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US 7,232,677 B2Granted
Phytase expression systems and methods of making and using them
AssigneeDiversa Corporation
InventorsJay M. Short, Keith A. Kretz
Life of this patent
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Abstract
The invention provides a purified or recombinant phytase enzyme (SEQ ID NO:2) initially derived from Escherichia coli B. The enzyme has a molecular weight of about 47.1 kilodaltons and has phytase activity (SEQ ID NO:2). The enzyme can be produced from native or recombinant host cells and can be used to aid in the digestion of phytate where desired. In particular, the phytase of the present invention can be used in foodstuffs to improve the feeding value of phytate rich ingredients.
Claims54
A recombinant expression system comprising an isolated host cell comprising a nucleic acid encoding a phytase enzyme
- (i) comprising the amino acid sequence of SEQ ID NO:2,
- (ii) comprising the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432;
- (iii) comprising (A) the amino acid sequence of SEQ ID NO:2 but lacking a homologous signal sequence, or (B) the amino acid sequence of (A) further comprising a heterologous signal sequence,
- (iv) comprising (A) the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432 but lacking a homologous signal sequence, or (B) the amino acid sequence of (A) further comprising a heterologous signal sequence, or
- (v) comprising the amino acid sequence of (i), (ii), (iii) or (iv) and further comprising a heterologous amino acid sequence,
- wherein the nucleic acid encoding the phytase is operably linked to a transcription control sequence.
3 more elements
A vector comprising a nucleic acid
- (i) comprising the nucleotide sequence of SEQ ID NO:1,
- (ii) comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide residue 1 to 1296,
- (iii) comprising (A) the nucleotide sequence of (i) or (ii) lacking a nucleotide sequence encoding a homologous signal sequence, or (B) the nucleotide sequence of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence, or
- (iv) comprising the nucleotide sequence of (i), (ii), or (iii), further comprising a heterologous nucleotide sequence.
1 more element
A prokaryotic cell comprising an exogenous nucleic acid encoding a phytase enzyme, wherein the nucleic acid encoding the phytase is operably linked to a transcriptional control sequence and the phytase enzyme
- (i) is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1,
- (ii) is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide residue 1 to 1296,
- (iii) is encoded by (A) the nucleic acid of (i) or (ii), lacking a nucleotide sequence encoding a homologous signal sequence, or (B) the nucleotide sequence of (A) that further comprises a nucleotide sequence encoding heterologous signal sequence,
- (iv) comprises the amino acid sequence of SEQ ID NO:2,
- (v) comprises the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432,
- (vi) comprises (A) the amino acid sequence of (iv) or (v), lacking a homologous signal sequence, or (B) the amino acid sequence of (A) and further comprising a heterologous signal sequence,
- (vii) is encoded by the nucleic acid of (i), (ii), or (iii) that further comprises a heterologous nucleotide sequence, or
- (viii) comprises the amino acid sequence of (iv), (v) or (vi) that further comprises a heterologous amino acid sequence.
5 more elements
An isolated eukaryotic cell comprising an exogenous nucleic acid encoding a phytase enzyme, wherein the nucleic acid is operably linked to a transcriptional control sequence and the phytase enzyme
- (i) is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1,
- (ii) is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide residue 1 to 1296,
- (iii) is encoded by (A) the nucleic acid of (i) or (ii), lacking a nucleotide sequence encoding a homologous signal sequence, or (B) the nucleotide sequence of (A) that further comprises a nucleotide sequence encoding heterologous signal sequence,
- (iv) comprises the amino acid sequence of SEQ ID NO:2,
- (v) comprises the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432,
- (vi) comprises (A) the amino acid sequence of (iv) or (v), lacking a homologous signal sequence, or (B) the amino acid sequence of (A) that further comprises a heterologous signal sequence,
- (vii) is encoded by the nucleic acid of (i), (ii), or (iii) that further comprises a nucleotide sequence encoding a heterologous amino acid sequence, or
- (viii) comprises the amino acid sequence of (iv), (v) or (vi) that further comprises a heterologous amino acid sequence.
5 more elements
An isolated cell comprising an exogenous nucleic acid encoding a phytase enzyme, wherein the nucleic acid is operably linked to a transcriptional control sequence and the phytase enzyme
- (i) is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1,
- (ii) is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide residue 1 to 1296,
- (iii) is encoded by (A) the nucleic acid of (i) or (ii), lacking a nucleotide sequence encoding a homologous signal sequence, or (B) the nucleotide sequence of (A) that further comprises a nucleotide sequence encoding heterologous signal sequence,
- (iv) comprises the amino acid sequence of SEQ ID NO:2,
- (v) comprises the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432,
- (vi) comprises (A) the amino acid sequence of (iv) or (v), lacking a homologous signal sequence, or (B) the amino acid sequence of (A) that further comprises a heterologous signal sequence,
- (vii) is encoded by the nucleic acid of (i), (ii), or (iii) and further comprises a nucleotide sequence encoding a heterologous amino acid sequence, or
- (viii) comprises the amino acid sequence of (iv), (v) or (vi) that further comprises a heterologous amino acid sequence.
5 more elements
An expression system for making a polypeptide having phytase activity, comprising an isolated host cell and an exogenous nucleic acid, wherein the exogenous nucleic acid codes for the polypeptide having phytase activity and wherein the isolated host cell is capable of expressing the polypeptide, and the polypeptide having phytase activity
- (i) is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1,
- (ii) is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide residue 1 to 1296,
- (iii) is encoded by (A) the nucleic acid of (i) or (ii), lacking a nucleotide sequence encoding a homologous signal sequence, or (B) the nucleotide sequence of (A) that further comprises a nucleotide sequence encoding heterologous signal sequence, or
- (iv) is encoded by the nucleic acid of (i), (ii) or (iii) and further comprises a heterologous nucleotide sequence.
1 more element
A vector comprising a nucleic acid
- (i) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2;
- (ii) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432;
- (iii) encoding a polypeptide (A) comprising the amino acid sequence of SEQ ID NO:2 and lacking a homologous signal sequence, or (B) comprising the amino acid sequence of (A) and further comprising a heterologous signal sequence;
- (iv) encoding a polypeptide (A) comprising the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432 and lacking a homologous signal sequence, or (B) comprising the amino acid sequence of (A) and further comprising a heterologous signal sequence;
- (v) encoding a polypeptide comprising the amino acid sequence of (i), (ii), (iii), or (iv), and further comprising a heterologous nucleotide sequence;
- or
- (vi) completely complementary to any of the nucleic acids of (i)–(v).
4 more elements
An isolated cell comprising a vector comprising a nucleic acid
- (i) comprising the nucleotide sequence of SEQ ID NO:1;
- (ii) comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide residue 1 to 1296;
- (iii) comprising (A) the nucleotide sequence of (i) or (ii) lacking a nucleotide sequence encoding a homologous signal sequence, or (B) the nucleotide sequence of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (iv) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2;
- (v) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432;
- (vi) encoding (A) the polypeptide of (iv) or (v) lacking a homologous signal sequence, or (B) the polypeptide of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (vii) comprising the nucleotide sequence of (i), (ii), or (iii) and further comprising a heterologous nucleotide sequence;
- (viii) encoding the polypeptide of (iv), (v), or (vi) and further comprising a nucleotide sequence encoding a heterologous amino acid sequence;
- or
- (ix) comprising a nucleotide sequence that is the complete complement of (i), (ii), (iii), or (vii), or the complete complement of the nucleotide sequence encoding the polypeptide of (iv), (v), (vi) or (viii).
7 more elements
A cloning vector comprising a nucleic acid
- (i). comprising the nucleotide sequence of SEQ ID NO:1;
- (ii) comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide residue 1 to 1296;
- (iii) comprising (A) the nucleotide sequence of (i) or (ii) lacking a nucleotide sequence encoding a homologous signal sequence, or (B) the nucleotide sequence of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (iv) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2;
- (v) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432;
- (vi) encoding (A) the polypeptide of (iv) or (v) lacking a homologous signal sequence, or (B) the polypeptide of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (vii) comprising the nucleotide sequence of (i), (ii), or (iii) and further comprising a heterologous nucleotide sequence;
- (viii) encoding the polypeptide of (iv), (v), or (vi) and further comprising a nucleotide sequence encoding a heterologous amino acid sequence;
- or
- (ix) comprising a nucleotide sequence that is the complete complement of (i), (ii), (iii), or (vii), or the complete complement of the nucleotide sequence encoding the polypeptide of (iv), (v), (vi) or (viii).
7 more elements
An expression vector comprising a nucleic acid
- (i) comprising the nucleotide sequence of SEQ ID NO:1;
- (ii) comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide residue 1 to 1296;
- (iii) comprising (A) the nucleotide sequence of (i) or (ii) lacking a nucleotide sequence encoding a homologous signal sequence, or (B) the nucleotide sequence of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (iv) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2;
- (v) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432;
- (vi) encoding (A) the polypeptide of (iv) or (v) lacking a homologous signal sequence, or (B) the polypeptide of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (vii) comprising the nucleotide sequence of (i), (ii), or (iii) and further comprising a heterologous nucleotide sequence;
- (viii) encoding the polypeptide of (iv), (v), or (vi) and further comprising a nucleotide sequence encoding a heterologous amino acid sequence;
- or
- (ix) comprising a nucleotide sequence that is the complete complement of (i), (ii), (iii), or (vii), or the complete complement of the nucleotide sequence encoding the polypeptide of (iv), (v), (vi) or (viii).
7 more elements
A bacterial vector comprising a nucleic acid
- (i) comprising the nucleotide sequence of SEQ ID NO:1;
- (ii) comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide residue 1 to 1296;
- (iii) comprising (A) the nucleotide sequence of (i) or (ii) lacking a nucleotide sequence encoding a homologous signal sequence, or (B) the nucleotide sequence of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (iv) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2;
- (v) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432;
- (vi) encoding (A) the polypeptide of (iv) or (v) lacking a homologous signal sequence, or (B) the polypeptide of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (vii) comprising the nucleotide sequence of (i), (ii), or (iii) and further comprising a heterologous nucleotide sequence;
- (viii) encoding the polypeptide of (iv), (v), or (vi) and further comprising a nucleotide sequence encoding a heterologous amino acid sequence;
- or
- (ix) comprising a nucleotide sequence that is the complete complement of (i), (ii), (iii), or (vii), or the complete complement of the nucleotide sequence encoding the polypeptide of (iv), (v), (vi) or (viii).
7 more elements
A plasmid comprising a nucleic acid
- (i) comprising the nucleotide sequence of SEQ ID NO:1;
- (ii) comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide residue 1 to 1296;
- (iii) comprising (A) the nucleotide sequence of (i) or (ii) lacking a nucleotide sequence encoding a homologous signal sequence, or (B) the nucleotide sequence of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (iv) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2;
- (v) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432;
- (vi) encoding (A) the polypeptide of (iv) or (v) lacking a homologous signal sequence, or (B) the polypeptide of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (vii) comprising the nucleotide sequence of (i), (ii), or (iii) and further comprising a heterologous nucleotide sequence;
- (viii) encoding the polypeptide of (iv), (v), or (vi) and further comprising a nucleotide sequence encoding a heterologous amino acid sequence;
- or
- (ix) comprising a nucleotide sequence that is the complete complement of (i), (ii), (iii), or (vii), or the complete complement of the nucleotide sequence encoding the polypeptide of (iv), (v), (vi) or (viii).
7 more elements
A viral particle comprising a nucleic acid
- (i) comprising the nucleotide sequence of SEQ ID NO:1;
- (ii) comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide residue 1 to 1296;
- (iii) comprising (A) the nucleotide sequence of (i) or (ii) lacking a nucleotide sequence encoding a homologous signal sequence, or (B) the nucleotide sequence of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (iv) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2;
- (v) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432;
- (vi) encoding (A) the polypeptide of (iv) or (v) lacking a homologous signal sequence, or (B) the polypeptide of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (vii) comprising the nucleotide sequence of (i), (ii), or (iii) and further comprising a heterologous nucleotide sequence;
- (viii) encoding the polypeptide of (iv), (v), or (vi) and further comprising a nucleotide sequence encoding a heterologous amino acid sequence;
- or
- (ix) comprising a nucleotide sequence that is the complete complement of (i), (ii), (iii), or (vii), or the complete complement of the nucleotide sequence encoding the polypeptide of (iv), (v), (vi) or (viii).
7 more elements
A phage comprising a nucleic acid
- (i) comprising the nucleotide sequence of SEQ ID NO:1;
- (ii) comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide residue 1 to 1296;
- (iii) comprising (A) the nucleotide sequence of (i) or (ii) lacking a nucleotide sequence encoding a homologous signal sequence, or (B) the nucleotide sequence of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (iv) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2;
- (v) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432;
- (vi) encoding (A) the polypeptide of (iv) or (v) lacking a homologous signal sequence, or (B) the polypeptide of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (vii) comprising the nucleotide sequence of (i), (ii), or (iii) and further comprising a heterologous nucleotide sequence;
- (viii) encoding the polypeptide of (iv), (v), or (vi) and further comprising a nucleotide sequence encoding a heterologous amino acid sequence;
- or
- (ix) comprising a nucleotide sequence that is the complete complement of (i), (ii), (iii), or (vii), or the complete complement of the nucleotide sequence encoding the polypeptide of (iv), (v), (vi) or (viii).
7 more elements
A recombinant expression system comprising a nucleic acid encoding a phytase enzyme
- (i) having (A) the amino acid sequence of SEQ ID NO:2 lacking a homologous signal sequence, or (B) the amino acid sequence of (A) further comprising a heterologous signal sequence;
- or
- (ii) having (A) the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432 lacking a homologous signal sequence, or (B) the amino acid sequence of (A) further comprising a heterologous signal sequence.
A method for making a phytase in a cell, wherein the phytase is encoded by an exogenous nucleic acid, comprising culturing the cell under conditions wherein the phytase is expressed,
- wherein the exogenous nucleic acid
- (i) comprises the nucleotide sequence of SEQ ID NO:1;
- (ii) comprises the nucleotide sequence of SEQ ID NO:1 from nucleotide residue 1 to 1296;
- (iii) comprises (A) the nucleotide sequence of (i)or (ii) lacking a nucleotide sequence encoding a homologous signal sequence, or (B) the nucleotide sequence of (A) and further comprises a nucleotide sequence encoding a heterologous signal sequence;
- or
- (iv) comprises the nucleotide sequence of (i), (ii), or (iii) and a heterologous nucleotide sequence.
3 more elements
An expression system for making a polypeptide having phytase activity, comprising an isolated host cell and an exogenous nucleic acid, wherein the exogenous nucleic acid codes for the polypeptide having phytase activity, wherein the isolated host cell is capable of expressing the polypeptide, and the polypeptide having phytase activity
- (i) comprises the amino acid sequence of SEQ ID NO:2;
- (ii) comprises the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432;
- (iii) comprises (A) the amino acid sequence of (i) or (ii), wherein the amino acid sequence lacks a homologous signal sequence, or (B) the amino acid sequence of (A) further comprising a heterologous signal sequence;
- (iv) comprises the amino acid sequence of (i), (ii) or (iii) and a heterologous amino acid sequence;
- or
- (v) comprises enzymatically active fragments of (i), (ii), (iii) or (iv).
3 more elements
A recombinant expression system comprising an isolated host cell comprising a variant nucleic acid encoding a polypeptide having phytase activity, wherein the variant nucleic acid
- (1) encodes a polypeptide
- (i) comprising the amino acid sequence of SEQ ID NO:2 and further comprising at least one conservative amino acid substitution;
- (ii) comprising the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432 and further comprising at least one conservative amino acid substitution;
- (iii) comprising (A) the amino acid sequence of SEQ ID NO:2 and further comprising at least one conservative amino acid substitution, wherein the amino acid sequence lacks a homologous signal sequence, or (B) the amino acid sequence of (A) and further comprising a heterologous signal sequence;
- (iv) comprising (A) the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432 and further comprising at least one conservative amino acid substitution, wherein the amino acid sequence lacks a homologous signal sequence, or (B) the amino acid sequence of (A) and further comprising a heterologous signal sequence;
- or
- (v) comprising the amino acid sequence of (i), (ii), (iii), or (iv), and further comprising a heterologous amino acid sequence;
- wherein the conservative amino acid substitution comprises (a) a replacement, one for another, among the aliphatic amino acids Ala, Val, Leu, and Be, (b) an interchange of the hydroxyl residues Ser and Thr, (c) an exchange of the acidic residues Asp and Glu, (d) a substitution between the amide residues Asn and Gln, (e) an exchange of the basic residues Lys and Mg, (f) a replacement among the aromatic residues Phe, Tyr, or (g) any combination of (a), (b), (c), (d), (e), or (f);
- (2) encodes enzymatically active fragments of (1);
- or
- (3) comprises a nucleotide sequence that is the complete complement of the nucleotide sequence encoding the polypeptide of (1) or (2);
- wherein the nucleic acid encoding the polypeptide having phytase activity is operably linked to a transcriptional control sequence, and the amino acid sequence of the polypeptide having phytase activity has at least 95% sequence identity to SEQ ID NO:2.
9 more elements
An isolated prokaryotic cell comprising a variant nucleic acid encoding a polypeptide having phytase activity, wherein the variant nucleic acid
- (1) encodes a polypeptide
- (i) comprising the amino acid sequence of SEQ ID NO:2 and further comprising at least one conservative amino acid substitution;
- (ii) comprising the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432 and further comprising at least one conservative amino acid substitution;
- (iii) comprising (A) the amino acid sequence of SEQ ID NO:2 and further comprising at least one conservative amino acid substitution, wherein the amino acid sequence lacks a homologous signal sequence, or (B) the amino acid sequence of (A) and further comprising a heterologous signal sequence;
- (iv) comprising (A) the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432 and further comprising at least one conservative amino acid substitution, wherein the amino acid sequence lacks a homologous signal sequence, or (B) the amino acid sequence of (A) and further comprising a heterologous signal sequence;
- or
- (v) comprising the amino acid sequence of (i), (ii), (iii), or (iv), and further comprising a heterologous amino acid sequence;
- wherein the conservative amino acid substitution comprises (a) a replacement, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile, (b) an interchange of the hydroxyl residues Ser and Thr, (c) an exchange of the acidic residues Asp and Glu, (d) a substitution between the amide residues Asn and Gln, (e) an exchange of the basic residues Lys and Arg, (f) a replacement among the aromatic residues Phe, Tyr, or (g) any combination of (a), (b), (c), (d), (e), or (f);
- (2) encodes enzymatically active fragments of (1);
- or
- (3) comprises a nucleotide sequence that is the complete complement of the nucleotide sequence encoding the polypeptide of (1) or (2);
- wherein the nucleic acid encoding the polypeptide having phytase activity is operably linked to a transcriptional control sequence, and the amino acid sequence of the polypeptide having phytase activity has at least 95% sequence identity to SEQ ID NO:2.
9 more elements
A vector comprising a variant nucleic acid encoding a polypeptide having phytase activity, wherein the variant nucleic acid
- (1) encodes a polypeptide
- (i) comprising the amino acid sequence of SEQ ID NO:2 and further comprising at least one conservative amino acid substitution;
- (ii) comprising the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432 and further comprising at least one conservative amino acid substitution;
- (iii) comprising (A) the amino acid sequence of SEQ ID NO:2 and further comprising at least one conservative amino acid substitution, wherein the amino acid sequence lacks a homologous signal sequence, or (B) the amino acid sequence of (A) and further comprising a heterologous signal. sequence;
- (iv) comprising (A) the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432 and further comprising at least one conservative amino acid substitution, wherein the amino acid sequence lacks a homologous signal sequence, or (B) the amino acid sequence of (A) and further comprising a heterologous signal sequence;
- or
- (v) comprising the amino acid sequence of (i), (ii), (iii), or (iv), and further comprising a heterologous amino acid sequence;
- wherein the conservative amino acid substitution comprises (a) a replacement, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile, (b) an interchange of the hydroxyl residues Ser and Thr, (c) an exchange of the acidic residues Asp and Glu, (d) a substitution between the amide residues Asn and Gln, (e) an exchange of the basic residues Lys and Mg, (f) a replacement among the aromatic residues Phe, Tyr, or (g) any combination of (a), (b), (c), (d), (e), or (f);
- (2) encodes enzymatically active fragments of (1);
- or
- (3) comprises a nucleotide sequence that is the complete complement of the nucleotide sequence encoding the polypeptide of (1) or (2);
- wherein the nucleic acid encoding the polypeptide having phytase activity is operably linked to a transcriptional control sequence, and the amino acid sequence of the polypeptide having phytase activity has at least 95% sequence identity to SEQ ID NO:2.
9 more elements
An isolated host cell comprising a vector comprising a variant nucleic acid encoding a polypeptide having phytase activity, wherein the variant nucleic acid
- (1) encodes a polypeptide
- (i) comprising the amino acid sequence of SEQ ID NO:2 and further comprising at least one conservative amino acid substitution;
- (ii) comprising the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432 and further comprising at least one conservative amino acid substitution;
- (iii) comprising (A) the amino acid sequence of SEQ ID NO:2 and further comprising at least one conservative amino acid substitution, wherein the amino acid sequence lacks a homologous signal sequence, or (B) the amino acid sequence of (A) and further comprising a heterologous signal sequence;
- (iv) comprising (A) the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432 and further comprising at least one conservative amino acid substitution, wherein the amino acid sequence lacks a homologous signal sequence, or (B) the amino acid sequence of (A) and further comprising a heterologous signal sequence;
- or
- (v) comprising the amino acid sequence of (i), (ii), (iii), or (iv), and further comprising a heterologous amino acid sequence;
- wherein the conservative amino acid substitution comprises (a) a replacement, one for another, among the aliphatic amino acids Ala, Val, Leu, and lie, (b) an interchange of the hydroxyl residues Ser and Thr, (c) an exchange of the acidic residues Asp and Glu, (d) a substitution between the amide residues Asn and Gln, (e) an exchange of the basic residues Lys and Arg, (f) a replacement among the aromatic residues Phe, Tyr, or (g) any combination of (a), (b), (c), (d), (e), or (f);
- (2) encodes enzymatically active fragments of (1);
- or
- (3) comprises a nucleotide sequence that is the complete complement of the nucleotide sequence encoding the polypeptide of (1) or (2);
- wherein the nucleic acid encoding the polypeptide having phytase activity is operably linked to a transcriptional control sequence, and the amino acid sequence of the polypeptide having phytase activity has at least 95% sequence identity to SEQ ID NO:2.
9 more elements
An isolated host cell comprising a variant exogenous nucleic acid encoding a polypeptide having phytase activity, wherein the variant nucleic acid
- (1) encodes a polypeptide
- (i) comprising the amino acid sequence of SEQ ID NO:2 and further comprising at least one conservative amino acid substitution;
- (ii) comprising the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432 and further comprising at least one conservative amino acid substitution;
- (iii) comprising (A) the amino acid sequence of SEQ ID NO:2 and further comprising at least one conservative amino acid substitution, wherein the amino acid sequence lacks a homologous signal sequence, or (B) the amino acid sequence of (A) and further comprising a heterologous signal sequence;
- (iv) comprising (A) the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432 and further comprising at least one conservative amino acid substitution, wherein the amino acid sequence lacks a homologous signal sequence, or (B) the amino acid sequence of (A) and further comprising a heterologous signal sequence;
- or
- (v) comprising the amino acid sequence of (i), (ii), (iii), or (iv), and further comprising a heterologous amino acid sequence;
- wherein the conservative amino acid substitution comprises (a) a replacement, one for another, among the aliphatic amino acids Ala, Val, Leu, and lie, (b) an interchange of the hydroxyl residues Ser and Thr, (c) an exchange of the acidic residues Asp and Glu, (d) a substitution between the amide residues Asn and Gin, (e) an exchange of the basic residues Lys and Mg, (f) a replacement among the aromatic residues Phe, Tyr, or (g) any combination of (a), (b), (c), (d), (e), or (f);
- (2) encodes enzymatically active fragments of (1);
- or
- (3) comprises a nucleotide sequence that is the complete complement of the nucleotide sequence encoding the polypeptide of (1) or (2);
- wherein the nucleic acid encoding the polypeptide having phytase activity is operably linked to a transcriptional control sequence, and the amino acid sequence of the polypeptide having phytase activity has at least 95% sequence identity to SEQ ID NO:2.
9 more elements
An isolated eukaryotic cell comprising a variant nucleic acid encoding a polypeptide having phytase activity, wherein the variant nucleic acid
- (1) encodes a polypeptide
- (i) comprising the amino acid sequence of SEQ ID NO:2 and further comprising at least one conservative amino acid substitution;
- (ii) comprising the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432 and further comprising at least one conservative amino acid substitution;
- (iii) comprising (A) the amino acid sequence of SEQ ID NO:2 and further comprising at least one conservative amino acid substitution, wherein the amino acid sequence lacks a homologous signal sequence, or (B) the amino acid sequence of (A) and further comprising a heterologous signal sequence;
- (iv) comprising (A) the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432 and further comprising at least one conservative amino acid substitution, wherein the amino acid sequence lacks a homologous signal sequence, or (B) the amino acid sequence of (A) and further comprising a heterologous signal sequence;
- or
- (v) comprising the amino acid sequence of (i), (ii), (iii), or (iv), and further comprising a heterologous amino acid sequence;
- wherein the conservative amino acid substitution comprises (a) a replacement, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile, (b) an interchange of the hydroxyl residues Ser and Thr, (c) an exchange of the acidic residues Asp and Glu, (d) a substitution between the amide residues Asn and Gln, (e) an exchange of the basic residues Lys and Arg, (f) a replacement among the aromatic residues Phe, Tyr, or (g) any combination of (a), (b), (c), (d), (e), or (f);
- (2) encodes enzymatically active fragments of (1);
- or
- (3) comprises a nucleotide sequence that is the complete complement of the nucleotide sequence encoding the polypeptide of (1) or (2);
- wherein the nucleic acid encoding the polypeptide having phytase activity is operably linked to a transcriptional control sequence, and the amino acid sequence of the polypeptide having phytase activity has at least 95% sequence identity to SEQ ID NO:2.
9 more elements
An expression system comprising a nucleic acid:
- (i) comprising the nucleotide sequence of SEQ ID NO:1;
- (ii) comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide residue 1 to 1296;
- (iii) comprising (A) the nucleotide sequence of (i) or (ii) lacking a nucleotide sequence encoding a homologous signal sequence, or (B) the nucleotide sequence of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (iv) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2;
- (v) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432;
- (vi) encoding (A) the polypeptide of (iv) or (v) lacking a homologous signal sequence, or (B) the polypeptide of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (vii) comprising the nucleotide sequence of (i), (ii), or (iii) and further comprising a heterologous nucleotide sequence;
- (viii) encoding the polypeptide of (iv), (v), or (vi) and further comprising a nucleotide sequence encoding a heterologous amino acid sequence;
- or
- (ix) comprising a nucleotide sequence that is the complete complement of (i), (ii), (iii), or (vii), or the complete complement of the nucleotide sequence encoding the polypeptide of (iv), (v), (vi) or (viii).
7 more elements
An isolated, synthetic or recombinant nucleic acid:
- (i) comprising the nucleotide sequence of SEQ ID NO:1;
- (ii) comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide residue 1 to 1296;
- (iii) composing (A) the nucleotide sequence of (i) or (ii) lacking a nucleotide sequence encoding a homologous signal sequence, or (B) the nucleotide sequence of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (iv) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2;
- (v) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432;
- (vi) encoding (A) the polypeptide of (iv) or (v) lacking a homologous signal sequence, or (B) the polypeptide of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (vii) comprising the nucleotide sequence of (i), (ii), or (iii) and further comprising a heterologous nucleotide sequence;
- (viii) encoding the polypeptide of (iv), (v), or (vi) and further comprising a nucleotide sequence encoding a heterologous amino acid sequence;
- or
- (ix) comprising a nucleotide sequence that is the complete complement of (i), (ii), (iii), or (vii), or the complete complement of the nucleotide sequence encoding the polypeptide of (iv), (v), (vi) or (viii).
7 more elements
A method for glycosylating a polypeptide comprising:
- (a) providing a nucleic acid comprising a sequence encoding a polypeptide having phytase activity to a cell, wherein the nucleic acid has a nucleotide sequence:
- (i) comprising the nucleotide sequence of SEQ ID NO:1;
- (ii) comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide residue 1 to 1296;
- (iii) comprising (A) the nucleotide sequence of (i) or (ii) lacking a nucleotide sequence encoding a homologous signal sequence, or (B) the nucleotide sequence of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (iv) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2;
- (v) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432;
- (vi) encoding (A) the polypeptide of (iv) or (v) lacking a homologous signal sequence, or (B) the polypeptide of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (vii) comprising the nucleotide sequence of (i), (ii), or (iii) and further comprising a heterologous nucleotide sequence;
- (viii) encoding the polypeptide of (iv), (v), or (vi) and further comprising a nucleotide sequence encoding a heterologous amino acid sequence;
- or
- (ix) comprising a nucleotide sequence that is the complete complement of (i), (ii), (iii), or (vii), or the complete complement of the nucleotide sequence encoding the polypeptide of (iv), (v), (vi) or (viii);
- and
- (b) expressing the polypeptide in the cell wherein the cell is capable of glycosylating the polypeptide.
10 more elements
An isolated transformed cell or an isolated host cell comprising an exogenous nucleic acid:
- (i) comprising the nucleotide sequence of SEQ ID NO:1;
- (ii) comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide residue 1 to 1296;
- (iii) comprising (A) the nucleotide sequence of (i) or (ii) lacking a nucleotide sequence encoding a homologous signal sequence, or (B) the nucleotide sequence of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (iv) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2;
- (v) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432;
- (vi) encoding (A) the polypeptide of (iv) or (v) lacking a homologous signal sequence, or (B) the polypeptide of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (vii) comprising the nucleotide sequence of (i), (ii), or (iii) and further comprising a heterologous nucleotide sequence;
- (viii) encoding the polypeptide of (iv), (v), or (vi) and further comprising a nucleotide sequence encoding a heterologous amino acid sequence;
- or
- (ix) comprising a nucleotide sequence that is the complete complement of (i), (ii), (iii), or (vii), or the complete complement of the nucleotide sequence encoding the polypeptide of (iv), (v), (vi) or (viii).
7 more elements
A vector comprising an exogenous nucleic acid:
- (i) comprising the nucleotide sequence of SEQ ID NO:1;
- (ii) comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide residue 1 to 1296;
- (iii) comprising (A) the nucleotide sequence of (i) or (ii) lacking a nucleotide sequence encoding a homologous signal sequence, or (B) the nucleotide sequence of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (iv) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2;
- (v) encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to 432;
- (vi) encoding (A) the polypeptide of (iv) or (v) lacking a homologous signal sequence, or (B) the polypeptide of (A) and further comprising a nucleotide sequence encoding a heterologous signal sequence;
- (vii) comprising the nucleotide sequence of (i), (ii), or (iii) and further comprising a heterologous nucleotide sequence;
- (viii) encoding the polypeptide of (iv), (v), or (vi) and further comprising a nucleotide sequence encoding a heterologous amino acid sequence;
- or
- (ix) comprising a nucleotide sequence that is the complete complement of (i), (ii), (iii), or (vii), or the complete complement of the nucleotide sequence encoding the polypeptide of (iv), (v), (vi) or (viii).
7 more elements
Dependencies are inferred from the claim text.