Phytases, nucleic acids encoding them and methods for making and using them

An isolated, synthetic or recombinant phytase polypeptide comprising

• (a) (i) an amino acid sequence having at least 95%, 96% 97%, 98% or 99% sequence identity to SEQ ID NO:2, and encoded by a polynucleotide comprising at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or all fourteen of the nucleotide base pair sequence modifications to SEQ ID NO:1, selected from the group consisting of:
• the nucleotides at positions 139 to 141 are TTT or TTC;
• the nucleotides at positions 289 to 291 are GTT, GTC, GTA or GTG;
• the nucleotides at positions 289 to 291 are GAA or GAG;
• the nucleotides at positions 406 to 408 are CAT or CAC;
• the nucleotides at positions 475 to 477 are GTT, GTC, GTA or GTG;
• the nucleotides at positions 475 to 477 are GAA or GAG;
• the nucleotides at positions 487 to 489 are CGT, CGC, CGA, CGG, AGA or AGG;
• the nucleotides at positions 490 to 492 are CGT, CGC, CGA, CGG, AGA or AGG;
• the nucleotides at positions 502 to 504 are CGT, CGC, CGA, CGG, AGA or AGG;
• the nucleotides at positions 535 to 537 are CGT, CGC, CGA, CGG, AGA or AGG;
• the nucleotides at positions 676 to 678 are GAT or GAC;
• the nucleotides at positions 697 to 699 are TGG;
• the nucleotides at positions 823 to 825 are GTT, GTC, GTA or GTG;
• the nucleotides at positions 865 to 867 are GCT, GCC, GCA or GCG;
• the nucleotides at positions 1045 to 1047 are TAT or TAC;
• and
• the nucleotides at positions 1087 to 1089 are CCA, CCC, CCG or CCT;
• (ii) an amino acid sequence having at least 95%, 96% 97%, 98% or 99% sequence identity to SEQ ID NO:2, and encoded by a polynucleotide comprising at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or all fourteen of the nucleotide base pair sequence modifications to SEQ ID NO:1, selected from the group consisting of:
• the nucleotides at the equivalent of positions 139 to 141 are TTT or TTC;
• the nucleotides at the equivalent of positions 289 to 291 are GTT, GTC, GTA or GTG;
• the nucleotides at the equivalent of positions 289 to 291 are GAA or GAG;
• the nucleotides at the equivalent of positions 406 to 408 are CAT or CAC;
• the nucleotides at the equivalent of positions 475 to 477 are GTT, GTC, GTA or GTG;
• the nucleotides at the equivalent of positions 475 to 477 are GAA or GAG;
• the nucleotides at the equivalent of positions 487 to 489 are CGT, CGC, CGA, CGG, AGA or AGG;
• the nucleotides at the equivalent of positions 490 to 492 are CGT, CGC, CGA, CGG, AGA or AGG;
• the nucleotides at the equivalent of positions 502 to 504 are CGT, CGC, CGA, CGG, AGA or AGG;
• the nucleotides at the equivalent of positions 535 to 537 are CGT, CGC, CGA, CGG, AGA or AGG;
• the nucleotides at the equivalent of positions 676 to 678 are GAT or GAC;
• the nucleotides at the equivalent of positions 697 to 699 are TGG;
• the nucleotides at the equivalent of positions 823 to 825 are GTT, GTC, GTA or GTG;
• the nucleotides at the equivalent of positions 865 to 867 are GCT, GCC, GCA or GCG;
• the nucleotides at the equivalent of positions 1045 to 1047 are TAT or TAC;
• and
• the nucleotides at the equivalent of positions 1087 to 1089 are CCA, CCC, CCG or CCT;
• (iii) an amino acid sequence comprising at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or all fourteen of the amino acid residue modifications to SEQ ID NO:2, selected from the group consisting of:
• the amino acid alanine at amino acid position 47 of SEQ ID NO:2 is changed to a phenylalanine;
• the amino acid cysteine at amino acid position 97 of SEQ ID NO:2 is changed to a valine;
• the amino acid cysteine at amino acid position 97 of SEQ ID NO:2 is changed to a glutamic acid;
• the amino acid at the equivalent of the threonine at amino acid position 136 of SEQ ID NO:2 is changed to a histidine;
• the amino acid asparagine at amino acid position 159 of SEQ ID NO:2 is changed to a valine;
• the amino acid asparagine at amino acid position 159 of SEQ ID NO:2 is changed to a glutamic acid;
• the amino acid at the equivalent of the threonine at amino acid position 163 of SEQ ID NO:2 is changed to an arginine;
• the amino acid aspartic acid at amino acid position 164 of SEQ ID NO:2 is changed to an arginine;
• the amino acid glutamic acid at amino acid position 168 of SEQ ID NO:2 is changed to an arginine;
• the amino acid glycine at amino acid position 179 of SEQ ID NO:2 is changed to an arginine;
• the amino acid cysteine at amino acid position 226 of SEQ ID NO:2 is changed to an aspartic acid;
• the amino acid valine at amino acid position 233 of SEQ ID NO:2 is changed to a tryptophan;
• the amino acid glutamine at amino acid position 275 of SEQ ID NO:2 is changed to a valine;
• the amino acid arginine at amino acid position 289 of SEQ ID NO:2 is changed to an alanine;
• the amino acid threonine at amino acid position 349 of SEQ ID NO:2 is changed to a tyrosine;
• and
• the amino acid leucine at amino acid position 363 of SEQ ID NO:2 is changed to a proline;
• or
• (iv) an amino acid sequence comprising at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or all fourteen of the amino acid residue modifications to SEQ ID NO:2, selected from the group consisting of:
• the amino acid at the equivalent of the alanine at amino acid position 47 of SEQ ID NO:2 is changed to a phenylalanine;
• the amino acid at the equivalent of the cysteine at amino acid position 97 of SEQ ID NO:2 is changed to a valine;
• the amino acid at the equivalent of the cysteine at amino acid position 97 of SEQ ID NO:2 is changed to a glutamic acid;
• the amino acid at the equivalent of the threonine at amino acid position 136 of SEQ ID NO:2 is changed to a histidine;
• the amino acid at the equivalent of the asparagine at amino acid position 159 of SEQ ID NO:2 is changed to a valine;
• the amino acid at the equivalent of the asparagine at amino acid position 159 of SEQ ID NO:2 is changed to a glutamic acid;
• the amino acid at the equivalent of the threonine at amino acid position 163 of SEQ ID NO:2 is changed to an arginine;
• the amino acid at the equivalent of the aspartic acid at amino acid position 164 of SEQ ID NO:2 is changed to an arginine;
• the amino acid at the equivalent of the glutamic acid at amino acid position 168 of SEQ ID NO:2 is changed to an arginine;
• the amino acid at the equivalent of the glycine at amino acid position 179 of SEQ ID NO:2 is changed to an arginine;
• the amino acid at the equivalent of the cysteine at amino acid position 226 of SEQ ID NO:2 is changed to an aspartic acid;
• the amino acid at the equivalent of the valine at amino acid position 233 of SEQ ID NO:2 is changed to a tryptophan;
• the amino acid at the equivalent of the glutamine at amino acid position 275 of SEQ ID NO:2 is changed to a valine;
• the amino acid at the equivalent of the arginine at amino acid position 289 of SEQ ID NO:2 is changed to an alanine;
• the amino acid at the equivalent of the threonine at amino acid position 349 of SEQ ID NO:2 is changed to a tyrosine;
• and
• the amino acid at the equivalent of the leucine at amino acid position 363 of SEQ ID NO:2 is changed to a proline;
• (b) the polypeptide of (a) having a phytase activity but lacking:
• a signal sequence or proprotein sequence;
• (c) the polypeptide of (a) having a phytase activity and further comprising a heterologous sequence;
• (d) the polypeptide of (c), wherein the heterologous amino acid sequence comprises, or consists of a heterologous (leader) signal sequence, or a tag or an epitope, or the heterologous sequence comprises an identification peptide;
• (e)
• the polypeptide of any of (a) to (d), wherein (i) the polypeptide is glycosylated, or the polypeptide comprises at least one glycosylation site, (ii) the polypeptide of (i) wherein the glycosylation is an N-linked glycosylation or an O-linked glycosylation;
• (iii) the polypeptide of (i) or (ii) wherein the polypeptide is glycosylated after being expressed in a yeast cell;
• or (iv) the polypeptide of (iii) wherein the yeast cell is a P. pastoris or a S. pombe;
• or
• (f) the polypeptide of any one of (a) to (e), wherein the polypeptide further comprises additional amino acid residues between a signal sequence (leader sequence or leader peptide) and the enzyme.