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

Abstract

The invention provides isolated and recombinant phytase enzymes. In one aspect, the phytases are produced by modification of the wild type appA of E. coli. The enzyme can be produced from recombinant host cells. The phytases of the invention can be used to aid in the digestion of phytate where desired. In particular, the phytases of the invention can be used in foodstuffs to improve the feeding value of phytate rich ingredients. The phytases of the invention can be thermotolerant and/or thermostable. Also provided are methods for obtaining a variant polynucleotide encoding a phytase and for obtaining a phytase with thermostability or thermotolerant at high or low temperatures.

1 . An isolated, synthetic, or recombinant nucleic acid comprising:
 (a) a nucleic acid having at least 98.5%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO:1, wherein the nucleic acid encodes a polypeptide having a phytase activity;   (b) a nucleic acid encoding a polypeptide having an amino acid sequence having at least 98.5%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO:2, wherein the polypeptide has a phytase activity;   (c) a nucleic acid encoding a polypeptide having an amino acid sequence of SEQ ID NO:2 and having an amino acid substitution selected from the group consisting of W68E, Q84W, A95P, K97C, S168E, R181Y, N226C, Y277D, and any combination thereof, wherein the polypeptide has a phytase activity;   (d) the nucleic acid of any of (a) to (c) encoding a polypeptide lacking a signal (leader) sequence;   (e) the nucleic acid of any of (a) to (d) encoding a polypeptide having a heterologous sequence;   (f) the nucleic acid of (e), wherein the heterologous sequence comprises a heterologous signal (leader) sequence; or   (g) a nucleic acid having a sequence completely complementary to the nucleic acid sequence of any of (a) to (f).   
     
     
         2 . A nucleic acid probe for identifying a nucleic acid encoding a polypeptide with a phytase activity, wherein the probe comprises the nucleic acid of  claim 1 , wherein the probe identifies the phytase-encoding nucleic acid by binding or hybridization. 
     
     
         3 . An expression cassette, a vector, or a cloning vehicle comprising:
 (a) the nucleic acid of  claim 1 ; or,   (b) the nucleic acid of  claim 1 , wherein (i) the expression cassette, cloning vehicle or vector is a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificial chromosome; (ii) the viral vector of (iii) comprises an adenovirus vector, a retroviral vector or an adeno-associated viral vector; or (iv) the expression cassette, cloning vehicle or vector is a bacterial artificial chromosome (BAC), a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome (YAC) or a mammalian artificial chromosome (MAC).   
     
     
         4 . An isolated host cell comprising:
 (a) the expression cassette, vector, or cloning vehicle of  claim 3 , or the nucleic acid of  claim 1 ; or   (b) the expression cassette, vector, or cloning vehicle of  claim 3 , or the nucleic acid of  claim 1 , wherein the host cell is a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell.   
     
     
         5 . A method of producing a recombinant polypeptide comprising:
 (i) (a) providing the nucleic acid of  claim 1 ; and
 (b) expressing the nucleic acid of step (a) under conditions that allow expression of the polypeptide, thereby producing a recombinant polypeptide; or 
   (ii) the method of (i), further comprising transforming a host cell with the nucleic acid of (a) followed by expressing the nucleic acid of (a), thereby producing a recombinant polypeptide in a transformed cell.   
     
     
         6 . A method for overexpressing a recombinant phytase in a cell comprising expressing a vector, expression cassette or a cloning vehicle comprising the nucleic acid of  claim 1 , wherein overexpression is effected by use of a high activity promoter, a dicistronic vector or by gene amplification of the vector. 
     
     
         7 . A phytase made by the method of:
 (i) (a) providing the nucleic acid of  claim 1 ; and
 (b) expressing the nucleic acid of step (a) under conditions that allow expression of the polypeptide, thereby producing a recombinant polypeptide; or 
   (ii) the method of (i), further comprising transforming a host cell with the nucleic acid of (a) followed by expressing the nucleic acid of (a), thereby producing a recombinant polypeptide in a transformed cell.   
     
     
         8 . A composition comprising the nucleic acid of  claim 1 . 
     
     
         9 . The nucleic acid of  claim 1 , wherein the nucleic acid is immobilized. 
     
     
         10 . An isolated, synthetic, or recombinant polypeptide comprising an amino acid sequence encoded by the nucleic acid of  claim 1 .