IE83483B1 - Selection marker gene free recombinant strains: a method for obtaining them and the use of these strains - Google Patents

Description

Title Selection marker gene free recombinant strains, a method for obtaining them and the use of these strains Technical field The present invention discloses selection marker gene free recombinant filamentous fungal strains, a method for obtaining these strains and the use of these strains.

Furthennore, the method of the present invention is used for performing strain improvement.

Background of the invention There is an increasing social concern about the use of recombinant DNA technology. One of the promising application areas of recombinant DNA technology is strain improvement.

Starting from the early days of fermentative production processes there has been a demand for the improvement of the productivity of the strains used for production.

Classical strain improvement programs for industrially employed microorganisms are primarily based on random mutagenesis followed by selection. Mutagenesis methods have been described extensively; they include the use of UV light, NTG or EMS as mutagens. These methods have been described extensively for example in "Biotechnology : a comprehensive treatise in 8 vol." Volume I, Microbial fundamentals, Chapter Sb, Verlag Chemie GmbH, Weinheim, Germany.

Selection methods are generally developed around a suitable assay and are of major importance in the discrimination between wild type and mutant strains.

It has turned. out that these classical methods are limited in their potential for improvement. Generally improvement_ yield speaking consecutive‘ rounds of strain diminishing increases in yield of desired products. This is at least partially due to the random character of the mutagenesis methods employed. Apart from desired mutations these methods also give rise to mutations which are undesirable and which may negatively influence other characteristics of the strains.

In view of these drawbacks it can be understood that the use of recombinant DNA methods was hailed as a considerable improvement.

In general, recombinant DNA methods used in strain increased improvement programs aim at the expression of desired gene products.

The gene products may be proteins that are of interest themselves, on the other hand it is also possible that the encoded gene products serve as regulatory proteins hi the synthesis of other products.

Strains can be improved by introducing multiple copies of desired protein encoding genes into specific host organisms. However, it is also possible to increase expression levels by introducing regulatory genes. that Such Vectors may be Genes are introduced using vectors serve as vehicles for introduction of the genes. plasmids, cosmids or phages. The vector may be capable of expression of the genes in which case the vector generally is self—replicating. The vector may however also only be capable of integration. Another characteristic of the vector is that, when the expression product cannot be selected easily based on altered phenotypic properties, the vector is equipped with a marker that can easily be selected for.

Vectors have not been isolated from all known microorganisms either since no vector could be found in the available vectors organism or since could be used with little from other organisms or no modification. The same applies to selection marker genes.

Widespread use and the subsequent spreading of specific marker genes has recently become debatable. This is especially due to the finding that the use of antibiotics and selection markers rise to an undesired antibiotic gives spread of strains that have become antibiotic resistant. This '4.) necessitates the continued development. of novel ever more potent antibiotics.

It is therefore not surprising that there is a to microorganisms containing no antibiotic resistance genes or general tendency in ‘large scale production use recombinant more generally as little as possible of foreign DNA.

Ideally the only the desired gene(s), transformed microorganism would contain fragments thereof or modifications in the gene and as little as possible or no further remnants of the DNA Used for Cloning. VVO93KH283 dmdommiecmnbnmnt marker gene free plant cciis from which marker genes are excised using the bacteriophage Pl cre/lox system.

Summary of the invention The present invention discloses a selection marker gene that can easily be deleted again from the recombinant host organism. The deletion of the said marker gene is based on dominant selection.

The marker is used in filamentous fungi The advantageous activity of the selection markers used herein is based on the following two step principle: a) the gene is integrated into the genome of the host organism and recombinant cells are selected, b) the transformed Cell is grown on a substrate, which is converted by the marker gene encoded activity to a product that is lethal to the cell.

Selected will be deleted the selection marker gene. cells recombinant and will haV€ In general terms the present invention discloses fibnmnmusfimgflcdk that have a modification in the genome characterized in that the alteration is introduced using the gmgs gene ogjthe CDNA derived therefrom. fl An example of a selection marker gene that can be Used in this way is the acetamidase gene. Preferably, this gene ig obtainable from filamentous fungi, more preferably from Aspergilli, most preferably from Asperqillus nidulans.

The invention further shows the introduction, deletion or modification of desired heterologous or homologous genes or DNA elements in the (E15) gene is deleted. fimmmnmmfimgm using the acetamidase gene as a marker. Subsequently the amds Preferably, the gmgs and the desired genes are introduced site—specifically.

The invention discloses a vector containing: a) a desired DNA fragment destined for introduction into the host genome, b) optionally a DNA sequence that enables the vector to integrate (site—specifically) into the genome of the host strain, c) a gene encoding an acetamidase (e.g. the gmgs gene from A.nidulans) between DNA repeats.

The transformed with the said vector. invention further discloses fihnwnmusfimg The invention further discloses selection marker gene free recombinant fihnmnmusfimgi.

Specifically, the invention disclesee flbnmnwusfimg containing sitewspecifically introduced genes without any further foreign DNA being present. The method is therefore also suited for repeated modifications of the host genome, e.g. the sequential introduction of multiple gene copies at predetermined loci.

The invention provides a method for obtaining selection marker gene free recombinant fibnwnmusfimgmsuanscmnpnnngflmibflowhg steps: a) integration into the genome of the strain of a desired DNA fragment and a selection marker, b) selection of the recombinants, c) deletion of the selection marker preferably using internal recombination between selection marker flanking repeats, d) counter-selection based on the absence of the selection marker.

Although. this is the preferred method for obtaining ‘selection marker gene free recombinant filmmymousfungfl The desired DNA fragment zumi the selection. marker‘ may’ be different which two The integrate into the genome of the strain but may be present on present on DNAJ molecules are co- transformed. selection marker does necessarily an episomal DNA molecule which can be cured.

The present invention further illustrates that this marker gene can be deleted from the genome of the transformed organisms without leaving a trace i.e. DNA used for cloning. present invention discloses the use of the ands gene from Asoergillus as a marker in bacteria and yeast.

The invention discloses also the use of the amgs gene for deleting a desired gene from the chromosome of a filamentous fungal host organism In embodiments the following strains are employed. Asoeroilli, specific Trichoderma, Penicillium The method of the present invention provides recombinant strains with genomic modifications obtained by repeating the procedure with the same or other vectors.

Brief description of the drawings Abbreviations used in the figures: Restriction enzvmes and restriction sites: T. = gggg terminator sequence Figure 1: shows the restriction map of plasmid pangs-1. This plasmid contains the CDNA of the gmgs gene from A.nidulans.

Figure 2: shows schematically the marker gene free deletion of the"glgA locus from A.niger using the gene replacement Vector pGBDEL4L. The essential part of the gene replacement vector pGBDEL4L contains the gmgs gene under control of the gpgA promoter cloned between repeats (3’—non-coding region of the glaA gene).

Figures 3-9: show schematically the construction pathway of pGBDEL4L as further outlined in Example 1.

Figure 10 Figure 11: A. Schematic presentation of EQEHI and Kpnl fragment lengths of the wild—type glaA locus in Aspergillus giger CBS 513.88. and GBA-108).

Figure 12: A: shows schematically the integration of the g;gA gene into the 3' non-coding region of truncated g1aA locus of Ame; GBA—lO7 .

B: shows the result of the internal recombination between the 3’ glaA repeats, flanking the amgs gene.

Figures 13-24: show schematically the construction pathway of the integration vector pGBGLA30 as further outlined in Example 2.

Figure 25: figlll digests of pGBGLA3O transformants #107—9 (lane 1), #107—7 (lane 2) and #107-5 (lane 3), the host strain A.niger GBA—107 (lane 4) and the parental strain A.niger CBS 513.88 (lane 5) and Kpnl digests of pGBGLA30 transformants #107-9 (lane 6), #107—7 {lane 7) and #107~5 (lane 8), the host strain A.niger GBA—107 (lane 9) and. the parental strain A.niger' CBS 513.88 (lane 10), probed with ”P—labelled glgA promoter fragment.

Figure 26: A: Schematic presentation of the gpnl and gglll fragment lengths of the wild—type glaA locus in Aspergillus niger CBS 513.88.

B: Schematic presentation of the fipgl and gglll fragment lengths of the truncated glaA locus in Aspergillus niger GBA~107.

C: Schematic presentation of the fipgl and Bdlll fragment lengths of the truncated glgA locus with. a single copy pGBGLA3O integrated into the glgA 3’—non— coding region as in transformants #107—5 (= GBA~119) and #lO7—9 (= GBA-122).

D: Schematic presentation of the gpgl and gglll fragment lengths of the truncated glaA locus in GBA—119 and. GBA-122 transformants after removal of the amds gene (= GBA-120, GBA-121, GBA-123 and GBA—l24).

Figure 27: A: gglll digests of A.niger CBS 513.88 (lane 10), GEA- 107 (lane 9), GBA—1l9 (lane 8) and the GBA—119 strains after fluoracetamide selection: #AG5—7 (= GBA~120) (lane 5), #AG5-5 (= GBA—l2l) (lane 6) and #AG5-6 (lane 7); GBA-122 (lane 4) and the GBA—122 strains after fluoracetamide selection: #AG9-1 (= GBA—123) (lane 3), #AG9—2 (lane 2) and #AG9-4 (= GBA-I24) (lane 1), probed with “P-labelled 3"g;aA non—coding fragment.

B: KEQI digests of A.niger CBS 513.88 (lane 10), 107 (lane 9), after fluoracetamide (lane 5), #AG5—5 (= GBA-121) 7); GBA—122 (lane 4) fluoracetamide selection: GEA- GBA-l19 (lane 8) and the GBA-119 strains #AG5-7 (= GBA-120) (lane 6) and #AG5—6 (lane and the GBA-122 strains after #AG9-1 (= GBA-123) (lane 3), #AG9—2 (lane 2) and #AG9-4 (= GBA—124) (lane 1), probed with 32P-labelled 3" g_l_aA non-coding fragment. selection: Figure 28: shows schematically the construction pathway of pGBGLA50.

Figures 29~33: show schematically the construction pathway of pGBGLA53. r Detailed description of the invention I he present invention discloses the use of a marker gene for preparing selection marker free recombinant filamentous fungal host strains. The selection marker gene can be used on an episomal DNA Vector. However, in the present invention, the marker gene is preferably Integrated into the genome of the filamentous fungal host strain. The advantage of the selection marker of the present invention is that it is a non—antibiotic dominant selection marker. Another advantage of the selection marker of the present invention is that it can be easily deleted from the transforming filamentous fungal host organism. The deletion of the marker is based on dominant selection. As such the selection marker of the present invention is a dominant and bi-directional selection marker. To our lmowledge it is the only selection-marker available which is bi- directional and dominant in both directions. _ 9 _ In the present description we use the term ’selection marker gene’. with this term we mean the DNA coding for the marker protein in a functional form irrespective of whether it is the actual gene or the cDNA derived therefrom. The gene or CDNA is used dependent on the host organism and the expected splicing problems.

In the present invention we use the term ’vector’. By this is intended any DNA molecule that can be introduced into a selected filamentous fungal host irrespective of whether the Vector integrates into the genome of the filamentous fungal host cell or remains episomal. The vector contains a selectable marker gene functional in the selected host or can be co—transformed with another DNA molecule containing such a selection marker gene.

The the heterologous or homologous genes or DNA fragments’. ’desired By this present description uses term is intended a DNA fragment that may be obtained from the filamentous fiingal host strain or from another species or strain. The desired DNA fragment may contain any genetic element, parts thereof or combinations thereof, a cDNA, such as a gene (coding part or complete locus), a promoter, a terminator, an intron, a signal sequence, any regulatory DNA sequence or recognition sequence of DNA~binding‘ proteins. The fragment may also be a DNA sequence that has been modified i.e. contains one or more nucleotide alterations (e.g. insertions, deletions, substitutions).

The present description further uses the term ’introduction’ of a desired gene or DNA fragment. By this is intended an insertion, deletion, substitution of desired DNA sequences in a selected filamentous fungal host 0611.

The term ‘genetic modification’ used in the present invention refers to any modification of DNA sequences in a selected filamentous fungal host cell which is the result of the introduction of ; ? any one of the above mentioned desired DNA fragments into the host cell, preferably by transformation or co—transformation. all performed using ‘Una method of the present In general these genetic modifications can be invention with subsequent deletion of the selection marker gene. Due to the _]()_ thctthatthereconfinnantfflarncntousfhngalsuaniconunnhngsuchzagenefic the so that combined in the modification does not contain the selection marker gene, procedure of the present invention can be repeated, the modifications suggested above (uni be recombinant filamentous fungal strain. Ultimately, the procedure of the present 3 invention can be used repeatedly up to the point that a ramnmnmntfibnmnmusflmgdsumnisommnaifimnvmmhafltmdmmed activities have been removed by deletion or inactivation of the the desired corresponding genetitt elements and which. contains acivities at desired levels by sequentiar introduction of the corresponding ciesired DNA fragments at desired oopynumbers and preferably at desired and defined :: g‘: j _,. ,,,,, The a_m_d_S gene from A.nidulans is capable of converting acetamide to ammonia and acetic acid. This property enables A.nidulans to grow on a medium containing acetamide as the sole N—source'or C—source.

Another property of the amds gene is that it is also able to convert fluoracetamide to ammonia and fluoracetic acid. Fluoracetic acid however is toxic to the cell.

It allows the production of marker gene free recombinant filamentous fimgal strains. The tluoracetamide converting property enables the counter-selection of transformed filamentous fungal cells. The % gene is introduced into the filamentous fungal host strain and integrated into the genome through homologous recombination. The transfonned filamentous fungal strains are selected on a medium containing acetamide as the sole N-source. Subsequently the selected strains are grown on a medium containing fluoracetamide and urea (or other preferably defined N—sources) as the sole N—sources.

The surviving filamentous fungal strains will have deleted the amds gene.

The present invention uses the A.nidulans amds gene as acetamidase marker gene. The relevant properties provided by the acetamidase encoded by the A.nidulans _a_n_1gS gene, i.e. the ability to hydrolyse acetamide into ammonia and acetate as the ability to can also be provided by acetamidases from well as liberate fluoracetic acid from fluoracetamide, other sources. Use of an acetamidase marker gene is therefore not restricted to the A.nidulans amds gene but includes any DNA sequence encoding a functional acetamidase.

The substantially the To achieve this frequency of marker deletion is increased by increasing the capacity of gene for intrachromosomal homologous recombination. the amds gene is preferably placed between DNA repeats. These repeats are not necessarily both present in the vector but may also be created by a single cross-over integration.

Alternatively, one may omit flanking repeats and rely on other mechanisms for removal or inactivation of the marker gene. In that case, however, the outcome may be less . predictable and may not result in removal but rather in mere inactivation of the marker gene.

The vector may be Constructed in such a way that, after deletion of ‘the marker gene, no extraneous foreign DNA (except the DNA of interest) remains in the chromosome of the filamentous fimgal host strain. The invention discloses a vector comprising: a) a desired DNA fragment destined for introduction into the filamentous fungal genome, b) optionally a DNA sequence that enables the vector to integrate (site-specifically) into the genome of the filamentous fungal host strain, c) a gene encoding an acetamidase (e.g. the _a_nid_S gene from _A.nidulans) between DNA repeats.

Identical results may be obtained when the DNA—fragment destined for introduction into the filamentous fungal host genome and the selectable ‘marker the gene (e.g. acetamidase gene) are present on two different DNA molecules which are co- transformed, in which case the DNA molecule containing the selectable marker does not necessarily integrate into the filamentous fungal host genome but may be present on an episomal DNA molecule which can be cured. 4 The sequences used for integration as mentioned under b) are used if site—specific (or better locus specific) integration is desired. If such a sequence is not present the vector nevertheless may integrate into the genome. This does not influence the ability to delete the selection marker gene.

The dominant counter-selection described above can be employed in the development of industrial filamentous fungal production strains in various ways. The use of a dominant selection marker is especially advantageous in the development of improved filamentous fimgal production strains due to the fact that these strains are often diploid or polyploid.

The vector used for integration of the aI_n_d_S gene preferably contains another gene of interest. The invention’ thus further enables the introduction of desired foreign or homologous genes or DNA elements in the filamentous fungal host organisms of _ 13 - choice using the amds gene as a marker. subsequently the amds gene is deleted. Preferably, the amdS and the desired genes or DNA elements are introduced site—specifically, whereafter the amgs gene is deleted.

Specifically, the invention discloses filamentous fungal organisms containing site—specifically introduced genes without any further foreign DNA being present. The invention is used for integration of multiple copies of a desired gene or a DNA element at predetermined genomic loci.

The invention provides a method for obtaining selection of marker gene free filamentous fungal recombinant strains comprising the following steps: ~ integration of a desired gene or DNA element and a selection marker by homologous recombination between sequences incorporated in an expression cassette and sequences on the filamentous fungal host chromosome, ~ selection using the selection marker gene that is dominant, — deletion of the selection marker gene using selection marker gene flanking regions, A " selection based on the absence of the selection marker gene (counter-selection).

The present invention further shows that this marker gene can be deleted from the chromosomes of the transformed filamentous fungal organisms without leaving a trace i.e. DNA used for cloning the that identical results can be obtained when the desired gene or Moreover, invention also shows similar if not DNA element and the selection marker are present on two different DNA molecules which are co—transformed.

Finally the invention discloses the use of the amdS gene for deleting a desired gene from the chromosome of a filamentous fungal ‘host’ organsim. of the suited for, the ideally method of the but not limited to the In view above, present invention is cloning and expression of genes coding for proteins used in W4. _ 14,.,___-,,._ food, feed or pharmaceutical applications or genes involved in biosynthesis of antibiotics and other bio-active compounds, i.e. recombinant proteins and/or hosts—organisms that are subject to strict registration requirements.

Examples of such proteins are well known in the art and include chymosin, phytase, xylanases, amylases, cellulases and hemicellulases, cytokines and other pharmaceutical proteins, etc.

The coding for proteins same method is employed for deletion of genes that desired proteins again without leaving a marker gene in the influence production levels of genome. Such proteins include proteases which actively digest the desired products that are highly expressed in the host and that therefore producing and or secreting the desired proteins. A preferred have a strain reduced potential of method for the deletion of a given gene would use a DNA construct containing the following elements in aa 5’ to 3’ order: sequences 5’ of the gene to be deleted, directly fused to sequences 3' of the gene to be deleted, followed downstream by a functional selection marker gene (preferably an acetamidase gene), followed downstream by again sequences 3’ of the gene to be deleted. In this Case both sequences 3' of the gene to Ix: deleted are chosen such that they form repeats flanking the selection marker gene. Transformation of this DNA replacement of the deleted by the DNA construct and subsequent chrmmxmmal copy of the gene to be construct with cross—over points in the sequences 5’ and 3’ of the gene to be deleted results in deletion of the given gene. Subsequent intrachromosmal recombination between the repeats flanking the selection marker gene and counter- selection for these recombinants finally results in a selection marker free filamentous fungal strain with the given gene deleted. The DNA construct used for this deletion can be constructed such that no DNA or the modification are left in the strain carrying the deletion. foreign other traces of genetic ' 135 The invention discloses selection marker gene free recombinant filamentous fungal microorganisms. Such microorganisms can be organisms that, after the use of the disclosed technology, contain an extra copy of a desired gene (either homologous or heterologous). Such filamentous fungal microorganisms can be re-transformed over and over by sequential application of the same technology to insert or delete additional copies of the same or other gene(s) of interest. flwfimmmmwmgmmmmmmmmmmwdmbmmmammflmmm they have (a) predetermined gene(s) deleted or altered in any desired way.

The method of the present invention makes possible the of the This possibility is based on the ease with which repeated rounds The method makes insertion or delethmi of a desired number of Thus the fine~tuning production of desired proteins. of insertion and deletion can be performed. possible the gene copies. proteins are produced in desired amounts and in desired ratios. This is especially useful for the production of mixtures of proteins or enzymes.

Whereas it is known that the acetamidase gene is capable of conversion of acetamide as the sole N—source in Aspergillus easily deleted from the genome of transformed Aspergilli. To it is here shown that the acetamidase gene is achieve this the amds gene is cloned between direct repeats.

In principle any direct repeat which allows for internal recombination can be employed. In the present examples this the amds non—coding DNA sequences. is demonstrated by cloning gene between 3’ amyloglucosidase (glaA) It is shown that the gmgs gene can be integrated and deleted upon plating on medium containing fluoracetamide and urea as N—sources.

It is further" demonstrated. that the amyloglucosidase gene can be deleted from the genome of Aspergillus. A replacement vector is constructed containing :1 part of the glaA promoter, a synthetic DNA sequence containing stop codons in all three reading frames, the amds gene from A. nidulans under the control of the A.nidulans glyceraldehyde- -phosphate dehydrogenase promoter and wherein the amds gene is flanked by 3’ glaA non—coding sequences. After transformation of A.niger the vector is integrated by double crossing-over thereby effectively replacing the amyloglucosidase gene. After selection for amgs activity the transformed strains are plated on fluoracetamide and urea.

Selection resulted in strains wherein the deleted.

This example is an illustration of the possibility of amds gene was using the amds gene for deletion of a desired gene from the Aspergillus Other eliminated or modified in a similar manner. genome of an strain. genes can be The construct is shown to integrate at the amyloglucosidase locus. After selection on fluoracetamide the amgs gene is deleted. In this way a gene copy is integrated at a specific locus without leaving marker DNA.

It is evident from the above that the procedures described herein enable one of skill in the art to integrate or delete desired genes at predetermined loci without leaving selection marker DNA behind.

This method can be employed for gene amplification and gene replacement. important would be Followed by classical strain that affected by the classical strain improvement techniques are An especially application the integration <3f desired genes. improvement whereafter the genes may be adversely replaced with fresh unaffected copies of the gene of interest without loss of expression level.

The system as described for above is Aspergillus expected to give the same results when other filamentous fungal strains _ 17‘_ are employed, which are known to be incapable of growth on acetamide as the sole N-source. The use of the amgs gene as a selection marker has been described for among others Penicillium and Trichoderma. Moreover, the amgs gene can even be used in filamentous fungi which are capable of using acetamide as sole N-source albeit poorly. In this case the background of poorly growing untransformed cells can be repressed by the inclusion of CsCl in the selection media (Tilburn, J. et al. (1983) _2_6_, 205-221). system is expected to be applicable to filamentous fungi in Gene. Hence the general.

The advantages of the system of the present invention are manifold. The most striking advantages are given below: ~ It is demonstrated that the amgs system is universally applicable in filamentous “fungi etc.), requiring only that the host in question cannot or only poorly grow on acetamide as sole C- or N~source but can utilize either acetate or ammonia as sole C— or N—source, respectively.

The amgs system represents the only bi-directional and This dominant selection system. feature is extremely convenient for use in poly— or aneuploid strains which often is the case with natural isolates and/or industrial filamentous fungal strains.

~ After classical strain improvement any mutated copies of the desired gene can be easily replaced by unmutated copies by gene replacement due to the fact that the desired genes have been integrated at well—defined loci. The genes are thus with without affecting the unmutated genes replaced expression level.

— Due to the ability to introduce multiple integrations at well—defined and therefore non—random loci one can be assured that no undesirable traits arise in the filamentous fungal strain upon gene amplification. amplification.

— The growing concern about the release of various selection by the No selection marker‘ gene or other unnecessary Or markers in the environment is overcome presented system. undesired DNA sequences need to be present in the filamentous fungal production — 18 — e— strains after introduction of the desired‘ genes or other genetic modifications.

Experimental General molecular cloning techniques In the examples described herein, standard molecular cloning techniques such as isolation and purification of nucleic acids, electrophoresis of nucleic acids, enzymatic modification, cleavage and/or amplification of nucleic acids, transformation of E.coli, etc., were performed as described (Sambrook et al: (1989) Cold Spring Harbour Laboratories, (eds.) (1990) and applications” in the literature "Molecular Cloning: Cold "PCR a laboratory manual", New York; Innis et al. methods Synthesis of oligo-deoxynucleotides and Spring Harbour, protocols, a guide to Academic Press, San Diego).

DNA sequence analysis were performed on an Applied Biosystems 3808 DNA synthesizer and 373A DNA sequencer, respectively, according to the user manuals supplied by the manufacturer" Transformation of A.niqer Transformation of gigiqgr was performed according to the method described by Tilburn, J. et.al. (1983) -221 and Kelly, J. (1985) EMBO J., 3, Gene gg, & Hynes, M. 475-479 with the following modifications: m spores were grown for 16 hours at 30°C in a rotary shaker at 300 rpm in Aspergillus minimal medium. minimal medium consists of the following Per liter: 6 g NaNO3; 0.52 g KCl,' 1.52 g ml 4M KOH: 0.52 g MgSO4JNgO; 10 g glucose; 22 mg ZnSO4.7Hg); 1.7 mg CoCl2.6H2O; Aspergillus components: KHQPO4; 1.12 1 g casamino acids; 11 mg }gBO3; 5 mg 1.6 mg CuSO4JfigO; 5 mg FeSO,. 7H2O,’ MnCl2.4H2O,' 1. 5 riboflavin; 2 mg thiamine.HCl; 2 mg nicotinamide; 1 mg pyridoxine.HCl; ml Penicillin (5000 IU/ml)/Streptomycin (5000 UG/ml) .2 mg panthotenic acid; 4 pg biotin; solution (Gibco). — 20-—- _,__ only Novozym 234 (Novo Industri), and no helicase, was used for formation of protoplasts; after protoplast formation (60-90 minutes), KC buffer (0.8 M RC1, 9.5 mM citric acid, pH6.2) was added to a volume of 45 ml. and the protoplast suspension was centrifuged at 2500 g at 4°C for 10 minutes in a swinging-bucket rotor. The protoplasts were resuspended in 20 ml. KC buffer. Then, 25 ml of STC buffer (1.2 M sorbitol, 10 mM Tris—HCl pH7.5, 50 mM CaCl2) was added and subsequently the protoplast suspension was centrifuged at 2500 g at 4°C for 10 minutes in a swinging-bucket rotor, washed in STC—buffer and resuspended in STC—buffer at a concentration of 108 protoplasts/ml; to 200 pl of the protoplast suspension the DNA fragment, in a volume of 10 )u.l in TE buffer (10 mM Tris~HCl pH7.S, 0.1 mM EDTA), was added and subsequently 100 pl of a PEG solution (20% PEG 4000 (Merck), 0.8 M sorbitol, 10 mM Tris—HCl pH7.5, 50 mM CaCl2): after incubation of ‘the ,DNA—protoplast suspension at room temperature for 10 minutes, 1.5 ml PEG solution (60% PEG 4000 (Merck), 10 mM Tris-HCl pH7.5, 50 IIIM Caclg was added slowly, with repeated mixing of the tubes, After incubation at room temperature for 20 minutes, the suspensions were diluted. with 5 nu. STC buffer, mixed by inversion and centrifuged at 2000 g at room temperature for 10 minutes. The protoplasts were resuspended gently in 1 ml 1.2 M sorbitol and plated onto selective regeneration medium consisting of Aspergillus minimal medium without riboflavin, thiamine.HCl, nicotinamide, pyridoxine.HCl, panthotenic acid, biotin, casamino acids and glucose but with 10 mM acetamide as the sole nitrogen source, 1 M sucrose, solidified with 2% bacteriological agar #1 (Oxoid, England). Following growth for 6-10 days at 30“C, the plates were replica plated onto selective acetamide -~21— plates consisting of Aspergillus selective regeneration medium with 2% glucose instead of sucrose and 1.5% agarose instead of agar. Single transformants were isolated after 5-10 days of growth at 30°C.

Transformation of A. orvzae Transformation of A. oryzae was performed according to the method described by Christensen, T. et al.

Patent Application 0 238 023 A2. in European Transformation of T. reesei Transformation of T. reesei was performed according to the method described by Penttilla M., (1987) Gene gi 155-154.

Knowles, J.

Transformation of P. chrysoqenum The Ca—PEG mediated protoplast transformation procedure is used. Preparation of protoplasts and transformation of P.chrysogenum was performed according to the method described by Gouka et al., Journal of Biotechnology ;Q(l991), 189-200 with the following modifications: ~ After transformation, the protoplasts were plated onto selective regeneration medium plates consisting of Aspergillus minimal medium, osmotically stabilized with .2 M sucrose, containing 0.1% acetamide as sole nitrogen source and solidified with 1.5% bacteriological agar #1 (Oxoid, ~ After 5~8 days of England). incubation at 25°C transformants appeared. ' rd” 7 _:Q _ M “_ ., Isolation of DNA from Asperqillusl Trichoderma, Penicillium The isolation of DNA from Penicillium was performed according to the procedure described by Kolar et alg, Gene Q; (1988), l27~l34.

Pemo\al of the amds selection marker The amds marker" in most examples relating to Asperqillus, Trichoderma and Penicillium is cloneda between repeats consisting of a part of the 3’ nonmcoding region of amyloglucosidase genel Removal of the amgs selection marker is achieved either by internal recombination between the 3’ glaA nonmcoding repeats that flank the gmgs selection marker or by homologous recombination between the repeats that are created by integration via a single cross~over event» Selection of cells that have lost the amgs selection marker is achieved kn! growth on plates containing fluoracetamide.

Cells harbouring the amgs gene metabolize fluoracetamide to to the cell.

Consequently, only cells that have lost the amds gene are ammonium and fluoracetate which is toxic able to grow on plates containing fluoracetamide.

In case of removal of the amds marker from Aspergillus transformants, spores from these transformants were plated onto selective regeneration medium (described above) containing 32 mM fluoracetamide and 5 mM ureum instead of 10 mM acetamide, 1.1% glucose instead of 1M sucrose and 1.1% \ 23 __,,_,,:,.__ instead of 2% bacteriological agar #1 (Oxoid, England). After 7—10 days of growth at 35°C single colonies were harvested and plated onto 0.4% potato dextrose agar (oxoid, England).

In case of removal of the ands marker from Trichoderma transformants, spores of these transformants were plated onto non selective minimal medium plates (per liter: 20 g. glucose, 5 g. (NH4)2SO4, 15 g. KH2PO,, 0.6 g. Mgscn, 0.6 g.

CaCl2, 0.005 g. Feso,.7H2o, 0.0016 g. MnSO4.I-I20, 0.0014 g.

ZnSO,.7H2O, 0.002 g. CoCl2; pH5.5) supplemented with 10 mM fluoracetamide. After 5-10 days at 30°C, colonies were harvested and plated onto 0.4% potato dextrose agar (oxoid, England).

In case of removal of the ands marker from Penicillium transformants, spores from these transformants were plated on Aspergillus medium with 10 mM fluor—acetamide and 5% glucose, solidified After 5» selective medium plates consisting of minimal with 1.5% bacteriological agar #1 {Oxoid, England}, :0 days of growth at 25“C resistant colonies appeared.

Determination of qlucoamylase production by A.niqer transformants Of recombinant and control A.nigeg strains spores were collected by plating spores or mycelia onto FDA-plates (Potato Dextrose Agar, Oxoid), prepared according to the supplier’s instructions. After growth for 3M7 days at 30°C spores were collected after adding 0,01% Triton X~10O to the plates. After washing with sterile water approximately 107 spores of selected transformants and control strains were inoculated into shake flasks, containing 20 ml of liquid pre- culture medium containing per litre: 30 g maltoseJgO; 5 g yeast extract; 10 g hydrolysed casein; 1 g KHQPO4; 0.5 g MgSO4JNgO; 3 g Tween 80; 10 ml Penicillin (5000 IU/ml)/Streptomycin (5000 UG/ml); pH 5.5. These cultures were grown at 34°C for 20~24 hours. 5-10 ml of this culture was inoculated into 100 ml of fermentation medium containing per 12.5 g .5 g MgSO4/Hgo; 0.03 g litre: 70 «g maltodextrines; 25 <3 hydrolysed casein; yeast extract; 1 g KHQXQ; 2 g Kgfly; - 24 — ml adjusted ZnCl2; 0.02 g CaCl2; Ovol g MnSO4uMgO; 0.3 g FeSO4.7HgN (5000 IU/ml)/Streptomycin (5000 UG/ml): to pH 5.6 with 4 N'IgSO4. These cultures were grown at 34°C penicillin for 5-10 days. Samples were taken for the analysis of the glucoamylase production at different 'time points during fermentation. Fermentation broth samples were centrifuged (10 minutes, l0.000xg) and supernatants collected.

The glucoamylase activity was determined by incubating ul of a diluted the supernatant in 0.032 M NaAC/HAC pH4.05 with 115 pl of 0.2% six times sample of culture (w/V) pwNitrophenyl a-Dmglucopyranoside (Sigma) in 0.032 M NaAc/HAC pH 4.05. After a 30 min incubation at room temperature, 50 #1 of 0.3 M Na2CO3 was added and the absorption at a wavelength of 405 nm was measuredg The Awflfi is a measure for the AG production. ~ 25 Example 1 Marker gene free deletion of an A.niqer gene by using the amds gene‘ In this example a genomic target gene in A.niger will be replaced by transforming A.niger with a replacement vector which integrates into the A.niger genome via a double cross~ over homologous recombination“ The replacement vector comprises a DNA region homologous to the target locus interrupted by a selectable marker gene flanked by DNA A.niger wherein the glgA coding sequences as well as a region, This vector“ comprises a part of the glaA genomic locus, with part of the glae. promoter sequences are replaced by transformants as sequences as direct repeats: Transformation of amdS counterw marker gene. By performing fluoracetamide selection on these described in experimental procedures, the amds marker gene will be deleted Short description of the glaA gene replacement gyector PGBDELGL The gene replacement vector pGBDEL4L contains 5’—part of the (glgA) synthetic DNA sequence of 16 bp providing stopcodons in all (QEQS) glyceraldehyde—3—phosphate A.nige;j amyloglucosidase promoter region, a three reading frames, the A.nidulans acetamidase gene under control of the A.nidulans — %3~. dehydrogenase promoter, flanked at both sides by 3’ (EIRQA) glaA non—coding sequences.

Construction pathway of pGBDEL4L In order to obtain the final deletion vector pGBDEL4L several subclones of the glaA locus were derived first. A schematic View is presented in figure 3. The glgA locus of A.niger was molecular cloned and described previously (EP 0 463 706 Al). Plasmid pAB6~l contains the entire glaA locus from A.niger on a 15.5 kb fiigglll fragment cloned in the fiindlll site of pUCl9 (Yanisch~Perron et“al;, Gene 33 (1983) lO3~ll9, and is obtainable Germany). pAB6~l was digested with EcoRI and the l.8 kb EcoRl from e.g. Boehringer Mannheim, DNA fragment just upstream of the glaA gene was isolated by agarose gel electrophoresis and ligated into pUCl9 digested molecular cloned. The resulting plasmid was designated pAB6«3 (Figure 3A). To construct plasmid pAB6~4, which is another subclone of pAB6-1, pAB6~l was digested with fiindlll and gglll. The 4.6 kb sized DNA fragment comprising the glaA destroyed appropriately by this cloning procedurei (Figure 3E)" As a sites in pAB6M4 were Subsequently, after digesting plasmid pAB6v4 with indlll and EcoRI and filling in the S’ sticky ends using E; coli DNA polymerase, the 1.8 kb glaA promoter DNA fragment was isolated. by agarose gel electrophoresis, ligated into which was partially digested with EcoRI and treated blunt the pAB6~3 transferred to EL coli for molecular ligation mixture was cloning. The derived plasmid (designated pAB6~31) contains a 3.6 kb glaA promoter fragment with a destroyed 5ggRI site in the middle, but still possessing the EcoRl site the (now unique in this fragment) just upstream of glaA ATG initiation site (Figure 4). approximately 4 kb sized EcoRI—KpnI in plasmid pGW325 University, Wageningen, The Netherlands). This EcoRI—KpnI flanked by fragment thesis (1986) Agricultural (Wernars et al., containing the amgs its own the appropriate sites of pUC19 as described by Verdoes et al.

(Transgenic Res. ; pp 84~92, 1993) resulting in pAN4wl. pAN4~ digested with EQQRI and KQQI, the 4 kb DNA fragment containing the amds gene was isolated by agarose fragment gene, regulatory sequences, was molecular cloned into was sized gpnl and the ligation mixture was transferred to E. coli for molecular cloning" The obtained plasmid was designated pAB6S {Figure 5; and contains a 3&8 Kb glaA promoter DNA fragment and the 4 Kb amds fragmente Plasmid pAB6S was first partially digested with gall, and ligated to the synthetic derived oligonucleotide TNGQOI (SEQ ID NO: 3) having the following sequence; TCGATTAACTAGTTAA 3’ TNOOOl (SEQ ID NO: 3): 5’ and secondly digested with ECOR:w The DNA fragment comprising the pUCl9, the glaA promoter and the amds gene sequences was purified and isolated by agarose gel electrophoresisa From plasmid pAB6«l, digested with Sall, the 2W2 Kb 3’ flanking glaA DNA fragment was isolated as well by agarose gel electrophoresis and ligated to the above mentioned synthetic oligonucleotide, treated with T4 polynucleotide Kinase, subsequently digested with EQQRI and ligated. to the above mentioned DNA fragment isolated of pAB6S. The DNA ligation derived plasmid was designated pGBDELl and is shown in Figure 6. By this procedure simultaneously the Sall restriction site was destroyed and stopcodons in all reading frames were introduced.

To obtain an approximately’ 1 kb large DNA fragment, containing 3’ glaA non~coding DNA sequences positioned just — fla- downstream the stop codon of the glaA gene and flanked by suitable restriction sites, a PCR amplification was performed. In this PCR amplification, the plasmid pAB6~1 was and as primers two synthetical derived used as template oligonucleotides: Oligo AB2l54 (SEQ ID NO: 4): ’AACCATAGGGTCGACTAGACAATCAATCCATTTCG 3’ (a 3’g1aA non—coding sequence just downstream of the stopcodon) and Oligo AB2155 (SEQ ID NO: 5): ’GCTATTCGAAAGCTTATTCATCCGGAGATCCTGAT 3’ site (a 3’glaA non~coding sequence around the ECORI approx, 1 kb downstream of the stopcodon}.

The PCR was performed as described by Saiki et alr {Science 239; 487~491, 1988} and according to the supplier of TAQ~pciymerase (Cetus). Twenty five amplification cvcles (each 2 minutes at 55 °C: 3 minutes at 72 “C and 2 minutes at °C) were performed in a DNA~amplifier (PerkinwElmer/Cetus)” The 1 Kb amplified DNA fragment was digested with fiigdlll lad Sall, purified by agarose gel electrophoresis, ethanol precipitated and subsequently cloned into the fligdlll and gall restriction sites of pGBDEL1° The thus obtained plasmid TO obtain the final _q1aA :r'ep]_a(j-ement ve,c'Z*_'r,3r the amds promoter region in pGBDEL2 was exchanged gene pGBDEL4L, by the stronger A.nidulans gpgA promoter. Fusion of the gpgA promoter sequence to the coding sequence of the gmgs gene was performed by the Polymerase Chain Reaction (PCR) method. For this PCR fusion two different templates were used: ) plasmid control of the Aynigplans gpgA promoter and plasmid pAN4—l, Etggli heh gene Under and the A nigulans trpc terminator regulatory sequences. As primers four synthetic _ g)_ oligonucleotides were used, possessing the following sequences: Oligo AB 2977 (SEQ ID NO: 6): ' TATCAGGAATTCGAGCTCTGTACAGTGACC 3’ (a 5’ gpgA promoter specific oligo nucleotide, positioned at approximately 880 bp upstream of the ATG startcodon of the E. coli hph gene) Oligo AB2992 (SEQ ID NO: 7); ’ GCTTGAGCAGACATCACCATGCCTCAATCCTGGGAA 3' Oligo AB2993 (SEQ ID NO: 8): : TTCCCAGGATTGAGGCATGGTGATGTCTGCTCAAGC )sequences are complementary to each other‘ and contain 18 bp of the 3’ end of the gpdA promoter and 18 b of the 5’ part of the ands coding region) 9 - r , , Oligo AB2994 (SEQ ID NO: 9): ’ CTGATAGAATTCAGATCTGCAGCGGAGGCCTCTGTG 3’ {an amds specific sequence around the Bglll site approximately 175 bp downstream of the ATG initiation codon) To fuse the 880 hp gpgA promoter region to the a coding sequence two separate PCR’s were carried out? first amplification witha pAN7—l as template and the oligo and AB2993 (SEQ ID NO: 8) as primers to amplify the 880 bp DNA fragment comprising the nucleotides AB 2977 (SEQ ID NO: 6) gpdA promoter flanked at the 3’ border by 18 nucleotides complementary to the 5’ end of the amds gene, and the second PCR reaction with pAN4—1 as template and the oligo nucleotides AB2992 (SEQ ID NO: 7) and AB2994 (SEQ ID NO: 9) as primers to amplify a 200 bp sized DNA fragment comprising the 5’ part of the amds gene flanked at the 5’ border by 18 nucleotides Complementary to the 3’ end of the gpgA promoteru —f%)— A schematic View of these amplifications is presented in Figure 8A. The two fragments generated were subsequently purified by agarose gel electrophoresis, ethanol precipitated and used. as ‘templates in a third PCR reaction. with oligo nucleotides AB 2977 (SEQ ID NO: 6) and AB2994 (SEQ ID NO: 9) as primers. The resulting DNA fragment was digested with EcoRI, purified by agarose gel electrophoresis and ethanol precipitation, and cloned into the EcoRI site of pTZ18R (United States Biochemicals). The resulting plasmid was designated pGBGLA24 To exchange the amds promoter sequence (Figure 8B). in pGBDEL2 by and ligated into the EQQRI and Bglll restriction »enzymes te The resulting glaA gene replacement vector si s of pGBDEL2, was designated pGBDEL4L (Figure 9).

Deletion of qlaA promoter and coding sequences in A.nioer Prior to transformation of A.niger with pGBDEL4L, the were removed by flindlll electrophoresis. The (deposited October 10, 1988) .5, 5 ug DNA and Xhgl digestion A.niger CBS was E coli sequences and gel strain l3»88 agarose transformed with or 10 either fragment by procedures as described in experimental procedures using acetamide as sole £~sourue in selective plates, Single ALniger‘ transformants were purified several times onto selective acetamide containing minimal plates. Spores of individual transformants were collected by growing for about 5 days at 30°C on 0.4% potato-dextrose (Oxoid, England) agar plates. Southern analyses were performed to verify the presence of the truncated glaA locus. High molecular weight DNA of several digested with BamHI and KpnI and 0.7% transformants was isolated, fractionated by electrophoresis on a After subsequently agarose gel. transfer to nitrocellulose hybridization was performed according to standard procedures using two ”P—labelled probes: a fihgl/Sall glaA promoter filters} _.

UV -» fragment isolated from plasmid pAB6—4 (described above, Figure 3A) and a probe recognizing endogenous xylanase sequences (European Patent. Application. (3 463 706 A). The results of only 4 transformants (#19, #23, #24, #41) and the control strain A.niger CBS 531.88 are shown as examples in 10A. better understanding of this Figure For a autoradiograph, a schematic presentation is presented in Figure 11 showing the size of the hybridizing fragments in intact and truncated glaA locie Characteristic for the intact is a 3g5 kb Bamfil glaA locus hybridizing fragment in a digest and a 495 Kb hybridizing fragment in a Kpnl digest (see figure 11A), In a Rb gpnl hybridizing fragment are absent and gpnl hybridizing fragment. In this example, as can be seen in ?igure lOA, transformant #19 shows the expected pattern of a truncated glaA locus (Figure 118), This transformant was designated GBA—lO2.

No replacement of the glaA gene had occurred in the other transformantsi The poorly hybridizing bands: 4, R and kg in the 3931 digest and 7 and 12 kb in the fiamfii digest, refer to the xylanase sequences as internal control.

Removal of the amds gene fIOHl A.niqer' GBA»lO2 by’ counter— selection on fluoracetamide containing_platesu The amdS gene in the transformant Ainiger GBAwlO2 was removed again as described in the Experimental section. The removal of the amgs selection marker gene in only 2 surviving recombinant strains was verified by Southern analysis of the High molecular weight DNA was chromosomal DNA. isolated, digested with gamfil and Kpnl and subsequently separated by electrophoresis on a 0.7% agarose gel. Following transfer to according to hybridization was nitrocellulose performed standard procedures using probes described in previous section. A schematic presentation of the hybridizing fragments is shown in Figure 11C. The results of the Southern _ffl _ analyses are presented in Figure 108. The presence of a 5.2 kb hybridizing BamHI fragment and a 3.4 kb hybridizing Kpnl fragment, with the concomitant loss of the 5.5 kb BamHI and the 6.3 kb hybridizing Kpnl fragments is specific for the absence of the amds selection marker. The weaker hybridizing and 12 kb fragments in a BamHI digest and the 4, 8 and 15 kb Kpnl fragments again refer to the endogenous xylanase locus. Both strains show the expected pattern. In these recombinant strains, which were designated GBA—lO7 and SBA- 108, that possess finally no selection marker gene at all, the preferred glaA sequences are removed correctly and Both strains can be reused again to delete or insert other genes or DNA elements by using the same type of vector.

Example 2 Marker gene freewintroduction of the glaA gene targeted at the 3’glaA non~codinq region of the truncated glaA locus in A.niger GBA~lO7 In this example the introduction of 61 gene into the genome of A.niger is described by using approximately the same approach and procedures as described ixm the previous examplei Besides the desired gene or DNA element the vector contains DNA sequences homologous to t.e host genome tn target the vector at a predefined genomic locus of the host. by a single cross—over event. This type of vector comprises a selection marker gene flanked by DNA repeats as well. The selection marker gene in transformants derived with this vector can be removed properly again by applying the counter- As an example the introduction of a glaA at the selection procedure. gene copy is described which becomes integrated truncated glaA locus in the recombinant AglaA A.niger GBA—lO7 strain derived in Example I (for a schematic drawing see Figure 12) — 53; Description of the qlaA integration vector: pGBGLA30 The integration Vector pGBGLA3O consists of the A.niger (QLQA) promoter and the A.nidulans gmds gene under control of the gpgA flanked by‘ 3’glgA sequences to direct. integration at the 3’ amyloglucosidase gene under control of the native A.nidulans promoter non—coding glaA non—coding region and to remove the amds selection marker gene via the countereselection.

Construction of the integration vector A 1.8 kb ghgl/EQQRI glaA promoter fragment from pAB6m1 (Figure was subcloned into the gmal and EQQRI sites of pTZ]9R (United States Biochemicals). the g;aA using the Klenow fragment of Eicoli DNA polymerase I The protru ing 5’ end of Xhol site of the promoter fragment was filled in prior to cloning in pTZ19R. The Smal site is destroyed and the Xhol site is restored by this cloning procedure, The thus obtained plasmid was designated pGBGLA5 (Figure 13). was inserted into the HindIII and Xhoi sites of pGBGLA5u The thus obtained plasmid was designated pGBGLA26 (Figure 14).

Next, the 3.4 kb EcoRI fragment from pAB6—1 containing the remaining 3’ part of the g1aA promoter, the glaA coding sequence and part of the 3’ glgA non—coding sequence, was cloned into the EQQRI site of pGBGLA26. pGBGLA27 digested with EQQBI and the synthetic fragment consisting of (SEQ ID NO: 12) and AB3780 (SEQ This new plasmid was designated (Figure 15); This plasmid was partially the oligonucleotides AB3779 ID NO; 13): , , _ H-.. , z4._ ’ AATTGGGCCCCATTAACTCGAGC 3’ AB3779 lllllllllllllllllll Illllllllllllllllll ’ CCCCGGGTAATTGAGCTCGTTAA 5' AB378O was inserted into the Egggl site at the end of the 3’ glgA non—coding sequence from the g;gA gene. By this cloning step, the EQQRI site was destroyed and an Apgl and ghgl restriction site were introduced. The resultant plasmid was designated pGBGLA42 (Figure 16).

Amplification of the 2.2 kb 3' g1aA non-coding sequences and concomitant adjustment of appropriate restriction sites was performed by the Polymerase Chain Reaction (PCR) method.

In these PCR reactions, plasmid pAB6~l containing the entire g;aA locus was used as template and as primers four nucleotides were designed possessing the synthetic oligo following sequence: Oligo AB3449 (SEQ ID NO: 15); ’ ATGGTTCAAGAACTCGGTAGCCTTTTCCTTGATTCT 3’ (a 3’ g;gA non~coding specific sequence around the K nI site approx; 1 kb downstream of the stop codon) Oiigo AS3450 (SEQ in N0: M3): %’ AGAATCAAGGAAAAGGCTACCGAGTTCTTGAACCAT 3’ (a 3’ glaA non~coding specific sequence around the fipgl site approx. 1 kb downstream of the stop codon) Oligo AB352O (SEQ ID NO: 17): ’ATCAATCAGAAGCTTTCTCTCGAGACGGGCATCGGAGTCCCG 3’ (a 3' glaA non-coding specific sequence approx. 2.2 kb downstream of the stopcodon) To destroy the Kpnl site approximately 1 kb downstream of the stop codon from the glaA gene and to alter the Sall site approximately 2.2 kb downstream the stop codon from the glaA gene into a hoI site two separate polymerase chain reactions were performed: the first reaction with oligonucleotides AB3448 (SEQ ID NO: 14) and AB3449 (SEQ ID NO: 15) as primers to amplify an approximately 1 kb DNA fragment just downstream the stopcodon of the glaA gene, and the second reaction with oligonucleotides AB345O (SEQ ID NO: 16) and. AB352O (SEQ ID NO: 17) approximately 1.2 kb DNA fragment just downstream the Kpnl as primers to amplify an glaA non—coding region both with pAB6-1 as these site in the 3’ templateo A schematic View of amplifications is presented in Figure l7A0 The PCR was performed as described in example I; Twenty~five amplification cycles (each 1 minute .3 minutes at 72°C? and 1 minute at 94°C; were The two generated PCR DNA fragments were purified by and ethanol precipitation and third agarose gel electrophoresis template in the PCR with (SEQ ID NO: subsequently used as oligonucleotides AB3448 14) and AB332O {SEQ ID NO: 17) as primers to generate the fusion fragment. Twenty“ five amplification cycles (each: 2 minutes at 55“C: 3 minutes at ;2 S; 2 minutes at 34‘£; were ca“riedl mat in a gNA= amplifier (Perkin~Elmer/Cetus). The amplified DNA fragment was purified by agarose gel electrophoresis and ethanol precipitation and subsequently subcloned in the gmgl site of pTZ18R. The obtained plasmid was designated pGBGLAl7 (Figure 1?B}~ amgs/3’glaA non—coding fragment was isolated by agarose gel electrophoresis and subcloned into the appropriate sites of pSP73 The pGBGLA21 (Figure l8)n The approximately 1 kb sized 3’ glaA non—coding region (Promega). resulting plasmid was designated in this plasmid was exchanged by the 2.2 kb 3’ pGBGLAl7 pGBGLA21 glaA non- coding region of pGBGLAl7. and were digested with Kpgl and fiindlll. The 2.2 kb 3’ glgA nonwcoding region DNA fragment from pGBGLA17 and the 4.9 kb DNA fragment of pGBGLA21 were isolated by agarose gel electrophoresis, ligated and subsequently molecular cloned by transferring the ligation mixture to E. cmli. designated pGBGLA22 (Figure 19).

The with the The thus‘derived plasmid was amds gene extended 3’glaA non~coding region was completed with the gpgA promoter and fused to the remaining part of the amgs gene. pGBGLA22 was digested with Bglll and HindIII, the 4,4 Kb ands/3’glaA non-coding region DNA fragment isolated by agarose gel electrophoresis, subsequently ligated with plasmid pGBGLA24 digested with Bglll and HindIII and transferred to E. colil The thus pGBGLA25 was (Figure 20}. partially digested with ECORI and in the {Figure 21}. Due to this cloning step, the EQQRI restriction site just in front of the gpdA promoter was destroyed by the introduction of an gpgl restriction site.

The plasmid pGBGLA43 was digested with Apgl and Xhgl, and the 5.3 kb DNA fragment comprising the gpgA promoter/amgs region was gene/3’glaA non—coding isolated by agarose gel electrophoresis, subsequently ligated with pGBGLA42 digested with Apal and XhoI, and transferred to E.coli. The derived plasmid was designated pGBGLA28 (Figure 22).

Prior to cloning, the 3’glaA non-coding region DNA fragment (positioned at approximately 2.2 kb downstream the stop codon of the glaA gene, designated 3”glaA non—coding -I ~ _- ~37; region), was amplified and provided with suitable restriction sites using the PCR method.

For this PCR reaction, the plasmid pAB6-1 was used as template and as primers two synthetic oligonucleotides were designed possessing the following sequence: AB3746 (SEQ ID NO: 20): ' TGACCAATAAAGCTTCTCGAGTAGCAAGAAGACCCAGTCAATC 3' Oligo (a partly 3”glaA non—coding specific sequence around the gall site positioned at about 2.2 Kb downstream the stop codon of the glaA gene) AB3747 (SEQ ID NO: 21): ’ CTACAAACGGCCACGCTGGAGATCCGCCGGCGTTCGAAATAACCAGT3 ’ Oligo {a partly 3"glaA nonwcoding specific sequence around the XQQI site located at about 4.4 Kb downstream the stop codon of the glaA gene) (each: il minute 55”C: DNA= Twentywfive amplification cycles ,5 minutes 72“C: 1 minute 94°C) were carried out in a amplifier (Perkin~Elmer/Cetus). A schematic representation of this amplification is shown in figure 23A. The thus obtained DNA fragment was digested with fiindlll, purified by agarose gel electrophoresis and ethanol precipitation and subcloned flindlll site <3f pTZl9R. The resulting plasmids were designated pGBGLA29A and pGBGLA29B in both orientations into the (Figure 23). non~coding sequenoe from pGBGlA29A into the plasmid pGBsLA28.

To achieve this, pGBGLA29A was digested with. gindlll and NotI. The 2.2 kb sized 3’glaA non—coding region fragment was isolated by agarose gel electrophoresis, subsequently ligated to pGBGLA28 digested with HindIII and NotI and transferred to pGBGLA3O (Figure 24)‘ Transformation of agniger GBA—107 with the integration vector EQBQLAJQ — $3- Prior to transformation, E.coli sequences were removed from the integration vector pGBGLA3O by XhoI digestion and agarose gel electrophoresis. The A.niger strain GBA~l07 was transformed with either 5 or 10 ug DNA fragment by procedures as described in the experimental section. Single A.niger transformants were purified several times on selective acetamide containing plates. Spores of individual transformants were collected following growth for about 5 days at 30°C on 0.4% potato dextrose agar (Oxoid, England) plates, Southern analyses were performed to verify whether integration into the 3’ glaA non coding region of the endogenous truncated glaA locus had occurred. High molecular weight DNA of several transformants was isolated, digested with either Kpnl, or Bglll and subsequently fractionated by electrophoresis on a O,7% agarose gel. After transfer to nitrocellulose filters, hybridization was performed according to standard procedures. As probe a 3%%labelled approx. 0.7 kb Xhol/gall glaA promoter fragment isolated from plasmid pAB6—4 (described in example 1) (#lO7~5, #107»? and #lO7w7) and the reference was used. The results of only 3 transformants strain g.nigeg GBAlO7 and its ancestor ALgiger Q83 ‘3l.88 are shown as example in Figure 25. For a better understanding of the Figure 26A,B,C showing the sizes of the hybridizing fragments autoradiograph, a schematic presentation is given in of the intact glaA locus, the truncated glaA locus and of the truncated glaA locus with a single pGBGLA3O copy integrated into the predefined 3’ olah non~coding region.

Characteristic for the intact glaA locus is a 4.5 kb hybridizing fragment in a Kpnl digest and a 10 kb hybridizing Characteristic for the truncated .4 kb fragment in a BglII digest.

A.niger GBA—lO7 is a a Kpnl digest and a 13 kb hybridizing fragment in glaA locus of hybridizing fragment in a figlll digest. In case of integration of the pGBGLA3O vector into the 3’ region of the truncated glaA locus, in a Kpnl digest an additional 6.7 kb hybridizing fragment is expected besides the 3.4 kb hybridizing fragment and. in a figlll digest the 13 kb hybridizing fragment is absent and ?- replaced by a l4¢5 kb hybridizing fragment. As can be seen in Figure 25, transformants #107—5 and #107-9 show the expected hybridization pattern of 21 single pGBGLA3O copy integrated into the predefined 3’ non-coding’ region of the truncated glgA locus. The hybridization pattern of transformant #107-7 indicates integration of the pGBGLA3O copy elsewhere into the A.niger GBA-107. The with the correctly integrated pGBGLA3O copy’ were designated. GBA~ll9 and GBAwl22 and were used to genome of transformants remove subsequently the amds selection marker gene properly.

Removal of the amds selection marker gene from A niger GBA= GBA-l22 by and counter~selection on fluoracetamide The amds selection marker gene in the transformants gnniger GBA~ll9 and GBA~l22 was removed again as described in the experimental sectionw The removal of the amdS selection marker gene in several surviving recombinant strains was verified by Southern analysis of the chromosomal DNA. High molecular weight DNA was isolated, digested either with Kbnl or Bglll .7% and subsequently separated by electrophoresis on a agarose gel. Following transfer to nitrocellulose, hybridization was performed according to standard procedures.

As probe the ”P labelled 2.2 kb }UndIII/Notl 3”glaA non“ coding fragment isolated from plasmid pGBGLA29A (described previously, Figure 24) was used.

A schematic presentation of the hybridizing fragments is shown in Figure 26. The results of only 3 surviving recombinant strains from A.niger GBA—ll9 (#AG5—5, #AG5—6 and surviving recombinant strains from #AG9—2 and #AG9-4) #AG5—7) as well as 3 A.niger GBA-122 (#AG9-l, and the reference strains A.niger CBS 531.88 and A.niger GBA—lO7 are shown in Figure 27A,B. lu88 a 6.9 kb A.niger CBS hybridizing fragment is present in a Kpnl digest and a 6.9 kb hybridizing In strain fragment in a Bglll digest. In the A.niger GBA—107 strain a .9 kb hybridizing fragment is present in a Kpnl digest and a ,_4,,_, _ , ~40 Q kb hybridizing fragment in a BglII digest.

GBA-119 and GBA~122 In the A.niger single pGBGLA3O copy glaA non-coding region an 8 kb and a of the amgs selection 6.7 kb 8.5 kb hybridizing fragment in a gpnl digest and concomitant loss of .5 kb Specific for correct removal the of a and a marker gene is presence the 8 kb hybridizing fragment‘ In a gglll digest, a and a 6.9 kb hybridizing fragment with concomitant loss or the of the gmgs selection marker gene.

#AG5~7, #Ae5-5, #AG9~l .6 kb hybridizing fragment is specific for the absence As can be seen in Figure and #AG9—4 the ‘.7’ 3. strains ghow expected hybridizing pattern of the correctly removed eggs selection marker gene. These strains were designated GBAwl20, GBA=12l. GBA~l23 and GBAwl24 #AG5w6 and #AG9~2 respectively» The hybridizing patterns of strains indicate loss of the entire pGBGLA3O copy resulting in the parental A.niger GBAW strain with only a truncated glaA locus. were tested in shake flask fermentations for the ability to produce glucoamylase. As reference strains A.niger CBS 531.88, GBA«lO7, GBA~1l9 and GBA~122 were tested; Shake flask Eermentations and the glucoamylase assay were performed as In the strains GBA«al9 l50"2OO described in the experimental section. till GBA~l24 levels varying‘ between U/ml could be measured. These glucoamylase levels were to be expected and comparable to levels obtained with the parental untransformed wild-type strain A.niger CBS 531.88.

Example 3 Marker gene free introduction of the phytase gene targeted at the 3’glaA non~coding region of the truncated glaA locus in A.niger GBA—l K) ”-~41 - In this example describes the introduction of a gene into the genome of A.niger by using approximately the same approach and procedures as described in the previous example.

The main difference is that the gene of interest and the selection marker gene are located on two'separate vectors and that these vectors are co-transformed to A.niger. Besides the gene of interest or the marker gene, the vectors contain DNA sequences homologous to the host genome to target the Vectors at a predefined genomic locus of the host, by a single cross— over event. By performing the fluoracetamide counter- selection CH1 these (co)=transformants (as described in the will be internal recombination event between experimental procedures), amdS marker gene deleted properly by an the DNA repeats that are created by integration via a single crosswover event.

Description of the vectors used for co—transformation The vector with the gene of interest pGBGLA53 consists of the A.ficuum phytase gene under control of the A.niger glucoamylase (glaA) promoter flanked by 3’glaA nonwcoding sequences to direct integration at the 3’glaR nonwcoding region. The vector with the selection marker gene pGBGLA50 consists of the A.nidulans amds gene under control of the A.nidulans gpgA promoter flanked by 3’glaA non-coding sequences to direct integration at the 3’glaA nonacoding regiong Construction pathway of pGBGLA5Q The of pGBGLA5O Plasmid. pGBGLA29A was digested. with fiindlll and the filled Next, construction comprises one cloning step. sticky ends were in using the Klenow fragment of E.coli DNA polymerase. the 2.2 kb 3”glaA non-coding region fragment was isolated by agarose gel—electrophoresis, subsequently ligated into pGBGLA43 digested with Apal and treated with T4 DNA polymerase to generate blunt ends, and transferred to E.coli. The derived plasmid with the 3”glaA —42;i non—coding region DNA fragment in the correct orientation was designated pGBGLA5O (Figure 28).

Construction pathway of pGBGLA53 The first step in the construction pathway of pGBGLA53 is the subcloning of two fragments, comprising’ the g1aA promoter fused to almost entire coding sequence of the A,ficuum phytase gene; To achieve this, plasmid pGBGLA42 was digested with flindlll and ECQRI and the 1.8 kb HindIII/EcoRI isolated by agarose gel~electrophoresis and ligated together with the 1,8 kb fligdlll/EQQRI 5’glgA promoter fragment isolated from pGBGLA42 into the fiindlll and Eglll sites of pSp73 (Promega)» The resulting plasmid was designated pGBGLA49 (Figure 29).

The next step is the cloning of a 3’glaA nonwcoding region DNA fragment. intc‘ pGBGLA49¢ Prior‘ to cloning, this 3’qlaA non~coding region DNA fragment (positioned at approximately 2.2 kb downstream the stop codon of the glaA gene) was amplified and provided with suitable restriction sites using the PCR methods For this PCR reaction, the plasmid pAB6~1 was used as template and as primers two synthetic oliqonucleotides wit the following sequence were designed: Oligo AB4234 (SEQ ID NO: 22): ’ GAAGACCCAGTCAAGCTTGCATGAGC 3’ (a 3’glgA non-coding sequence located approximately 292 kb downstream the stopcodon of the glaA gene) Oligo AB 4235 (SEQ ID NO: 23): ’TGACCAATTAAGCTTGCGGCCGCTCGAGGTCGCACCGGCAAAC 3' (a 3’glgA non—coding sequence located approximately 4.4 kb downstream the stopcodon of the glaA gene) (K) L7’ — 43- Twenty—five amplification cycles (each: 1 minute 94°C: minute 55°C: 1.5 minutes 72°C) were carried out in a DNA~ amplifier (Perkin—Elmer). A schematic representation of this amplification‘ is shown in figure 30A. The thus obtained fragment was digested with fiindlll, purified by agarose gel~ the fligdlll pTZl9R. The resulting plasmid was designated pGBGLA47 (Figure electrophoresis and subcloned into site of the isolated by Plasmid pGBGLA47 was digested with HindIII en Notl, 2:2 Kb 3”glaA agarose gel—electrophoresis and cloned into the fligdlll and non—coding DNA fragment was Notl sites of pGBGLA49. The resulting plasmid was designated pGBGLA5l (Figure 31)" The last step in the construction pathway of pGBGLA:5 is the cloning of the DNA fragment comprising the remaining part of the phytase coding sequence fused to the 3’glaA non" coding DNA fragment located just downstream the stop codon of the remaining part of the the glaA gene. Prior to cloning, phytase gene and the 3’glaA non~coding DNA fragment located just downstream the stopcodon of the glgA gene were fused and provided with suitable restriction sites using the FTP method; In the PCR, plasmid pAB6 l was used as template and as primers two synthetic oligonucleotides were used, having the following sequences: Oligo AS4236 (SEQ ID NO: 24): ’ TGACCAATAAAGCTTAGATCTGGGGGTGATTGGGCGGAGTGTTTTGFT? AGACAATCAATCCATTTCGC 3’ (36 bp of the phytase coding sequence, starting at the figlll site until the stopcodon fused to the 3’glaA non~ coding region, starting just downstream the stopcodon of the glaA gene) AB4233 (SEQ ID NO: 25): ’ TGACCAATAGATCTAAGCTTGACTGGGTCTTCTTGC 3’ Oligo (a 3’glaA non—coding sequence located approximately 2&2 kb downstream the stopcodon of the glaA gene) _ ii _.

Twenty-five amplification cycles (each: 1 minute 94°C; minute 55°C; 1.5 minutes 72°C) were carried out in a DNAw amplifier (Perkin-Elmer). A schematic representation of this The thus fragment was digested with HindIII, purified by agarose gelw amplification‘ is shown in figure 32A. obtained electrophoresis and subcloned in both orientations into the HindIII of pTZl9R. The designated pGBGLA48 and pGBGLA52 resulting (figure 32B).

Plasmid pGBGLA52 was digested with Bglll and partially site plasmids were digested with ggmfil, the 2H2 kb phytase/3’glgA non—coding DNA fragment was isolated by agarose gel-electrophoresis and l pg pGBGLA50 digestion agarose gelwelectrophoresisi M? pGBGLA5O fragment plus 1 pg pGBGLA53 fragment, fragment plus 5 pg pGBGLA53 fragment, or 1 pg pGBGLA5O fragment plus 10 pg pGBGLA53 fragment using the transformation procedure described in the experimental section» Single transformants were isolated, purified and Southern analysis was performed, using the same digests and probes as described in example 2, both pGBGLA5O and pGBGLA53. In about 10-20% transformants both pGBGLA5O and pGBGLA53 were integrated into the of the Agniger GBA~lO7 host The transformant showing the correct copy pGBGLA5O to verify integration of of the analyzed genome strain. integration pattern of a single and a single copy pGBGLA53, both integrated at the predefined 3’glgA non—coding region of the truncated glgA locus was used to remove subsequently the gmgs selection marker gene.

~ Q3~ Removal of the amdS marker gene by Counter-selection on fluoracetamide containing plates By performing the fluoracetamide counter—selection (as described in the experimental procedures), the amds marker gene was deleted by an internal recombination event between the DNA repeats that were created by integration via a single cross—over event (i.e. the 3’glaA non-coding sequences).

Proper removal of only the amds marker gene was verified by Southern analysis using the same digests and probes as in example 2.

Marker gene free introduction of the glaA gene and the phytase gene in A.oryzae This example describes the marker gene free introduction of the glaA gene or the phytase gene in A.oryzae NRRL3485w A.oryzae NRRL3485 was transformed as described in the experimental section using the same vectors and approach as described in examples 2 and 3. Single transformants were isolated, purified and Southern analysis of chromosomal DNA of several transformants was performed to verify integrations of respectively the pGBGLA30 vector or the pGBGLA5O and pGBGlA53 vectors. In the Southern analysis, the same digests and probes were used as described in example 2.

Removal of the amds gene by counter-selection _ on fluoracetamide containing plates In case of integration of the pGBGIA30 vector, a transformant with a single copy of the pGBGLA3O integrated into the genome of the host strain A.oryzae NRRL3485 was used to remove the amgs gene properly. The counterwselection on fluoracetamide containing plates was performed as described in the experimental section. Correct removal of the amgs gene was verified by Southern analysis of Chromosomal DNA of IQ VI‘ several fluoracetamide resistant strains. The same digests and probes were used as described in Example 2. the pGBGLA5O pGBGLA53 vector, a transformant with a single copy of both pGBGLA5O and pGBGLA53 In case of co—transformation of and integrated into the host genome was used to remove the amds marker gene properly. The counter- selection using fluoracetamide plates was performed as described in the experimental section. Correct removal of the the pGBGLA5O vector) was verified by DNA of amds marker gene (e.g.

Southern analysis of chromosomal several fluoracetamide resistant strains using the same digests and probes as described in example 2.

This example describes the marker gene free introduction of the glaa gene or the pbytase gene in Trichoderma reesei strain QM94l4 (ATCC 2692l}9 Egreesei QM94l4 was transformed as described in the experimental section using the same vectors and approach as described in examples 2 and 3. Single transformants were isolated; purified and Southern analysis of chromosomal DNA of several transformants was performed to verify whether integration of respectively the pGBGLA3O vector or the pGBGLA5O and pGBGLA53 vectors. In the Southern analysis, the same digests and probes were used as described in example 2.

Removal of the amds qene by counter—selection on fluoracetamide containing plates In case of integration of the pGBGLA3O vector, a transformant with a single Copy of the pGBGLA3O integrated into the genome of the host strain T.reesei QM94l4 was used to remove the amdS gene properly. The counter—selection on - 47 — fluoracetamide containing plates was performed as described in the experimental section. Correct removal of the gmgs gene was verified by Southern analysis of chromosomal DNA of several fluoracetamide resistant strains. co-transformation of the pGBGLA50 a transformant with a single copy of both In case of pGBGLA53 Vector, pGBGLA5O and pGBGLA53 used to remove the amds marker gene properly. integrated into the host genome was The counter~ selection using fluoracetamide plates was performed as described in the experimental section. Correct removal of the the pGBGLASO vector) was verified by DNA of amgS marker gene (e.g.

Southern analysis on chromosomal several fluoracetamide resistant strains using the same digests and described in example 2.

Examol Q Marker gene free introduction into P.chrysoqenum of a P.chrvsoqenum gene by coetransformation using the amdS—qene as a selection marker Tn this example the marker gene free introducticn Cf 3 gene into the genome of P.chrysogenum by cowtransformatiom is described.

In the cowtransformation procedure, 2 different pieces of DNA are offered to the protoplasts, one of them being the amgsuselection marker: on the presence of which, the 111st transformant selection takes place, as described in the experimental section, the second being another piece of DNA of interest, encoding a particular enzyme of interest. e.g.

In a certain number of transformants both pieces of DNA will integrate into the chromosomes and will be stably maintained and expressed.

The removed can then be by applying counter—selection procedure as described in the experimental amdS—selection marker gene selectively from purified transformants section, while the second piece of DNA will remain stably integrated into the chromosomes of the transformant.

D‘: KM _ 48 _ As an example to illustrate the general applicability of the method the stable, marker gene free introduction of a niaD- gene is described. which enables a niaD'~host to grow on nitrate as sole nitrogen-source.

Host for this co-transformation ‘is a P.ch;ysogenum giafiestrain which lacks nitrate reductase and therefore is unable to grow on plates containing nitrate as sole nitrogen These strains can be easily obtained by well known (Gouka et al., source. procedures Journal of Biotechnology ;Q(l991), 189-200 and references there in) During the co-transformation (procedure described in two pieces of DNA are simultaneously the 7.6 kb ECORI experimental section), offered to restriction from pGBGiA28 the protoplasts: fragment containing the ands selection marker gene and the 6&5 kb ECORI restriction fragment from pPC1»l, E,coli vector sequences by agarose gelselectrophoresis an purified from agarose gel by electro~elutionu The first selection of transformants took place on selective plates centaining acetamide as sole nitrogen source as described in the experimental section.

Among the transformants, co~transformants are found by replica plating spores of purified transformants to plates containing nitrate as sole nitrogen source.

Typically about 20—60% of the replica plated transformants were able to grow on this medium, indicating that in these transformants not only the amds selection marker gene but also the niaD—gene has integrated into the genome and is expressed.

Removal of the amds qene by counter~selection on fluoracetamide containing plates The amdS selection marker gene is subsequently removed from the co—transformants by counter~selection on fluor- acetamide. '\4 (I‘ — 49 ~ For direct selection on the amgS7niaDV1menotype the medium used contained 10 mM fluor—acetamide.

Spores were plated at a density of 104 spores per plate.

After 5-7 days of incubation at 25°C, fluor—acetamide solid colonies The phenotype of the recombinants is demonstrated by their growth resistant colonies could be identifiedi as clearly distinct from the faint background. niaD“— on the fluoracetamide—medium containing nitrate as sole DNA form confirmed that the several amds Southern analysis on chromosomal fluoracetamide resistant strains selection marker gene was removed from the P.chrysogenum genomea .4; I T1 ¥\‘ (IW SEQUENCE LISTINO.,", (1) GENERAL INFORMATION: (2) INFORMATION FOR SEQ ID NO: 1: (1 SEQUENCE CHARACTERISTICS? (A1 LENGTH: 26 base paxrs :8) TYPE: nucleic acid (C; STRANDEDNESS: single (D) TOPOLOGY: linear /wwv MOVWCULE TYPE: DNA lqennmjcn {" ) HYPOTHETICAL: NO (ii: ANTI-SENSE: NO ‘w , .EM_‘r*iEDIATE SOURCE; .B) CLONE: ABEICU Xx} SEQUENCE DESCRIPTION: SEQ ID NO: CTAATCTAGA ATGCCTCAAT CCTGAA {2} INFORMATION FOR SEQ ID NO; 2; (ii) (Xi) APPLICANT: (A) NAME: Gist—brocades B.V.

(B) STREET: Wateringseweg 1 (C) CITY: Delft (E) COUNTRY: The Netherlands (F) POSTAL CODE (ZIP): 2611 XT TITLE OF INVENTION: Strains: Strains NUMBER OF SEQUENCES: 37 COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (8) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: :9; SOFTWARE; SFQUENCE CHARACTERISTICS: (A) LENGTH: 28 base palre (B) TYPE: nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear MOLECULE TYPE: DNA (genomic) HYPOTHETICAL: NO ANTI~SENSE: YES IMMEDIATE SOURCE: (B) CLONE: AB3101 SEQUENCE DESCRIPTION: GACAGTCGAC AGCTATGGAG TCACCACA SEQ ID NO: PC-DOS/MS-DOS Patentln Release #1.0, Version #l.25 Selection Marker Gene Free Recombinant a Method for Obtaining Them and the Use of These (EPO) Pu 0\ CD (2) INFORMATION FOR SEQ ID NO: 3: " (ii) (111) (iii) (vii) (Xi) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) HYPOTHETICAL: NO ANTI-SENSE: NO IMMEDIATE SOURCE: (B) CLONE: TNOOOI SEQUENCE DESCRIPTION: SEQ ID NO: 3: TCGATTAACT AGTTAA (2) INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs {8} TYPE: nucleic acid {C} STRANDEDNESS: single (D) TOPOLQGY: linear MOLECULE TYPE: DNA (genomic) HYPOTHETICAL' NO IMMEDIATE SOURCE: (B) CLONE: AS2154 SEQUENCE DESCRIPTION: SEQ ID NC: 4: .cGC TCCAQVAGPK 7iTCAATFCF TTTCV (2) INFORMATION FOR SEQ ID NO: 5: (Xi) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base paLr3 (8) TYPE: nucleLc acLd (C) JTRANDEDNESS; Single (D) TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) HYPOTHETICAL: NO ANTI-SENSE: YES IMMEDIATE SOURCE: (B) CLONE: AB21S5 SEQUENCE DESCRIPTION: SEQ ID NO: 5: GCTATTCGAA AGCTTATTCA TCCGGAGATC CTGAT L...

I'\.‘ 0 DJ —_-—\ .,,,, (2) INFORMATION FOR SEQ ID NO: 9: (ii) (iii) (iii) (vii) (Xi) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) HYPOTHETICAL: NO ANTI~SENSE: YES IMMEDIATE SOURCE: (B) CLONE: AB2994 SEQUENCE DESCRIPTION: SEQ ID NO: CTGATAGAAT TCAGATCTGC AGCGGAGGCC TCTGTG (2) INFORMATION FOR SEQ ID NO: 10: (ii) (iii; (Iii) {vii} (xi) UENCE CHARACTERISTICS: ) LENGTH: 31 base pairs , TYPE: nucleic acid ) STRANDEDNESS: single X TOPOLOCY: linear Q A B C SE ( MOLECULE TYPE: DNA (genomic) IMMEDIATE SOURCE: (B) CLONE: AB3657 SEQUENCE DESCRIPTION: SEQ ID NO: AGCTTGACGT CTACGTATTA ATGCGGCCGC T (2) INFORMATION FOR SEQ ID NO: 11: (ii) (iii) (:11) (vii) (Xi) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base paizs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) HYPOTHETICAL: NO ANTI~SENSE: YES IMMEDIATE SOURCE: (B) CLONE: AB3658 SEQUENCE DESCRIPTION: SEQ ID NO: TCGAAGCGGC CGCATTAATA CGTAGACGTC A ,i C} a ..

(M O\ LI 1 Na (J7 wk‘, (2) INFORMATION FOR SEQ ID N6: 12: (ii) (:11) (iii) (Vii) (Xi) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) HYPOTHETICAL: NO ANTI—SENSE: NO IMMEDIATE SOURCE: (B) CLONE: AB3779 SEQUENCE DESCRIPTION: SEQ ID NO: 12: AATTGGGGCC CATTAACTCG AGC (2) INFORMATION FOR SEQ ID NO: 13: QUENCE CHARACTERISTICS: A) LENGTH: 23 base pairs 8} TYPE: nucleic acid {C; STRANDEDNESS: Singie (D; TOPOLOGY: linear MOLECULE TYPE: DNA (gencmic) HTPOTHETICAL; ND ANTI~SENSE: YES IMMEDIATE SOURCE: (B) CLONE: AB378O sLQUENLa DLSCKIPTION: bug ID NU; Lg; AATTGCTCGA GTTAATGGGC CCC (2% INFORMATION FOR SEQ ID NO; 14. (ii) (:11) (:11) (vii) (Xi) SEQUENCE CHARACTERISTICS: {A} LENGTH: 30 base paxrs (8) TYPE: nucleic acid \C) STRANDEDNESS: sIng1e (D) TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) HYPOTHETICAL: NO ANTI—SENSE: NO IMMEDIATE SOURCE: (B) CLONE: AB3448 SEQUENCE DESCRIPTION: SEQ ID NO: 14: GTGCGAGGTA CCACAATCAA TCCATTTCGC i\) ( L} F 4; LT’ (ii) (iii) (1:1) (vii) (xi) (2) INFORMATION FOR SEQ ID NO: 15: SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) HYPOTHETICAL: NO ANTI-SENSE: YES IMMEDIATE SOURCE: (B) CLONE: AB3449 SEQUENCE DESCRIPTION: SEQ ID NO: 15: ATGGTTCAAG AACTCGGTAG CCTTTTCCTT GATTCT (2) INFORMATION FOR SEQ ID NO: I6: axi) P) LENGTH: 36 base pairs (5) TYPE: nuCl:'c =”‘H STRANDEDNESQ: single TOPOLOCY: linear MOLECULE TYPE: DNA (genomic) HYFOTHEIICRL: NW ANTI~SENSE: NO IMMEDIATE SOURCE: (B) CLONE: AB3450 SEQUENCE DESCRIFTION: SEQ ID NO: 15: AGAATCAAGG AAAAGGCTAC CGAGTTCTTG AACCAT (2) INFORMATION FOR SEQ ID NO: I?‘ (ii) (iii) (:11) (vii) (Xi) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: SLRQLB (D) TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) HYPOTHETICAL: NO ANTI—SENSE: YES IMMEDIATE SOURCE: (B) CLONE: AB3S2O SEQUENCE DESCRIPTION: SEQ ID NO: 17: ATCAATCAGA AGCTTTCTCT CGAGACGGCC ATCGGAGTCC CG (.17 N; Q)“. l’\) ‘C, (2) INFORMATION FOR SEQ ID NO: 18: (ii) (iii) (:11) (vii) (Xi) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear MOLECULE TYPE} DNA (genomic) HYPOTHETICAL: NO ANTI—SENSE: NO IMMEDIATE SOURCE: (B) CLONE: AB378l SEQUENCE DESCRIPTION: SEQ ID NO; AATTGGGGCC CAGCGTCC (2) INFORMATION FOR SEQ ID NO: 19: (XL) SEQUENCE CHARACTERISTICS; (A) LENGTH: 18 base pairs (8; TYPE: nucleic acid {C} STRANDEDNESS: Single (DE TOPOLOGY: linear MOLECULE TYPE; DNA (gEflCmLC) HYPOIHETlCALt NO RNT1~SENSE: YES IMMEDIRTE SOURCE: (B) CLONE: E83782 SEQUENCE DESCRIPTION: SEQ ID NO: AATTGGACGC TGGGCCCC (2) INFORMATION FOR SEQ ID NO; 25‘ (Xi) UENCE CHARACTERISTICS: ) LENGTH: 43 base pairs ) TYPE: nucleLc acid ) STRANDEDNESS: single GE ( ( ( ( TOPOLOGY: linear Q A B C D MOLECULE TYPE: DNA (genomic) HYPOTHETICAL: NO ANTI-SENSE: NO IMMEDIATE SOURCE: (B) CLONE: AB3746 SEQUENCE DESCRIPTION: SEQ ID NO: TGACCAATAA AGCTTCTCGA GTAGCAAGAA GACCCAGTCA ATC (_/L) UT (2) INFORMATION FOR SEQ ID NO{”2I: (ii) (iii) (iii) (vii) (Xi) SEQUENCE CHARACTERISTICS: (A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) HYPOTHETICAL: NO ANTI—SENSE: YES IMMEDIATE SOURCE: (B) CLONE: AB3747 SEQUENCE DESCRIPTION: SEQ ID NO: 21: CTACAAACGG CCACGCTGGA GATCCGCCGG CGTTCGAAAT AACCAGT (2) INFORMATION FOR SEQ ID NO: 22: (viii (Xi) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs {8} TYPE: nucleic acld (C) STRANDEDNESS; single (D) TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) IMMEDIATE SOURCE: (B) CLONE: AB4234 SEQUENCE DESCRIPTION: SEQ ID NO: J2: GAAGACCCAG TCAAGCTTGC ATGAGC (ii) (111) (1:1) (vii) (Xi) (L) INFORMATION FOR SEQ ID NO: 23; SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) HYPOTHETICAL: NO ANTI—SENSE: YES IMMEDIATE SOURCE: (B) CLONE; AB4235 SEQUENCE DESCRIPTION: SEQ ID NO: 23: TGACCAATTA AGCTTGCGGC CGCTCGAGGT CGCACCGGCA AAC I) O\ (i) lN%ORMATlON FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (ii) (iii) (iii) (vii) (Xi) (A) LENGTH: 69 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear MOLECULE TYRE: DNA (genomic) HYPOTHETICAL: NO ANTI~SENSE: NO IMMEDIATE SOURCE: (B) CLONE: AS4236 SEQUENCE DESCRIPTION: SEQ ID NO: 24: TGACCAATAA AGCTTAGATC TGGGGGTGAT TGGGCGGAGT GTTTTGCTTA GACAATCAAT CCATTTCGC (2) INFORMATION FOR SEQ ID NO: 25: «Xi; SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base paire (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear MOLECULE TYPE: DNA (genomic; HYPOTHETlCA,: NO ANTI-SENSE: YES IMMED IATE SOURCE: .~ .a '3) CLONE’ ::\:;i&;’i.4 SEQUENCE DESCRIPTTON: SEQ ID NO: 25: TGACCAATAG ATCTAAGCTT GACTGGGTCT TCTTGC (2) lNFO"MATION FOR SEQ ID NO: 26: (ii) (iii) (iii) (vii) SEQUENCE CHARACTERISTICS: (A) LENGTH; 32 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) HYPOTHETICAL: NO ANTl—SENSE: NO IMMEDIATE SOURCE: (B) CLONE: AS35 (xi)~SEQUENCEkbESCRIPTIONa SEQ ID NO; 26: CTGCGAATTC GTCGACATGC CTCAATCCTG GG (2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: YES (Vii) IMMEDIATE SOURCE: (B) CLONE: AB3SlS (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: GGCAGTCTAG AGTCGACCTA TGGAGTCACC ACATTTC (2: INFORMATION FOR SEQ ID NO: 28: ;i) SEQUENCE CHARACTERISTICS; (A) LENGTH: 50 base pairs (8) TYPE: nucleic acid {C} STRANDEDNESS; Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO ~: (vii) IMMEDIATE SOURCE: (B) CLONE: AB370l CTGCGAATTC GTCGACACTR GTGGTRCCAT TATAGCCATA GGACAGCAAG {3} INFORMATION FOR s:Q iD NO: 29; (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 70 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) OJ UT (iii) HYPOTHETICAL: NO (ii ) ANTI—SENSE: YES (vii) IMMEDIATE SOURCE: (B) CLONE: AB37OO (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: GCTCTAGAGC GCGCTTATCA GCTTCCAGTT CTTCCCAGGA TTGAGGCATT TTTAATGTTA CTTCTCTTGC (2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base pairs ' (B) TYPE: nucleic acid (C) STRRNDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ll) MOLECULE TYPE: DNA (genomic) (:11) HYPOTHETICAL: NO (iii) (vii) (X1) ANTI-SENSE: NO IMMEDIATE SOURCE: (B) CLONE: AB3704 SEQUENCE DESCRIPTION: SEQ ID NO: 32: GCTCTAGAAG TCGACACTAGVTCTGCTACGT ACTCGAGAAT TTATACTTAG ATAAG (2) INFORMATION FOR SEQ ID NO: 33: SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) HYPOTHETICAL: NO ANTI-SENSE: YES IMMEDIATE SOURCE: (B) CLONE: AB37OS SEQUENCE DESCRIPTION: SEQ ID NO: 33: TGCTCTAGAI CTCAAGCCAC AATTC (2) INFORMATION FOR SEQ ID NO: 34: (Xi) SEQUENCE CHARACTERISTICS; (A) LENGTH: 31 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single '0) TOPOLOGY; linear MOLECULE TYPE: DNA (genomic) HYPOTHETICAL: NO ANTISSENSE: NO IMMEDIATE SOURCE: (5) LLONE; AB3965 SEQUENCE DESCRIPTION: SEQ ID NO: 34: CTGCTACGTA ATGTTTTCAT TGCTGTTTTA C (2) INFORMATION FOR SEQ ID NO: 35: (ii) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) '4 w (iii) (iii) (vii) (Xi) HYPOTHETICXE:”NO ANTI—SENSE: YES IMMEDIATE SOURCE: (B) CLONE: AB3966 SEQUENCE DESCRIPTION: SEQ ID NO: CCGCCCAGTC TCGAGTCAGA TGGCTTTGGC CAGCCCC (2) INFORMATION FOR SEQ ID NO: 36: (ii) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) HYPOTHETICAL; NO ANTI—SENSE: NO IMMEDIATE SOURCE: (B) CLONE: AB3825 (X1) SEQUENCE DESCRIPTION: SEQ ID NO: 36: CGCGCTTATC AGCGGCCAGT TCTTCCCAGG RTTCAGGCAT ATGT (2) INFORMATION FOR SEQ ID NO; 37: SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base pairs {B} TYPE: nucleic acid {C) SIRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (1:1) HYPOTHETICAL: NO (iii: ANTI—SENSE; YES (vii) IMMEDIATE SOURCE: {B} CLONE: AB3826 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: CTAGACATAT GCCTCAATCC TGGGAAGAAC TGGCCGCTGA TAAG .3; ;»

Claims (1)

1. CLAIMS A vector containing a dominant, bidirectional and counter-selectable marker gene which is flanked by direct DNA repeats, which allow for internal recombination in the genome of a filamentous fungus. A vector according to claim 1, wherein the direct DNA repeats are chosen from the 5’ or 3' of the DNA sequence to be deleted from the genome. A vector according to claim 1 or 2, wherein the selection marker gene is an acetamidase gene. A vector according to ciairn 3, wherein the acetamidase gene is of fungal origin, A vector according to claim 4, wherein the acetamidase gene is from an Aspergi/Ius species. A vector according to any one claim i to 5, wherein the vector also contgains ciéesired Bite; tragntent. A vector according to ciaim wherein the desired DNA tragrnent contains a gerietit: elements selected from the group consisting of: a gene, a CDNA, prornoter, a terminator, a regulatory eiement, an intron, a recognition sequence for a E'2i\.tA~~ binding protein, a translation—initiation site, a restriction site and combinations thereof according to claim wherein the desired DNA fragrnent «:;orit:;iir2s sequence encoding a chyrnosin, a phytase, a xyianase, a izpase, amylase, as protease, or a Bgalactosidase, A recornbinant filamentous. fungus transformed with the vector according to any one of claim 1 to 8 A :‘eccrn‘oiriant i(iiéai7"lF:i'ii€.‘»Li$ iurigus: at.;<;oi“ding to ;;iairn if'.;Qi’l'1pl'iSi§‘ifi at in,-.»». A recombinant filamentous fungus according to claim 9 or it), wherein the vector is integrated into the genome of said filamentous fungus through site-specific =»:>t;:;la:l2r= ts. homologous recombination. A recombinant filamentous fungus according to any one of claim 9 to it, wherein the filamentous fungus is an Aspergil/us, Trichoderma, or Penicillium species. A method for obtaining a selection marker gene free recombinant filamentous fungus comprising the following steps: (3) integration into the genome of the filamentous fungus a desired DNA fragment and a dominant and bidirectional selection marker gene, (b) selection of the transformants, (C) deletion of the selection marker gene by recombination between repeats flanking the selection marker gene, and d) counter-selection based on the absence of the selection marker gene. A method according to claim 13, characterized in that 5‘ or 3’ of the selection in-arl=<.er gene a sequence is cloned which forms a repeat with a sequence wrrich is: 3’ or 5' ofthe sequence to be deleted from the genome. is method according to claim 13 or 14, wherein the desired DNA fragment contains “ cen.e‘:i: zgiesnehte irr;.:.=‘:*' *:"‘:e group firtzesisting a gene 27;: ;i “remotes; a terminator‘, a regtilatczry element, an intron, a recograition sediieltoe tot a t3l\tA»biriding protein, a translatiominitiatiori site, a restriction site and cornbinatioris therer;n;’. fa rrzetisoo according ix; ears; on: ’<.,idllTi I: if-;.~ ‘;‘v§‘l‘;'£'€ilé 4,; :,.i,- repeated on the reccrnbinant filamentous fungus ot:>tained7 using either the same a different desired DNA fragment. A method according to any one or clairrzs ii» to wherein the selection rtieizkéés‘ is an eczetarriidase gene A method according to claim ‘l7, wherein the selection marker gene is an acetamidase gene is of fungal origin A method according to claim 18, wherein the selection marker gene is an acetamidase gene from an Aspergillus species. A method for the production of a bio-active compound, which method cornpnses the step of culturing a filamentous fungus produced according to the method of any one of claims 13 to 19. A method according to claim 20, wherein the bio—active compound is a protein. A method according to claim 20, wherein the bio—active compound is an antibiotic. Use of a filamentous fungus produced by the method of any one of claims 13 to 19 for the production of a bio-active compound. Use according to claim 23, wherein the bio—active compound is a protein. Use according to claim 23, wherein the bio-active compound is an antibiotic. A Vector as claimed in claim 1 substantially as described herein with reference to the examples and/or the accompanying drawings. A recombinant filamentous fungus transformed with the vector of claim 26. A method as claimed in claim 13 or claim 20 substantially as described herein with reference to the examples and/ or the accompanying drawings A selection marker whenever produced by a method as claimed in any one of claims 13 to 19 or 28. A bio-active compound whenever produced by a method as claimed in any of claims 20 to 22 or 28. Gene replacement via double cross—over Gene replacement vector pGBDEL4L Internal recombination on 3’gZaA repeats Genomic truncated gla/—\ locus