

TCA

Ser 250

OAC

Gin fl COT

'AC

yr

~TG

iet kCG hr

ATG

Met

ATG

Met

CAA

Gin

CCG

Pro 235

GCG

Ala

TCG

Ser TC1

GAO

Asp 140

CAC

Gin I

OCA

Ala

OAT

His

TCC

Ser

GGA

Cly 220

CAA

Gin

CAA

Glu

CCT

Pro Srr [CC OCT Mrr 3er Ala Phe CAT OCA AGO His Ala Ser CCC TOG CAT Pro Ser His 175 TTG AT OAT Leu Asn His 190 ATA TCC GAT lie Ser Asp 205 CTA AGC TCC Leu Ser Ser GAG COG OGC Glu Pro Gly AAC CCA ACC Asn Pro Thr 255 GCA TOG CTC Cly Trp Leu 270 AGC TTG CAT Ser Leu His 285 OTC CTO CCT Val Leu Pro 1440 1488 1536 1584 1632 1680 1728 1776 1824 1872 1920 1968 a Pro Ser Phe G=T TG CAG CCG r Thr Leu Arg Tyr 295 Pro Val Leu Gin Pro CAC ATC CCC TCC A77 AIT CC CAC TOG OTA GCC TOT GAC CT CT GAT His lie 305 Ala Ser lie lie Pro 310 Gin Ser Leu Ala Asp Leu Leu Asp 77

CAC

His GTT TAC TTC ACT ACT TCC TCT TCC TCC Val Tyr Phe Thr Ser Ser Ser Ser Ser CTG TOT CCC 1TC TCC CCA Leu Ser Pro Leu Ser Pro 2016 325 330 TAC C Tyr V AAA C Lys F CCC C Ala CGG C Arg C

COA

Arg I 400

GCG

Ala

TCC

Ser

CAT

Asp

TAC

Tyr

COT

Arg 480

CGG

Arg

CCT

Pro

TG

'al cc 'ro

CA

la

;GG

3ly 585

CG

?ro

GCG

Ala

ATG

Met

GTA

Val

AAG

Lys 465

GAC

Glu

CAI

Glr

CCC

Prc CTG GCC TAC A Val Gly Tyr 1 340 OGA ATA TGC I Arg lie Cys E 355 CAA ACC ACT Gin Thr Ser 370 CCT CTA TOC Arg Val Cys TTG OTC CAT Leu Val His M.T ATC CTC Asn Met Val 1420 CAT CAG CTC Asp Gin Leu 435 GCA ACT TAT Ala Thr Tyr 450' GCG CCC AGO Ala Ala Ser CTG AAG CTA Leu Lys Leu CAT GGA GAG Asp Gly Glu 500 3 ACC CTC ATC Thr Leu lie 515 GC CCC jer Pro GOT CTC CTG GCG

I

JAA C Au I ln 1

GOT

Cly 405

ATC

lie

GCC

Cly

CTC

Val

ATC

Met

GGC

Cly 485

CGA

Arg

ACG

Thr

;CT

la .ys 390

CCT

?ro

A.AT

Asn

CC

Ala

CAT

His

COC

Arg 470

CCT

Arg

CAI

AsF

TC~

SeI Cly Leu L 360 GCG 'TI C Ala'Phe L 375 CTG CTA C Leu Leu GOT ACC Ala Thr GGC GTC 4 Gly Val CAA AGT Gin Ser 440 CTT GCG Leu Ala 455 TOG TGG Trp Trp GAG CTC Glu Leu CG CAT Cly Asp CT0 GGT r Leu Cly eu

:TG

.eu

AA

3lu

'GA

Gly

GCT

Ala 425

AGC

Ser

ACA

Thr

ACT

Thr

CCA

Prc

GCC

cil 50~

CA"

Ala E ACA Thr

CTG

Leu

GAA

Glu 410

CTG

Leu

GCC

Ala

OTA

Val

CC

Ala

CCC

Pro 490

GAG

r Glu r GGA

OTI

er rCG C 3er I kCC fr 395

GCG

Ala

GGC

Cly

ACC

Thr

OTA

Val

CC

Ala 475

AAT

Asn

CCC

Ala

TCG

Ser

~TG

let

:CG

?ro 380

IT

ie Ile

TCG

Ser

GGA

Cly

GGC

Gly

TCC

Ser 460

TOO

Trp

CIM

Val

CAC

Asj

GO

012 rTC 'TC CCC AAC CAG TCT TTC CTT lie Phe Arg Lys Gin Ser Phe Leu CAC CCC A His Pro 7 350 CTC TOC C Leu Trp V 365 CCC 7CC C Pro Ser t COT 'TC C Cly Leu I CCC AAC Pro Asn TTT GGG Phe Cly 430 CCC GTG Ala Val 445 CCC AGO Ala Ser TCT CTA Ser Leu 7CC CAC Ser His AAA OCA Lys Arg 510 k AGC TCC y Ser Ser 525

CA

'hr

?TA

'al

CT

la .rc .eu rAT ryr 415

OTC

Val

CAT

Asp

GAG

Clu.

GCG

Ala

OCA

Ala 495

CAT

His

GGC

Cly 2064 2112 2160 2208 2256 2304 2352 2400 2448 2496 2544 2592 His Gly 520 ATT AAT OTC ACC CAA lie Asn Val Thr Glu 530 GAG GAG COT GAG GAG COT CGA CCC CTA 7CC TCC 2640 Clu Clu Arg Clu 535 Glu Arg Arg Arg 540 Leu Trp Trp CTC 'ITA TAT Leu Leu Tyr 545 070 ACG CTG Leu Thr Leu GCG-ACO SAT CGG CAC CTG Ala Thr Asp Arg His Leu 550 CG CTG TGC Ala Leu Cys 555 TAC AAC CGG CCC Tyr Asn Arg Pro CAG CCG ATG MAC Gin Pro Met Asn 575 2688 CTG SAC Leu Asp

AAG

Lys 565 GAA TGT SOC OGG CTG CTG Glu Cys Gly Sly Leu Leu 570 2736 560

GAT

Asp OT0 Val.

CTA

Leu

SAT

Asp

GA

Sly

CCG

Pro 075 Leu

COG

Pro

CTS

Leu

TO

Trp

CCC

Pro 595

ATO

Met

CAS

Sin 580 OTc Val

ACG

Thr 610 *0 .0

GAG

Glu~

/GAG

640

AGC

Ser

OCT

Ala Ser

TCC

Ser

CAC

His 720

TTG

kAT k -cm 625

COT

Arg TrG Leu

ACS

Thr

CCT

Pro

ATC

Ile 705

OTC

Val

GAA

CAT

H{is

CAG

Gin

MAG

Lys

SAT

Asp

TCG

Ser 690

OTC

Val 070 Lei

CAT

CS

Pro

STA

Val

GAA

Glu

MOC

Asn 675

OGG

Sly

CAC

His

CAT

His

CAT

;.c krg CT0 Leu

TO

Phe 660

GAS

Slu

CSC

Arg

ACC

Thr A71 le

GA

Asj 714 570 C Val C SAG 'I Giu C A77' Ile

TFTT

Phe

SAC

Asp 645

GAG

Glu

CCT

Pro

TCC

*Ser

ASS

*Arg

'TSG

Leu 725 r O

~GC

~ly

O

Al a 630 Val

AC

ATC

Mel 71(

CTC

Le~ ACO C Thr C 6 GSA C Sly C 615

CO

Leu

ACS

Thr

CCC

Arg

OTC

Val

AGO

*Ser 695 -Val .1 Ala

C

Lsp Phe Ala Ala Ala 585 'AC TIT SCA C SCT SOC TAO CCC CAC

GT

;iy )00 31y 3CO Arg

TAC

Tyr

SM.

Glu 680

ACC

Thr OT C Val

GG

12

CAC

His

ATC

Ile

TTO

Phe

CAG

Gin

ACC

Thr 665

GGT

Sly Val G CC Ale e Ly~ AGO Ser l Val CoC Arg 075 Leu 65.0

AGO

Ser

CC

Al a

GA

Sly

TAO

LTyr TOO75 STrp 730 e t 3AT

AAT

635

GAC

Asp

AAC

Asn

CAC

His Ala

TAT

Tyr

CTO

Leu 620

AGC

Ser

ACA

Thr Tra Leu :rc Leu Tyr Arg Gin 590 GSA TAC TTT Sly Tyr Phe 605 CAC CAC GOT His His Ala CCC SAG TG Pro Clu Trp TAT 555 CC Tyr Sly Arg 655 ACT CTG CCC Thr Leu Sly 670 CAT CAC ACS., Asp His Thr 685 3r"A TAGAG SVal Ser Clu G CAT ATO ATS rHis le Met G ars MAT OTS o Val Asn Leu 735 r arc TCC CC e Val Ser Ala 2784I 2832 2880 2928 2976 3024 3072 3120 3168 3216 3264

TOG

Ser

GS

Sly 715

GAO

Asp CcC Arj

ACC

Th~ ccC Pri TOG ATO TOO TOG SAG TO TT' Leu Glu Asp His Leu Trp Ile Ser Ser 745 SMA OCA Glu Ala 760 Glu Ser Ph 750 ATS AGO CAT Met Ser His

GC

Al a 755 OTC =GOTSC SCA Val Sly Ala Ala C SCA SMA Ala Ala 51u ATO 'rs GAS Ile Leu Slu 765 3312 TAC GAC CCC GAT CTC ACC 'FTC ATC Tyr Asp Pro Asp Leu Ser Phe Met CCC TrC TrC TTC GGG ATI TAC CTA Pro Phe Phe Phe Gly Ile Tyr Leu 780 3360 CTA CAG GCC Leu Gin Cly 785 CAT CCC ACT Asp Ala Ser 800 ACT 'FTC 'FTC CTC CTA Ser Phe Leu Leu Leu 790 CTG CC GCC Leu Ala Ala CCC ACT CTC Pro Ser Val 805 GTG CCC GCA TGC Val Arg Ala Cys

GAG

Giu 810 CAC AAG 'TCG CAG GCC Asp Lys Leu Gin G2Ly 795 ACO ATC CTC CCC C Thr Ile Val. Arg Ala 815 TAC CAG GTAGCITFC Tyr Gin CAT GAA CC TGC GTC GTG ACC 7IC AAC ACC GAG His Giu Ala Cys Val Val. Thr Leu Asn Thr Giu 820 825 TGTTTCTCT CCCTAGCTTG GCAATAGTAG CTAACACAAT GTAG ACC ACA 'FTC CC Arg Thr Phe Arg 830 GGA CCC ATC CCA GAG Gly Arg le Pro Giu MAG CTC ATC Lys Va). Met 835 GAC =F CCC .Asp Phe Gly 850 CCC TCC AGC Arg Trp Ser 865

TGATCATGAG

TTTGATACTA

ACCCCTGGCA

C'FTCCTGAT

AC'ITTGATAC

TGACCCCATC

CCAAAATCTT

TCCCCAAAAT

CGA TCC CC CTC Arg Ser Ala Leu CCA CAC CT1' CCA Ala Gin Vai Arg 8'40 CCC CGA CCC GAA Arg Arg Arg Clu 8'45 GAG CAC CA Ciu Gin Ci CCC CAT CC Gly Asp Gi 87 kTGCCATTTA

CT'FFTGGATT

rGCCGAATG

ATATGTGGAT

CACCTCAATC

CCTGAGATAA

C'FTCATATAA

GCCCTCCCTA

C

C ACT CCC CTC CCA CTC y Ser Cly Leu Ala Leu 0 875 TGCCCTCCA 'FTGACCGGTC CCCTA'F=TCA CTCCGGCTT AAATATGC'FT AC'ITCGTGTTI A TITTTGGCA TCTACACTAT TAA'FTGCGTT CTTCAT GCTGCCCATA AGCATTCCCA CCAATCCATC AA'FTCAACAT TACACTCCC TCCCCACTTC GTG C'FI CC CTA TAC Vai Leu Ala Leu Tyr 860 TAGITFCCA GTAACACGC AATGC'FCT TACA'FICTCA ATGCTGC'T CA'TCGTCAAG GATACCA'FT CGTACATATA GCCTGATCFI' TGGACATGAT GF1'GCCCAAC AGCCGACGTA 'FTCCATCCTC CATCGAAGCA TCGTAATGAC

AATAGTATAA

CCC

.3408 31456 3505 3561 3609 3657 3710 3770 3830 3890 3950 4010 4070 4130 4173

Claims

1. A combination nucleic acid cassette comprising 1) a promoter normally associated with a target gene of an activating regulator of an inducible enhancer or activator sequence, 2) the nucleic acid sequence encoding the regulator being operably linked to a further promoter, 3) a homologous or heterologous sequence encoding a homologous or heterologous protein or peptide, said promoter being operably linked to the homologous or heterologous encoding sequence, wherein said such activating regulator is involved in metabolism.

2. A combination nucleic acid cassette according to claim 1, wherein said activating regulator is involved in .a part of metabolism with an enzyme cascade or feed back loop or multiple feed back loops and such target gene 4 having a binding site for the expression product of the i regulator gene.

3. A combination nucleic acid cassette according to claim 1 or 2, wherein the further promoter operably linked to the regulator encoding nucleic acid sequence is a S. promoter associated natively with the regulator encoding nucleic acid sequence.

4. A combination nucleic acid cassette according to any one of claims 1 to 3, wherein the nucleic acid sequence encoding the regulator encodes a xylanolytic regulator. A combination nucleaic acid cassette according to claim 4, wherein the nucleic acid sequence encodes xylR. \\melbfiles\homeS\wendyS\Keep\species\65450-99 Danisco.doc 12/03/02 81

6. A combination nucleic acid cassette according to any one of claims 1 to 5, wherein the nucleic acid sequence encoding the regulator is any one of: a) xlnR encoding the expression product xylR according to the encoding nucleic acid sequence of SEQ ID NO. 9, or b) a nucleic acid sequence capable of hybridising under specific minimum stringency conditions as described in the examples to the encoding SEQ ID NO. 9 or primers or probes of nucleic acid SEQ ID NO. 9, said primers or probes being present in the non-zinc finger binding region and said primers or probes being at least 20 nucleotides in length, or c) a nucleic acid sequence encoding an expression product exhibiting 80% to 100% identity with the amino *acid sequence of xylR according to SEQ ID NO. 9 or as encoded by the nucleic acid sequence encoding xylR of SEQ ID NO. 9. S7. A combination nucleic acid cassette according to any one of claims 1 to 6, wherein the promoter is selected from a promoter associated with the target genes xlnA, xlnB, xlnC, xlnD and axeA.

8. A combination nucleic acid cassette according to any of claims 1 to 7, wherein the promoter is a promoter associated with the target gene xlnD.

9. A combination nucleic acid cassette according to any of claims 1 to 8, wherein the homologous or heterologous sequence encodes an enzyme. A combination nucleic acid cassette according to any of claims 1 to 9, wherein the homologous or \\melbfiles\home$\WendyS\Keep\species\65450-99 Danisco.doc 12/03/02 82 heterologous sequence encodes a xylanase, glucanase, a-glucuronidase, lipase, esterase, ferulic acid esterase, a protease or an oxidoreductase such as hexose oxidase.

11. A vector comprising a combination nucleic acid cassette according to any one of claims 1 to

12. A host cell comprising a combination nucleic acid cassette according to any of claims 1 to 10 and/or a vector according to claim 11.

13. A host cell comprising the components of claim 1, said host cell comprising a target gene of the regulator either natively or through recombinant DNA technology with the proviso that when the target gene and the regulator are native to the host cell, the regulator is present in multiple copies.

14. A host cell comprising multiple copies of the nucleic acid sequence encoding the regulator as defined in claim 1.

15. A host cell according to claim 14, wherein the nucleic acid encoding the regulator is any one of: a) xlnR encoding the expression product xylR according to the encoding nucleic acid sequence of SEQ ID NO. 9, or b) a nucleic acid sequence capable of hybridising under specific minimum stringency conditions as described in the examples to the encoding SEQ ID NO. 9 or primers or probes of nucleic acid SEQ ID NO. 9, said primers or probes being present in the non-zinc finger binding region and said primers or probes being at least 20 nucleotides in length, or s. c) a nucleic acid sequence encoding an expression \\melb_files\home$\WendyS\Keep\species\65450-99 Danisco.doc 12/03/02 83 product exhibiting 80% to 100% identity with the amino acid sequence of xylR according to SEQ ID NO. 9 or as encoded by the nucleic acid sequence encoding xylR of SEQ ID NO. 9.

16. A host cell according to any of claims 10 to 12 comprising the nucleic acid sequence encoding the regulator as defined in claim 1.

17. A host cell according to claim 16, wherein the nucleic acid sequence encoding the regulator is any one of: a) xlnR encoding the expression product xylR according to the encoding nucleic acid sequence of SEQ ID NO. 9, or b) a nucleic acid sequence capable of hybridising under specific minimum stringency conditions as described in the examples to the encoding SEQ ID NO. 9 or primers or probes of nucleic acid SEQ ID NO. 9, said primers or probes being present in the non-zinc finger binding region and said primers or probes being at least 20 nucleotides in length, or c) a nucleic acid sequence encoding an expression product exhibiting 80% to 100% identity with the amino :acid sequence of xylR according to SEQ ID NO. 9 or as encoded by the nucleic acid sequence encoding xylR of SEQ ID NO. 9 and the promoter as defined in claim 1, said promoter being a promoter associated with the target gene xlnD.

18. A host cell according to any of claims 12 to 16 being selected from the group comprising microorganisms and plant cells. \\melb_files\home$\wendyS\Keep\species\65450-99 Danisco.doc 12/03/02 84

19. A host cell according to any of claims 12 to 18 being selected from fungal cells, preferably filamentous fungal cells. A host cell according to any of claims 12 to 19, said host cell being selected from the genus Aspergillus, Trichoderma, Penicillium and Fusarium.

21. A host cell according to any of claims 12 to said host cell being a strain selected from Aspergillus niger, Aspergillus tubigensis, Aspergillus aculeatus, Aspergillus awamori, Aspergillus oryzae, Aspergillus nidulans, Aspergillus foetidus, Aspergillus terreus, Aspergillus sydowii, Aspergillus kawachii, Aspergillus carbonarius and Aspergillus japonicus.

22. A host cell according to any of claims 12 to said host cell being a strain belonging to a genus selected from Saccharomyces, Kluvyeromyces and Lactobacillus.

23. A host cell according to claim 22, said host cell being a strain selected from Saccharomyces cerevisiae or Saccharomyces pombe.

24. A host cell according to any of claims 12 to 23, wherein the target gene is endogenous to the host cell. A host cell according to any of the claims 12 to 24, wherein the target gene is present in multiple copies.

26. Use of a combination nucleic acid cassette according to any of claims 1 to 10 for production of the homologous or heterologous protein or peptide in a manner known per se for producing protein or peptide from nucleic Sacid sequences encoding protein or peptide. \\melbfiles\homeS\WendyS\Keep\species\65450-99 Danisco.doc 12/03/02 85

27. Use of any one of a nucleic acid sequence a) xlnR encoding the expression product xylR according to the encoding nucleic acid sequence of SEQ ID NO. 9, or b) a nucleic acid sequence capable of hybridising under specific minimum stringency conditions as described in the examples to the encoding SEQ ID NO. 9 or primers or probes of nucleic acid SEQ ID NO. 9, said primers or probes being present in the non-zinc finger binding region and said primers or probes being at least 20 nucleotides in length, or c) a nucleic acid sequence encoding an expression product exhibiting 80% to 100% identity with the amino acid sequence of xylR according to SEQ ID NO. 9 or as encoded by the nucleic acid sequence encoding xylR of SEQ ID NO. 9 or overexpression of a target gene by expressing the nucleic acid sequence in a host cell comprising the target gene operably linked to a promoter normally '...associated with a target gene of the activating regulator of an inducible enhancer or activator sequence encoded by S: the nucleic acid sequence with the proviso that when the target gene and the nucleic acid sequence are native to the host cell the nucleic acid sequence is present in multiple copies in comparison to the wild type host cell.

28. Use of a host cell according to any one of claims 12 to 25, for production of the homologous or heterologous protein or peptide in a manner known per se for production of protein or peptide from a nucleic acid sequence encoding a protein or peptide. \\melb_files\home$\WendyS\Keep\species\65450-99 Danisco.doc 12/03/02 86

29. A combination nucleic acid cassette according to claim 1, substantially as herein described with reference to any one of the Examples. A host cell according to claim 12, substantially as herein described with reference to any one of the Examples.

31. Use according to claim 26 or 27, substantially as herein described with reference to any one of the Examples. Dated this 1 3 t h Day of March 2002 DANISCO INGREDIENTS A/S (DANISCO A/S) By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia 0 U \\melbfiles\homeS\Wendys\Keep\pecies\65450-99 Danisco.doc 12/03/02