Title: Novel phytases

FIELD OF THE INVENTION

The present invention is in the field of food and feed supplements. In particular, the present invention relates to phytase enzymes useful as a feed supplement, and to methods of producing said phytase enzymes by genetic engineering techniques.

BACKGROUND TO THE INVENTION

Phytic acid (known as inositol hexakisphosphate (IP6), or phytate when in salt form) is the principal storage form of phosphorus in many plant tissues, especially bran and seeds. Phosphorus in phytate form is, in general, not bioavailable to non-ruminant animals because they lack the digestive enzyme phytase, which is required to separate phosphorus from the phytate molecule. On the other hand, ruminants readily utilize phytate because of the phytase produced by rumen microorganisms. In most commercial agriculture, non-ruminant livestock such as swine and poultry are fed mainly grains such as soybeans and maize. Because phytate from these grains is unavailable for absorption, the unabsorbed phytate passes through the gastrointestinal tract, elevating the amount of phosphorus in the manure. Excess phosphorus excretion can lead to environmental problems such as eutrophication. The bioavailability of phytate phosphorus can be increased by supplementation of the diet with phytase enzyme.

Phytases are by far the most industrially relevant feed enzymes due to their nutritional and environment benefits in intensive animal breeding, and applications in fish feed are rapidly developing.

From the plant kingdom, e.g. a wheat-bran phytase is known (Thomlinson et al., Biochemistry 1 (1962), 166-171). An alkaline phytase from Lilly pollen has been described by Barrientos et al., Plant Physiol.106 (1994), 1489-1495. Amongst the bacteria, phytases have been described which are derived from Bacillus subtilis (Paver and Jagannathan, Journal of

Bacteriology 151 (1982), 1102-1108) and Pseudomonas (Cosgrove, Australian Journal of Biological Sciences 23 (1970), 1207-1220).

There are several descriptions of phytase producing filamentous fungi. In particular, there are several references to phytase producing ascomycetes of the Aspergillus genus such as Aspergillus terreus (Yamada et al., Agric. Biol. Chem. 322 (1986), 1275-1282). Also, the cloning and expression of the phytase gene from Aspergillus niger var. awamori has been described (Piddington et al., Gene 133 (1993), 55-62).

EP 0 420 358 describes the cloning and expression of a phytase of Aspergillus ficuum (niger). EP 0 684 313 describes the cloning and expression of phytases of the ascomycetes Myceliophthora thermophila and Aspergillus terreus.

EP 897 010 entitled "Modified phytases" discloses, i.a., certain variants of an Aspergillus fumigatus phytase. EP 897 985 entitled "Consensus phytases" discloses, i.a., a fungal consensus phytase which may be designed on the basis of, i.a., a multiple alignment of several ascomycete phytases. WO 99/48380 entitled "Thermostable phytases in feed preparation and plant expression" relates to certain aspects of using thermostable phytases. WO 00/143503 entitled "Improved phytases" relates inter alia to certain phytase variants of increased thermo- stability, which may be designed by a process similar to the one described in EP 897985.

A phytase derived from Peniophora lycii is disclosed in WO 98/28408, and certain variants thereof in WO 99/49022, as well as in WO 03/066847.

Phytase producing yeasts are also described: For example, EP 0 699 762 A2 describes the cloning and expression of a phytase of the yeast Schwanniomyces occidentalis.

There is however still need for additional sources of phytase. In addition there is need for phytases having improved properties, such as increased activity.

SUMMARY OF THE INVENTION

The present inventors have now discovered novel fungal phytases dissimilar to previously identified phytases, including two different groups.

One group including Phy 538 and Phyl900 (Figure 7, 8, 13, 14) consists of a new subclass of phytases with very low similarity to previously know fungal phytases.

The other class consists of a fungal gene having two tandemly linked phytase domains (such as Phy7789ab Fig 1, 2-5, 10). Both of these domains have high levels of homology to phytases, proteins to date only characterized from bacterial sources. The structure of these phytases is homologous to known bacterial phytases (vide Paver and Jagannathan supra, and Cosgrove, supra). Thus, the present inventors discovered the existence of a completely new class of fungal proteins with homology to bacterial phytases.

In a first aspect, the present invention provides an isolated polypeptide selected from the group consisting of:

(a) polypeptides having the amino acid sequence as shown in FIG. 2;

(b) polypeptides having an amino acid sequence which is at least 40, preferably at least 50% more preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%,

90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in FIG. 2 and having phytase activity; and

(c) fragments of the polypeptide of (a) or (b) having phytase activity.

In a preferred embodiment of a polypeptide of the present invention, said polypeptide is a double domain phytase comprising a first and second phytase domain,

- said first phytase domain having an amino acid sequence which is at least 40, preferably at least 50% more preferably at least 55%, 60%, 65%,

70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in FIG. 3, and

- said second phytase domain having an amino acid sequence which is at least 40, preferably at least 50% more preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in FIG. 4.

In a further preferred embodiment of a polypeptide of the present invention, said first and second phytase domains are separated by a cysteine- rich region having a sequence which is at least 40, preferably at least 50% more preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,

97%, 98% or 99% identical to the amino acid sequence shown in FIG. 5. In yet another aspect, the present invention provides an isolated nucleic acid molecule encoding a protein having phytase activity selected from the group consisting of: a) a nucleic acid molecule having a nucleotide sequence which is at least

50% identical to the nucleotide sequence shown in Fig. 1; b) a nucleic acid molecule hybridizing under stringent conditions to the nucleotide sequence shown in Fig. 1; c) a nucleic acid molecule encoding a polypeptide having the amino acid sequence which is at least 50% identical to the amino acid sequence shown in

Fig. 2 and having phytase activity; d) a nucleic acid molecule encoding a naturally occurring allelic variant of the polypeptide having the amino acid sequence shown in Fig.2; e) a nucleic acid molecule complementary to the nucleic acid molecules of any one of a) to d).

In yet another aspect, the present invention provides a recombinant nucleic acid molecule comprising a polynucleotide selected from the group consisting of:

(a) polynucleotides comprising a nucleotide sequence as disclosed in FIG. 1; (b) polynucleotides comprising a nucleotide sequence encoding a polypeptide as defined above;

(c) polynucleotides comprising a nucleotide sequence the complementary strand of which hybridizes to the polynucleotide of (a) or (b), wherein said nucleotide sequence encodes a polypeptide having phytase activity; and (d) polynucleotides comprising a nucleotide sequence that deviates from the nucleotide sequence defined in (c) by the degeneracy of the genetic code.

As indicated above a further embodiment of the present invention is provided by two new classes of fungal phytases, exemplified by Phy538 and Phyl900. Also included in said class are homologous proteins that are preferably at least 40, preferably at least 50% more preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence depicted in Fig. 7 or 8.

In yet another aspect, the present invention provides an isolated nucleic acid molecule encoding a protein having phytase activity selected from the group consisting of: a) a nucleic acid molecule having a nucleotide sequence which is at least 40, preferably at least 50% more preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to identical to the nucleotide sequence shown in Fig. 17 or 18; b) a nucleic acid molecule hybridizing under stringent conditions to the nucleotide sequence shown in Fig. 17 or 18; c) a nucleic acid molecule encoding a polypeptide having the amino acid sequence which is at least 40, preferably at least 50% more preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%

identical to identical to the amino acid sequence shown in Fig. 7 or 8 and having phytase activity; d) a nucleic acid molecule encoding a naturally occurring allelic variant of the polypeptide having the amino acid sequence shown in Fig.7 or 8; e) a nucleic acid molecule complementary to the nucleic acid molecules of any one of a) to d).

In yet another aspect, the present invention provides a recombinant nucleic acid molecule comprising a polynucleotide selected from the group consisting of: (a) polynucleotides comprising a nucleotide sequence as disclosed in FIG. 17 or 18;

(b) polynucleotides comprising a nucleotide sequence encoding a polypeptide as defined above;

(c) polynucleotides comprising a nucleotide sequence the complementary strand of which hybridizes to the polynucleotide of (a) or (b), wherein said nucleotide sequence encodes a polypeptide having phytase activity; and

(d) polynucleotides comprising a nucleotide sequence that deviates from the nucleotide sequence defined in (c) by the degeneracy of the genetic code.

In a preferred embodiment of a recombinant nucleic acid molecule of the present invention, said recombinant nucleic acid molecule further comprises expression control sequences operably linked to said polynucleotide.

In yet another aspect, the present invention provides a vector comprising the recombinant nucleic acid molecule of the invention as described above. In yet another aspect, the present invention provides a method for producing genetically engineered host cells comprising introducing the recombinant nucleic acid molecule of the present invention or the vector of the present invention into a host cell.

In yet another aspect, the present invention provides a host cell that is genetically engineered with the recombinant nucleic acid molecule or the

vector of the present invention or a host cell that is obtainable by the method of the present invention.

In a preferred embodiment, said host cell is a bacterial, yeast, fungus, plant or animal cell, most preferably a fungus. In yet another aspect, the present invention provides a method for the production of a polypeptide having phytase activity, comprising cultivating the host cell of the present invention under conditions that allow for the expression of the polynucleotide, and in which the polypeptide encoded by said polynucleotide is isolated from the cells and/or the culture medium. In yet another aspect, the present invention provides a polypeptide having phytase activity obtainable by a method of the present invention.

In yet another aspect, the present invention provides an antibody specifically recognizing the polypeptide of the present invention.

In yet another aspect, the present invention provides a composition comprising a polypeptide of the present invention. In a preferred embodiment, said composition is a feed, food or an additive for a feed or a food.

In yet another aspect, the present invention provides a process for preparing a feed or a food comprising the step of adding a polypeptide of the present invention to the feed or food components. In yet another aspect, the present invention provides the use of a polypeptide of the present invention for liberating inorganic phosphate from phytic acid.

BRIEF DESCRIPTION OF THE DRAWINGS: Figure 1 shows the complete nucleotide sequence of the ORF encoding the double domain phytase protein of the present invention. Also separately presented are the nucleotide sequences of the two domains.

Figure 2 shows the complete amino acid sequence of the double domain phytase protein of the present invention encoded by the gene in Fig. 1.

Figure 3 shows the sequence of the first phytase domain of the double domain phytase protein of the present invention the complete amino acid sequence of which is displayed in Fig. 2.

Figure 4 shows the sequence of the second phytase domain of the double domain phytase protein of the present invention.

Figure 5 shows the sequence of the cysteine-rich region that separates first and second phytase domains.

Figure 6: Homology tree of selected fungal phytases. The boxed entries are Magnaporthe sequences selected for expression. Figure 7 shows the amino acid sequence based on a manually corrected annotation of a DNA sequence of PhyO538 from Magnaporthe grisea. Figure 8 shows the amino acid sequence of Phyl900 based on the annotation of a DNA sequence from Magnaporthe grisea. Figure 9. Plug-in expression cassette based on the cbhl promoter and terminator sequences of Chrysosporium lucknowense.

Figure 10. Synthetic expression cassette for phy7789ab. Figure 11. Synthetic expression cassette for phy7789a. Figure 12. Synthetic expression cassette for phy7789b. Figure 13. Synthetic expression cassette for phyO538. Figure 14. Synthetic expression cassette for phyl900.

Figure 15. Glucoamylase phytase fusion protein (glucoamylase phy7789ab).

Figure 16. DNA sequence coding for the protein of Fig. 15. Figure 17. Nucleotide sequence of coding region of phyO538. Figure 18. Nucleotide sequence of coding region of phyl900.

Figure 19. Homologue protein sequences of phy7789ab Figure 20. Homologue protein sequences of phyO538

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids. The terms "polypeptide", "peptide" and "protein" are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP- ribosylation.

The term "sequence identity," as used herein, is generally expressed as a percentage and refers to the percent of amino acid residues or nucleotides, as appropriate, that are identical as between two sequences when optimally aligned. For the purposes of this invention, sequence identity means the sequence identity determined using the well-known Basic Local Alignment Search Tool (BLAST), which is publicly available through the National Cancer Institute/National Institutes of Health (Bethesda, Maryland) and has been described in printed publications (see, e.g., Altschul et al., J. MoI. Biol, 215(3), 403-10 (1990)). Preferred parameters for amino acid sequences comparison using BLASTP are gap open 11.0, gap extend 1, Blosum 62 matrix.

The term "phytase" is defined herein as a protein or polypeptide which is capable of catalysing the hydrolysis of phytate (inositol hexakisphosphate (IP6) or phytic acid and releasing inorganic phosphate. The term encompasses both 3-phytase (E. C.3.1.3.8), 4-phytase (also referred to as 6-phytase, E. C.3.1.3.26), or 5-phytase (E. C.3.1.3.72) as classified in accordance with the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology. Preferably, the phytase is 3-phytase (E. C.3.1.3.8), 4-

phytase (E.C.3.1.3.26) or 5-phytase (E.C.3.1.3.72). The term phytase includes reference to the protein that exhibits peroxidase activity when the vanadate ion is incorporated into the active site of the phytase enzyme. (Van de Velde et al. 2000, Biotech. Bioeng. 67:87-96). The term "phytase activity" as used herein, refers to the specific enzyme activity of a phytase to catalysing the hydrolysis of phytate or phytic acid expressed in μmol substrate/min/mg protein.

The term "double domain" as used herein, refers to the presence of two related domains in a single protein molecule resulting in an active protein. As used herein, the term "cysteine-rich region" refers to a protein domain which has an amino acid sequence of about 10-200, preferably about 40-90, more preferably about 50-80 amino acid residues, and even more preferably at least about 60-65 amino acids of which at least about 8-30, preferably about 10-15, and more preferably about 11, 12, 13, or 14 of the amino acids are cysteine residues.

The term "signal peptide" or "signal sequence" as used herein, refers to an amino acid sequence, typically located at the amino terminus of an immature protein or polypeptide (e.g., prior to secretion from a cell and associated processing and cleavage), which directs the secretion of the protein or polypeptide from the cell in which it is produced. The signal peptide typically is removed from an immature protein or polypeptide prior to or during secretion and, thus, is not present in the mature, secreted polypeptide. As used herein, the term "recombinant nucleic acid molecule" refers to a recombinant DNA molecule or a recombinant RNA molecule. A recombinant nucleic acid molecule is any synthetic nucleic acid construct or nucleic acid molecule containing joined nucleic acid molecules from different original sources or and not naturally occurring or attached together and prepared by using recombinant DNA techniques.

The term "recombinant host cell" as used herein, refers to a host cell strain containing nucleic acid not naturally occurring in that strain and which has been introduced into that strain using recombinant DNA techniques. The term "nucleic acid" as used herein, includes reference to a deoxyribonucleotide or ribonucleotide polymer, i.e. a polynucleotide, in either single-or double- stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single- stranded nucleic acids in a manner similar to naturally occurring nucleotides (e. g., peptide nucleic acids). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" (a polymeric form of nucleotides, either ribonucleotides or deoxyribonucleotides, double- or single- stranded of any length) as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including among other things, simple and complex cells. Every nucleic acid sequence herein that encodes a polypeptide also, by reference to the genetic code, describes every possible silent variation of the nucleic acid. The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids which

encode identical or conservatively modified variants of the amino acid sequences due to the degeneracy of the genetic code.

The term "degeneracy of the genetic code" refers to the fact that a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations" and represent one species of conservatively modified variation. The term "gene", as used herein, refers to a nucleic acid sequence containing a template for a nucleic acid polymerase, in eukaryotes, RNA polymerase II. Genes are transcribed into mRNAs that are then translated into protein.

"Expression" refers to the transcription of a gene into structural RNA (rRNA, tRNA) or messenger RNA (mRNA) with subsequent translation into a protein.

The term "complementary", as used herein, refers to a sequence of nucleotides which forms a hydrogen-bonded duplex with another sequence of nucleotides according to Watson- Crick base-paring rules. For example, the complementary base sequence for 5'-AAGGCT-3' is 3'-TTCCGA-5'. As used herein, "substantially complementary" means that two nucleic acid sequences have at least about 40, preferably about 50% more preferably at least 55%, more preferably about 60%, more preferably about 70%, more preferably about 80%, even more preferably 90%, and most preferably about 98%, sequence complementarity to each other.

The term "hybridise" refers to the process by which single strands of nucleic acid sequences form double-helical segments through hydrogen bonding between complementary nucleotides.

As used herein, the term "expression control sequence" refers to a nucleic acid sequence that regulates the transcription and translation of a gene

to which it is operatively linked. An expression control sequence is "operatively linked" to a gene when the expression control sequence controls and regulates the transcription and, where appropriate, translation of the gene. The term "operatively linked" includes the provision of an appropriate start codon (e.g. ATG), in front of a polypeptide-encoding gene and maintaining the correct reading frame of that gene to permit proper translation of the mRNA.

As used herein, the term "operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to another control sequence and/or to a coding sequence is ligated in such a way that transcription and/or expression of the coding sequence is achieved under conditions compatible with the control sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.

The term "vector" as used herein, includes reference to an autosomal expression vector and to an integration vector used for integration into the chromosome.

The term "expression vector" refers to a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest under the control of (i.e., operably linked to) additional nucleic acid segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. In particular an expression vector comprises a nucleotide sequence that comprises in the 5' to 3' direction and operably linked: (a) a fungal-recognized transcription and translation initiation region, (b) a coding sequence for a polypeptide of interest, and (c) a fungal-recognized transcription and translation termination region. "Plasmid"

refers to autonomously replicating extrachromosomal DNA which is not integrated into a microorganism's genome and is usually circular in nature.

An "integration vector" refers to a DNA molecule, linear or circular, that can be incorporated in a microorganism's genome and provides for stable inheritance of a gene encoding a polypeptide of interest. The integration vector generally comprises one or more segments comprising a gene sequence encoding a polypeptide of interest under the control of (i.e., operably linked to) additional nucleic acid segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and one or more segments that drive the incorporation of the gene of interest into the genome of the target cell, usually by the process of homologous recombination. Typically, the integration vector will be one which can be transferred into the host cell, but which has a replicon which is nonfunctional in that organism. Integration of the segment comprising the gene of interest may be selected if an appropriate marker is included within that segment.

"Transformation" and "transforming", as used herein, refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion, for example, direct uptake, transduction, f- mating or electroporation. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome.

By "host cell" is meant a cell which contains a vector or recombinant nucleic acid molecule and supports the replication and/or expression of the vector or recombinant nucleic acid molecule. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, fungus, plant, insect, amphibian, or mammalian cells. Preferably, host cells are fungal cells.

The term "fungus" or "fungi" includes a wide variety of nucleated, spore-bearing organisms which are devoid of chlorophyll. Examples of fungi include yeasts, mildews, molds, rusts and mushrooms. Preferred fungi in aspects of the present invention are organisms of the genera Aspergillus,

Trichoderma, Myceliophtora, Rhizopus, Magnaporthe, Chrysosporium, Gibberella, Phaeosphaeria, Chaetomium , Coprinopsis, Ajellomyces, Penicillium, Thielavia, Corynascus, Agrocybe, Peniophora, Trametes, Laccaria, Ceriphora, Neurospora, Humicola, Talaromyces, Neosartorya. The terms "isolated" or "purified" as used herein refer to a nucleic acid or protein or peptide that is removed from at least one component with which it is naturally associated. In the present invention, an isolated nucleic acid can include a vector comprising the nucleic acid. Purified as used herein to describe a polypeptide produced by cultivation of a recombinant host cell refers to removing that polypeptide from at least one component with which it is naturally associated in the host cell or culture medium.

Description of the preferred embodiments

The amino acid sequence of the double domain phytase protein of the present invention is, in a preferred embodiment, the polypeptide as shown in FIG. 2.

This double domain protein resembles a phytase with very low levels of sequence homology (sequence identity to Bacillus sequence in US6638746 is 36%) with the phytases presently known. Despite being encoded by a fungal gene, the protein exhibits the highest level of sequence homology with Bacillus phytases. The finding that genes in the natural fungal genome are present that encode proteins that resemble bacterial-type phytases is a surprising finding. The expression of such bacterial phytase genes in fungi has hitherto not been described. The gene as presently identified and presented in FIG. 1, comprises two phytase domains in a single open reading frame and phytase activity has yet only been observed for the double domain protein. It is, however, expressly noticed that the individual phytase domains are part of the present invention. Although known in bacteria, phytase genes having a double phytase domain structure are poorly studied and have hitherto not been reported in the fungal genome.

The phytases of the present invention may be expressed in any host cell, but are preferably expressed in fungal cells.

Phytase producing organisms may not only be obtained by using recombinant techniques and expression vectors. Alternatively, DNA fragments may be generated using restriction-endonuclease digestion or PCR the expression of the endogenous gene in an organism may be controlled by methods known in the art, which expression may be altered by any available method for genetic modification, for instance via alteration of the regulation of the expression. In another preferred embodiment, a polypeptide of the present invention has an amino acid sequence which is at least 40, preferably at least 50% more preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in FIG. 2 wherein said polypeptide exhibits phytase activity. The skilled person is well aware how phytase activity can be determined for such polypeptides. For instance, the polypeptide can be synthesized or expressed in a host cell and the optionally purified polypeptide can be brought in contact with a phytate or phytic acid substrate under conditions of temperature, salinity, pH and ions and the like that allow for the enzymatic hydrolysis of the substrate by phytases. As a reference enzyme a commercially available bacterial or fungal phytase can be used. The hydrolysis of phytate or phytic acid may be determined by measuring the liberation of inorganic phosphates by spectrophotometric, chromatographic or chemical assay methods. In another preferred embodiment, a polypeptide of the present invention may be a fragment of the polypeptide as shown in FIG. 2, such as for instance the fragment shown in FIG. 3 or the fragment shown in FIG. 4, preferably having phytase activity, or a polypeptide of the present invention may be a fragment of the polypeptide having an amino acid sequence which is at least 40, preferably at least 50% more preferably at least 55%, 60%, 65%,

70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in FIG. 2 as described above, having phytase activity.

Whether a polypeptide or fragment thereof exhibits phytase activity as required by the present invention may be established by using a suitable enzymatic assay. One suitable assay for the measurement or detection of phytase activity is provided in the Examples below.

In a highly preferred embodiment, a polypeptide of the present invention is a double domain phytase comprising a first and second phytase domain, wherein said first phytase domain has an amino acid sequence which is at least 40, preferably at least 50% more preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in FIG. 3, and wherein said second phytase domain has an amino acid sequence which is at least 40, preferably at least 50% more preferably at least 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in FIG. 4.

Alternatively, the double domain phytase comprises two or more copies of one of these domains.

The first phytase domain as described above is preferably located at the 5' end, while the second phytase domain as described above is preferably located at the 3' end of the gene encoding the phytase of the invention. It should be noted that the order in which the domains are ordered is not limiting, the domain ordering may be in principle be AB, BA, AA and BB (wherein A and B denote the first and second domain). Even proteins with more than two domains would be considered covered, and of course any permutation of these domains is intended.

The present invention also contemplates the use of fusion proteins, accomplished by the use of a gene fusion, for instance with a carrier protein such as glucoamylase in order to increase secretion levels of the phytase protein.

The first and second phytase domains may be separated by a cysteine- rich region. Such a region is generally characterized by an amino acid sequence having an unusual high level of cysteine residues, for instance more than 10%, preferably around 20%, of the amino acid residues being cysteine. The cysteine-rich region serves as a region connecting and separating the two phytase domains of the double domain phytase of the present invention. This region may suitably be between 10 and 200 amino acid residues in length. A fungal double domain phytase of the present invention preferably comprises a cysteine-rich region having a sequence which is at least 40, preferably at least 50% more preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in FIG. 5. Most preferably the region consists of the amino acid sequence shown in FIG. 5. It should be noted that all uncharacterized database examples of fungal proteins are proteins with double domains carrying a Cysteine linker. There are very few uncharacterized bacterial proteins from Pseudomonas or

Pseudoalteromonas with double domains but without a cysteine domain; e.g. emb I CAI85536.1 1 putative phytase domain protein [Pseudoalteromonas haloplanktis]. A putative fungal phytase protein that possesses a double domain according to the invention was disclosed as EMBL Ace. Nr. CH476828 (GenBank XP_367885.2). However, the fact that this protein obtained from

Magnaporthe grisea has a double domain was not recognized by the annotators of the sequence (Dean, R. et al,, 2005, Nature 434:980-986). Further, it has also appeared (see Experimental section) that a shorter version of this peptide, that still contains the two phytase domains, has phytase activity, while the phytase activity of the protein as disclosed in Dean et al., was only considered to be a phytase putatively, i.e. na actual phytase activity has been establishe for this protein. It is clear that the present invention does not comprise the protein as disclosed in GenBank XP_367885.2.

In another embodiment the present invention comprises two new classes of phytases, characterized by the two exemplary sequences phyO538

and phyl900 (of which the amino acid sequence is given in Figures 7 and 8 respectively, and of which the nucleotide sequence is given in Fig. 17 and 18, respectively). Both protein sequences are derived from the fungus Magnaporthe grisea. As indicated in the experimental part, these phytases do not resemble the currently known phytases. Further they form, together with the orthologues indicated in Example 1 two separate, new groups of phytases, as is indicated in Figure 6.

Although the orthologues listed have a relatively low identity with the sequences of phy538 and phyl900, they can be classified in distinct groups since the sequence identity with the classical phytases and acid phosphatases is even much lower (see fig. 6).

As indicated above a further embodiment of the present invention is provided by two new classes of fungal phytases, exemplified by Phy538 and Phyl900. Also included in said class are homologous proteins that are preferably at least 40, preferably at least 50% more preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence depicted in Fig. 7 or 8.

In yet another aspect, the present invention provides an isolated nucleic acid molecule encoding a protein having phytase activity selected from the group consisting of: a) a nucleic acid molecule having a nucleotide sequence which is at least 40, preferably at least 50% more preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to identical to the nucleotide sequence shown in Fig. 17 or 18; b) a nucleic acid molecule hybridizing under stringent conditions to the nucleotide sequence shown in Fig. 17 or 18; c) a nucleic acid molecule encoding a polypeptide having the amino acid sequence which is at least 40, preferably at least 50% more preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%

identical to identical to the amino acid sequence shown in Fig. 7 or 8 and having phytase activity; d) a nucleic acid molecule encoding a naturally occurring allelic variant of the polypeptide having the amino acid sequence shown in Fig.7 or 8; e) a nucleic acid molecule complementary to the nucleic acid molecules of any one of a) to d).

In yet another aspect, the present invention provides a recombinant nucleic acid molecule comprising a polynucleotide selected from the group consisting of: (a) polynucleotides comprising a nucleotide sequence as disclosed in FIG. 17 or 18;

(b) polynucleotides comprising a nucleotide sequence encoding a polypeptide as defined above;

(c) polynucleotides comprising a nucleotide sequence the complementary strand of which hybridizes to the polynucleotide of (a) or (b), wherein said nucleotide sequence encodes a polypeptide having phytase activity; and

(d) polynucleotides comprising a nucleotide sequence that deviates from the nucleotide sequence defined in (c) by the degeneracy of the genetic code.

A polypeptide of the present invention may comprise a signal sequence, or may be fused to additional polypeptide sequences, such as for instance to a signal peptide, which signal sequence or peptide may contribute to excretion of the produced polypeptide from the host cell of the invention. A preferred signal sequence is the signal sequence as shown in FIGS. 2, 3 and 4. Recombinant nucleic acid molecules of the present invention comprise polynucleotides that encode for polypeptides and phytases of the present invention.

One such polynucleotide comprises a nucleotide sequence as disclosed in FIG. 1. This polynucleotide encodes a polypeptide according to FIG. 2. The skilled person will understand that other polypeptides will also encode a

functional phytase as described in FIG. 2, for instance polynucleotides comprising a nucleotide sequence that deviates from the nucleotide sequence shown in FIG. 1 by the degeneracy of the genetic code will also encode a functional enzyme. In general all recombinant nucleic acid molecules comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide or phytase of the present invention as defined above are part of the present invention.

Also part of the present invention are recombinant nucleic acid molecules comprising a polynucleotide comprising a nucleotide sequence the complementary strand of which hybridizes to the polynucleotide encodes a polypeptide according to FIG. 2 or deviating therefrom due to the degeneracy of the genetic code.

Also part of the present invention are recombinant nucleic acid molecules comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide having phytase activity according to the present invention and polynucleotides deviating therefrom by the degeneracy of the genetic code.

The recombinant nucleic acid molecule of the present invention may further comprise one or more expression control sequences, such as a promoter, a ribosome binding site, a transcription initiation region, a transcription termination region, operably linked to said polynucleotide. As used herein "promoter" is a DNA sequence that directs the transcription of a gene. Typically, a promoter is located in the 5'-region of a gene, proximal to the transcriptional start site of a gene. Promoter sequences useful in aspects of the present invention may be constitutive, inducible or repressible. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. Preferred promoter sequences are sequences functional in fungi.

In order to achieve optimal expression in the host cell of interest, such as a fungal cell, the codon (pair) usage of the polynucleotides encoding the polypeptides and phytases of the present invention may be optimized by using any one of a variety of synthetic gene design software packages, for instance GeneOptimizer® from Geneart AG (Regensburg, Germany) for codon usage optimization or for codon pair usage optimization. Such adaptation of codon usage ensures that the gene, which is essentially of fungal origin, is effectively processed by the projected host cell's transcription and translation machinery. Optimization of codon (pair) usage will generally result in enhanced protein expression in the host cell.

The recombinant nucleic acid molecule of the present invention may for instance be cloned into a high-copy-number plasmid or other suitable vector system for transformation of and subsequent expression in the host cell.

Recombinant host cells can be obtained using methods known in the art for providing cells with recombinant nucleic acids. These include transformation, transconjugation, transfection or electroporation of a host cell with a suitable plasmid or vector comprising the nucleic acid construct of interest operationally coupled to a promoter sequence to drive expression. Host cells of the invention are preferably transformed with a recombinant nucleic acid molecule of the present invention and may comprise a single but preferably comprise multiple copies of the nucleic acid construct. The nucleic acid construct may be maintained episomally and thus comprise a sequence for autonomous replication, such as an ARS sequence. Suitable episomal nucleic acid constructs may e.g. be based on the yeast 2μ or pKDl (Fleer et al., 1991, Biotechnology 9: 968-975) plasmids. Preferably, however, the nucleic acid construct is integrated in one or more copies into the genome of the host cell. Integration into the host cell's genome may occur at random by illegitimate recombination but preferably the nucleic acid construct is integrated into the host cell's genome by homologous recombination as is well known in the art of

fungal molecular genetics (see e.g. WO 90/14423, EP-A-O 481 008, EP-A-O 635 574 and US 6,265,186).

Transformation of host cells with the nucleic acid constructs of the invention and additional genetic modification of the fungal host cells of the invention as described above may be carried out by methods well known in the art. Such methods are e.g. known from standard handbooks, such as Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987). Methods for transformation and genetic modification of fungal host cells are known from e.g. EP-A-O 635 574, WO 98/46772, WO 99/60102 and WO 00/37671.

Promotors suitable to drive expression of the genes in the hosts of the invention include e.g. promoters from glycolytic genes (e.g. from a glyceraldehyde-3-phosphate dehydrogenase gene), ribosomal protein encoding gene promoters, alcohol dehydrogenase promoters (ADHl, ADH4, and the like), promoters from genes encoding amylo- or cellulolytic enzymes (glucoamylase, TAKA-amylase and cellobiohydrolase). Other promoters, both constitutive and inducible and enhancers or upstream activating sequences will be known to those of skill in the art. The promoters used in the nucleic acid constructs of the present invention may be modified, if desired, to affect their control characteristics. Preferably, the promoter used in the nucleic acid construct for expression of the genes is homologous to the host cell in which genes are expressed. In the nucleic acid construct, the 3'-end of the coding nucleotide acid sequence(s) preferably is/are operably linked to a transcription terminator sequence. Preferably the terminator sequence is operable in a host cell of choice. In any case the choice of the terminator is not critical; it may e.g. be from any fungal gene, although terminators may sometimes work if from a

non-fungal, eukaryotic, gene. The transcription termination sequence further preferably comprises a polyadenylation signal.

Optionally, a selectable marker may be present in the nucleic acid construct. As used herein, the term "marker" refers to a gene encoding a trait or a phenotype which permits the selection of, or the screening for, a host cell containing the marker. A variety of selectable marker genes are available for use in the transformation of fungi. Suitable markers include auxotrophic marker genes involved in amino acid or nucleotide metabolism, such as e.g. genes encoding ornithine-transcarbamylases (argB), orotidine-5'-decaboxylases (pyrG, URA3) or glutamine-amido-transferase indoleglycerol-phosphate- synthase phosphoribosyl-anthranilate isomerases (trpC), or involved in carbon or nitrogen metabolism, such e.g. niaD or facA, and antibiotic resistance markers such as genes providing resistance against phleomycin, bleomycin or neomycin (G418). Preferably, bidirectional selection markers are used for which both a positive and a negative genetic selection is possible. Examples of such bidirectional markers are the pyrG (URA3, pyr4), pyrE (uraδ, pyrδ) facA and amdS genes. Due to their bidirectionality these markers can be deleted from transformed filamentous fungus while leaving the introduced recombinant DNA molecule in place, in order to obtain fungi that do not contain selectable markers. This essence of this MARKER GENE FREE™ transformation technology is disclosed in EP-A-O 635 574, which is herein incorporated by reference. Of these selectable markers the use of dominant and bidirectional selectable markers such as acetamidase genes like the amdS genes of A. nidulans, A. niger and P. chrysogenum is most preferred. In addition to their bidirectionality these markers provide the advantage that they are dominant selectable markers that, the use of which does not require mutant (auxotrophic) strains, but which can be used directly in wild type strains.

Optional further elements that may be present in the nucleic acid constructs of the invention include, but are not limited to, one or more leader

sequences, enhancers, integration factors, and/or reporter genes, intron sequences, centromere, telomere and/or matrix attachment (MAR) sequences. The nucleic acid constructs of the invention may further comprise a sequence for autonomous replication, such as an ARS sequence. Suitable episomal nucleic acid constructs may e.g. be based on the yeast 2μ or pKDl (Fleer et al., 1991, Biotechnology 9: 968-975) plasmids. Alternatively the nucleic acid construct may comprise sequences for integration, preferably by homologous recombination (see e.g. WO98/46772). Such sequences may thus be sequences homologous to the target site for integration in the host cell's genome. The nucleic acid constructs of the invention can be provided in a manner known per se, which generally involves techniques such as restricting and linking nucleic acids/nucleic acid sequences, for which reference is made to the standard handbooks, such as Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987).

The transformed host cell comprising the recombinant nucleic acid molecule or the vector of the present invention can be used to produce the polypeptides of the present invention. As stated above, the host cell is preferably a fungal cell, such as a yeast cell, but may also be a bacterial cell. The polypeptide of the present invention can then be produced by cultivating the host cell under conditions that allow said host cell to replicate and express the polynucleotide encoding the polypeptide of the invention. Such conditions depend on the specific host cell applied, but are generally are well known to those of skill in the art. The cultivation may be on solid media, but is preferably in liquid medium, the cells may be freely suspended or may, alternatively, be immobilized to a support. Suitably, the cultivation medium may comprise agents that induce the expression of the polynucleotide in the host cell.

A method for the production of a polypeptide having phytase activity further comprises the step of isolating the polypeptide from the cells and/or the culture medium, depending on whether the polypeptide is excreted or remains associated with the host cells. When a suitable signal sequence, such as MVASSIGVLLAAVLLGGTSTIVTA or other suitable signal sequence, is incorporated at for instance the N-terminus of the polypeptide, the polypeptide may be transported to the endoplasmatic reticulum and excreted from the cells.

Once excreted to the culture medium, the polypeptide can be easily purified therefrom by using chromatographic or immunocapture techniques well known in the art. Chromatographic techniques may include liquid chromatography such as HPLC, size exclusion chromatography or affinity chromatography.

If not excreted to the medium the polypeptides of the invention may be isolated form the host cell by disrupting or Iy sing the host cells, separating the membrane fragments from the cell contents and purifying the polypeptide from either of these using the same techniques as described above. Suitable alternative protein purification techniques are well known to the skilled person and may be employed in methods of the present invention. Once purified, the polypeptide of the present invention may be used for immunization of a mammal and the antibodies raised may in the mammal may be isolated and purified, optionally after producing hybridoma cell lines with isolated spleen cells producing specific antibodies, to produce polyclonal or monoclonal antibodies against the polypeptides of the invention. The phytases of the present invention can be incorporated into any composition, preferably an animal feed, a human food product, or can be used as an additive or supplement for a feed or food product, most preferably a feed for non-ruminant animals, most preferably poultry or swine. An effective amount of phytase in food or feed is from about 10 to 20,000 FTU/kg; preferably from about 10 to 15,000 FTU/kg, more preferably from about 10 to

10,000 FTU/kg, in particular from about 100 to 5,000 FTU/kg, especially from about 100 to about 2,000 FTU/kg feed or food. The analytical method for determining microbial phytase activity and the definition of a phytase unit has been published by Engelen et al. (Journal of AOAC International 77 (3): 760- 764 (1994).

In addition to its major application in animal nutrition (including livestock and crustaceans and fish), phytase is also used for processing of human food. Research in this field focuses on better mineral absorption and technical improvement of food processing such as bread making, production of plant protein isolates, corn wet milling and the fractionation of cereal bran. Also applications relating to biodefense and to biomass conversion for fuels development are foreseen. The protein may be provided in solution or in dry enzyme preparations.

The invention will now be illustrated in more detail by the following non-limiting Examples.

Examples

Example 1 Genome mining

Genome mining in Magnaporthe grisea with phytase of Aspergillus and of Bacillus resulted in the identification of three new classes of fungal phytases:

1) a completely new class of fungal phytases, resembling bacterial phytases. This phytase consist of a double phytase domain fused by a linker region. Further genome mining showed that also the genomes from Giberella zeae (hypothetical protein FG05062.1 Identities = 414/765 (54%), Positives = 508/765 (66%)), Aspergillus nidulans, (hypothetical protein AN4140.2 (Identities = 365/752 (48%), Positives = 486/752 (64%)), Aspergillus terreus (XP_001212017, Identities = 368/757 (48%), Positives = 484/757 (63%), Phaeosphaeria nodorum (hypothetical protein SNOG_10880 Identities = 330/737 (44%), Positives = 429/737 (58%), Chaetomium globosum

(hypothetical protein CHGG_04017 Identities = 288/590 (48%), Positives = 371/590 (62%)) and Coprinopsis cinerea (hypothetical protein CC1G_O5528, Identities = 301/744 (40%), Positives = 408/744 (54%), carry a similar gene. None of these genes have been previously described. The Magnaporthe gene was manually annotated. This polypeptide sequence is shown in Figure 2

(phy7789ab). It appears that a longer polypeptide, comprising the sequence of Figure 2, is already disclosed as GenBank XP_367885.2, but it was not recognized that this polypeptide would be a member of this new class of fungal phytases that comprise two phytase domains.

(2) In addition also two diverse fungal type phytases were identified from Magnaporthe grisea (Phy538 and Phyl900, Fig 6, 7 and 8). From Phy538, homologous proteins are present in Giberella zeae (hypothetical protein FG00530. ^Identities = 350/549 (63%), Positives = 404/549 (73%), Neurospora crassa, (hypothetical protein NCU03255, Identities = 324/502 (64%), Positives = 381/502 (75%), Botryotinia fuckeliana, (hypothetical protein BC1G_11835, Identities = 302/450 (67%), Positives = 350/450 (77%), Aspergillus terreus (XP 001210909.1, Identities = 263/544 (48%), Positives = 343/544; XP_001211533, Identities = 236/506 (46%), Positives = 320/506 (63%), Aspergillus niger (hypothetical protein Anl8g01340, Identities = 248/531

(46%), Positives = 334/531 (62%); hypothetical protein Anl5g07000 , Identities = 240/528 (45%), Positives = 313/528 (59%), Phaeosphaeria nodorum (hypothetical protein SNOG_11624, A.oryzae (BAE55917, Identities = 234/521 (44%), Positives = 312/521 (59%), and Sclerotinia sclerotiorum, (hypothetical protein SSlG_04790, Identities = 181/284 (63%), Positives = 214/284 (75%). See also Fig. 20.

Phyl900 is very dissimilar to all known phytases. In blasting the sequence, the highest scoring sequences were present in Sclerotinia sclerotiorum (hypothetical protein SS1G_O9959, Identities = 202/468 (43%), Positives =

282/468 (60%), Botryotinia fuckeliana, hypothetical protein BC1G_O7284, Identities = 197/442 (44%), Positives = 272/442 (61%), Aspergillus fumigatus, (CAD 12029, Identities = 176/436 (40%), Positives = 253/436 (58%), Thielavia heterothallica , (Identities = 187/463 (40%), Positives = 257/463 (55%), Aspergillus niger (Identities = 190/468 (40%), Positives = 269/468 (57%); XP_001401713 , (Identities = 186/467 (39%), Positives = 265/467 (56%)), Aspergillus ficuum, (Identities = 186/467 (39%), Positives = 265/467 (56%)), Aspergillus awamori , P34753 (Identities = 186/466 (39%), Positives = 262/466 (56%)), Aspergillus oryzae, (Identities = 176/463 (38%), Positives = 264/463 (57%),

From Magnaporthe grisea Phy 538 and Phy 1900 were annotated (Figure 7 (phyO538) and Figure 8 (phy 1900)).

Example 2 Phytase expression cassettes

A plug-in expression cassette based on the cbhl promoter and terminator sequences of Chrysosporium lucknowense was synthesized in vivo (GENEART). The Ncol and the EcoRV sites were present as cloning sites. Hundred fifty base pairs of the 3' end of the cbhl promoter (from Ncol until ATG) are introduced with the gene of interest. Fifteen hundred base pairs upstream the Ncol site a synthetic amdS gene was synthesized, based on the Aspergillus nidulans amdS gene. C. lucknowense strains are naturally amdS- negative. The amdS gene encodes an amidase, and introduction of this gene into C. lucknowense allows the strain to grow on acetamide as sole nitrogen source. The introns and most of the six-base restriction enzymes were removed in this amdS gene. Fivehundred basepairs of the cbhl promoter upstream the amdS were also introduced downstream the amdS marker to create a repeat flanked selection marker. This repeat flanked amdS marker can be removed with Stul. The sequence of this expression cassette including the amdS marker is shown in Figure 9.

From the polypeptide sequence of the bacillus type phytase phy7789ab three gene versions were designed and synthesized in vivo (GENEART). The codons were optimized for C. lucknowense. The double domain (phy7789ab, Figure 10)) and each of the separate mono-domain genes (phy7789a, (Figure 11) and phy7789b (Figure 12)) were made in all cases carrying the native signal sequence. The nucleotide sequence of these three genes can be found in Figure 10, 11 and 12. The genes for phyO538 and phyl900 were designed and synthesized in vivo (GENEART). The codons were optimized for C. lucknowense. The nucleotide sequence of these genes can be found in Figure 13, respectively Figure 14. To facilitate the construction of the final expression cassette 150 bp of the cbhl promoter were also synthesized at the 3' of the gene starting with an Ncol site. At the 5' end of the gene an EcoRV site was introduced for cloning purposes.

The phytase genes were cloned with Ncol and EcoRV in the expression vector. The expression cassettes were digested with NotII and isolated to obtain E.coli DNA free fragments for transformation of C. lucknowense.

Another non-limiting example of this invention encompasses these phytases linked to glucoamylase with the KEX processing site to increase the expression and production of these phytases. In Heerikhuisen (Heerikhuisen, M., van den Hondel, C.A.M.J.J. and P.J. Punt (2005) Aspergillus sojae. In: Production of Recombinant Proteins. Novel microbial and eukaryotic expression systems. (Gerd Gellissen, Ed). Wiley- VCH Verlag, Weinheim. pp. 191-214) is described that fusion of heterologous proteins to glucoamylase results in higher production of heterologous proteins. An example of a glucoamylase phytase fusion protein (glucoamylase phy7789ab) before processing of both KEX-sites is shown in Figure 15. The nucleotide sequence encoding this protein is shown in Figure 16.

Example 3 Transformation of Chrysosporium lucknownense

For transformation of Chrysoporium lucknowense low protease mutants were used. These protease mutants may be obtained not only by disrupting actual protease encoding genes. Exemplary low protease strains include, but are not limited to, a mutant of strain UV18-25, which has been mutated with ultraviolet light and selected for low protease activity, denoted herein as UV18#100.f; a strain denoted UV18#100.f δpyrδ δalpl, in which the protease gene alpl has been selectively disrupted; and a strain denoted UV18#100.f δpyrδ δalpl δpep4 δalp2, in which three protease genes, alpl, pep4, and alp2 have been disrupted.

The protocol is based on a procedure originally used for Aspergillus transformation (P. Punt, C. van den Hondel, Methods in Enzymology 1992 216:447-457). Rich medium (250 ml) was inoculated with 10 ⁶ spores/ml of a Chrysosporium lucknowense protease mutant in alL Erlenmeyer flask. The culture was grown for 24-36 hours at 35 ⁰ C in an air incubator 25 (300 rpm). The mycelium was filtered through a sterile Miracloth(TM) filter (Calbiochem) and washed with ca. 100 ml 1700 mOsmol NaCl/CaCl ₂ (0.27 M CaCl ₂ / 0.6 M NaCl). The mycelium was weighed and then kept on ice. Caylase(TM) (Cayla) was added (20 mg per gram mycelium) and 1700 mOsmol NaCl/CaCl ₂ (3.3ml/g mycelium) and the suspension was incubated in a 33 ⁰ C air incubator (100 rpm). The protoplasting was followed under the microscope. After 1-3 hours of incubation, most of the mycelium was digested, leaving mostly protoplasts in the microscopic view of the preparation. The protoplasts were filtered through a sterile Myracloth filter and the filter was washed with 1 volume cold STC1700 (1.2 M sorbitol/ 10 mM Tris.HCl pH 7.5/ 50 mM CaCl ₂ / 35 mM NaCl). The protoplasts were spun down at 2500 rpm for 10 minutes at 4 ⁰ C. The pellet was resuspended in STC 1700 and centrifuged again. After resuspending the pellet in STC 1700, the protoplasts were counted. STC 1700 was added to a final concentration of 2 x 10 ⁸ protoplasts per ml.

DNA fragment of the phytase expression vector (5-10 μg) was pipetted into the bottom of a sterile tube and 1 μl IM ATA (aurintricarbonic acid) and 100 μl protoplasts (ca. 2 x 107) were added to the DNA. A minus DNA negative control was included in the experiment. After mixing, the protoplasts were incubated at room temperature for 25 minutes. 1350 μl PEG6000 (60% PEG/50 niM CaCb/ 10 mM Tris pH 7.5) was added drop by drop and mixed. The solution was kept at room temperature for 20 minutes. The tubes were then filled with 8 ml STC1700, mixed and centrifuged at 2500 rpm for 10 minutes at 4°C, and the pellet was suspended in 250 μl STC1700. Aliquots of the sample were used for plating on selective medium. For amdS + selection, plates were prepared containing 1.5% Oxoid agar, 1.2 M sorbitol, 2mM MgSO4.7 H2O, Ix trace elements, 1% glucose, Ix AspA without nitrate, 15mM CsCl and 1OmM acetamide. The plates were incubated at 30 or 35°C. The spores and viable protoplasts before and after PEG6000 treatment were counted by plating dilutions in STC 1700 on minimal medium plates with nitrate and with or without sorbitol. 100 μl of 10 ¹ , 10 ² and 10 ³ dilutions were plated on plates without sorbitol to count for spores and 100 μl of 10 ² , 10 ³ , 10 ⁴ and IO ⁵ dilutions were plated on plates with sorbitol to count the viable protoplasts.

Example 4 Selection of transformants

Twelve transformants of UV18#100.f δpyrδ δalpl δpep4 δalp2 Phy7789ab, were purified. The transformants were grown in 3 ml cultures. Phytase activity (as described in Piddington et al, (1993) Gene 133. 55-62) was measured in these medium samples. Activity could be measured in some medium samples of the transformants. A transformant was selected for controlled fermentation (UV18#100.f δpyrδ δalpl δpep4 δalp2 Phy7789ab#3). Southern analysis showed that 2-3 phytase expression copies were integrated in each transformant.

Example 4 determination of phytase activity

The transformant was grown in controlled fed batch fermentation. Phytase activity was determined in end- of- fermentation samples as described in Piddington et al at pH5.5 and at pH 7.0 with the addition of ImMCaCb.

In another non-limiting example of this invention the phytase activity from phytase produced by glucoamylase phytase fusions was measured.