Alpha-Amylase Variants and Polynucleotides Encoding Same

Abstract

The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

1 . An alpha-amylase variant comprising a substitution at a position corresponding to position 176 of SEQ ID NO: 1, in particular the substitution K176L, in combination with an alteration at one or more positions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450 of SEQ ID NO: 1, in particular one or more alterations selected from the group consisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F, N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y, G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y, H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H, Y158W, E185P, I120F, I201Y, H205Y, K213T, S239A, S239Q, F279Y, F279W, H316F, H316Y, H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F, R437H, R437Y, R437W, H450F, H450Y and H450W, preferably one or more alterations selected from the group consisting of A1AH, A1AW, A1H, A1W, N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W; wherein the variant has at least 60% and less than 100% sequence identity to (i) the mature polypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or (ii) amino acids 1 to 483 of SEQ ID NO: 1, amino acids 1 to 483 of SEQ ID NO: 2, amino acids 1 to 485 of SEQ ID NO: 3, amino acids 1 to 482 of SEQ ID NO: 4, amino acids 1 to 484 of SEQ ID NO: 5, amino acids 1 to 483 of SEQ ID NO: 6, amino acids 1 to 485 of SEQ ID NO: 7, amino acids 1 to 485 of SEQ ID NO: 8, amino acids 1 to 485 of SEQ ID NO: 9, amino acids 1 to 485 of SEQ ID NO: 10, amino acids 1 to 485 of SEQ ID NO: 11, amino acids 1 to 480 of SEQ ID NO: 12, amino acids 1 to 483 of SEQ ID NO: 13 or amino acids 1 to 481 of SEQ ID NO: 14; and wherein the variant has alpha-amylase activity. 
     
     
         2 . The variant of  claim 1 , which is an isolated alpha-amylase variant. 
     
     
         3 . The variant of  claim 1 , which comprises a substitution at one or more positions corresponding to any of positions 68, 185, 201, 205, 213, 360, 416 and 437, in particular one or more substitutions selected from the group consisting of H68W, E185P, I201Y, H205Y, K213T, Q360S, D416V and R437W. 
     
     
         4 . The variant of  claim 1 , which comprises a substitution at one or more positions corresponding to any of positions 2, 68, 133, 142, 156, 158, 279 and 437, in particular one or more substitutions selected from the group consisting of N2H, H68W, H133Y, H142W, Y156W, Y158W, F279W and R437W. 
     
     
         5 . The variant of  claim 1 , which comprises an alteration at two or more positions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, in particular two or more alterations selected from the group consisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F, N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y, G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y, H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H, Y158W, E185P, I120F, I201Y, H205Y, K213T, S239A, S239Q, F279Y, F279W, H316F, H316Y, H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F, R437H, R437Y, R437W, H450F, H450Y and H450W, preferably two or more alterations selected from the group consisting of A1AH, A1AW, A1H, A1W, N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W. 
     
     
         6 . The variant of  claim 1 , which comprises a substitution at two or more positions corresponding to any of positions 68, 185, 201, 205, 213, 360, 416 and 437, in particular two or more substitutions selected from the group consisting of H68W, E185P, I201Y, H205Y, K213T, Q360S, D416V and R437W. 
     
     
         7 . The variant of  claim 1 , which comprises a substitution at two or more positions corresponding to any of positions 2, 68, 133, 142, 156, 158, 279 and 437, in particular two or more substitutions selected from the group consisting of N2H, H68W, H133Y, H142W, Y156W, Y158W, F279W and R437W. 
     
     
         8 . The variant of  claim 1 , wherein the variant has at least 65% sequence identity to (i) the mature polypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or (ii) amino acids 1 to 483 of SEQ ID NO: 1, amino acids 1 to 483 of SEQ ID NO: 2, amino acids 1 to 485 of SEQ ID NO: 3, amino acids 1 to 482 of SEQ ID NO: 4, amino acids 1 to 484 of SEQ ID NO: 5, amino acids 1 to 483 of SEQ ID NO: 6, amino acids 1 to 485 of SEQ ID NO: 7, amino acids 1 to 485 of SEQ ID NO: 8, amino acids 1 to 485 of SEQ ID NO: 9, amino acids 1 to 485 of SEQ ID NO: 10, amino acids 1 to 485 of SEQ ID NO: 11, amino acids 1 to 480 of SEQ ID NO: 12, amino acids 1 to 483 of SEQ ID NO: 13 or amino acids 1 to 481 of SEQ ID NO: 14. 
     
     
         9 . The variant of  claim 1 , which is a variant of a parent alpha-amylase selected from the group consisting of:
 (a) a polypeptide having at least 60% sequence identity to (i) the mature polypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or (ii) amino acids 1 to 483 of SEQ ID NO: 1, amino acids 1 to 483 of SEQ ID NO: 2, amino acids 1 to 485 of SEQ ID NO: 3, amino acids 1 to 482 of SEQ ID NO: 4, amino acids 1 to 484 of SEQ ID NO: 5, amino acids 1 to 483 of SEQ ID NO: 6, amino acids 1 to 485 of SEQ ID NO: 7, amino acids 1 to 485 of SEQ ID NO: 8, amino acids 1 to 485 of SEQ ID NO: 9, amino acids 1 to 485 of SEQ ID NO: 10, amino acids 1 to 485 of SEQ ID NO: 11, amino acids 1 to 480 of SEQ ID NO: 12, amino acids 1 to 483 of SEQ ID NO: 13 or amino acids 1 to 481 of SEQ ID NO: 14; or   (b) a fragment of the mature polypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, which has alpha-amylase activity.   
     
     
         10 . (canceled) 
     
     
         11 . An isolated polynucleotide encoding the variant of  claim 1 . 
     
     
         12 . A nucleic acid construct comprising the polynucleotide of  claim 11 . 
     
     
         13 . An expression vector comprising the polynucleotide of  claim 11 . 
     
     
         14 . A host cell comprising the polynucleotide of  claim 11 . 
     
     
         15 . A method of producing an alpha-amylase variant, comprising:
 a. cultivating the host cell of  claim 14  under conditions suitable for expression of the expression of the variant; and   b. recovering the variant.   
     
     
         16 . A method for obtaining an alpha-amylase variant, comprising (a) introducing into a parent alpha-amylase a substitution at one or more positions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450 of SEQ ID NO: 1; and (b) recovering the variant. 
     
     
         17 . A method of producing liquefied starch, comprising liquefying a starch-containing material with the variant of  claim 1 . 
     
     
         18 . A method of producing a fermentation product, comprising
 (a) liquefying a starch-containing material with the variant of  claim 1  to produce a liquefied mash;   (b) saccharifying the liquefied mash to produce fermentable sugars; and   (c) fermenting the fermentable sugars in the presence of a fermenting organism.   
     
     
         19 . The method of  claim 18 , wherein step (a) is performed at pH 4-5. 
     
     
         20 . A method of producing a fermentation product, comprising contacting a starch substrate with the variant of  claim 1 , a glucoamylase, and a fermenting organism.