Glucoamylase variants and polynucleotides encoding same

Patent metadata

Field	Value
Publication number	US-2017306310-A1
Application number	US-201515520567-A
Country	US
Kind code	A1
Filing date	Oct 23, 2015
Priority date	Oct 23, 2014
Publication date	Oct 26, 2017
Grant date	—

Abstract

The present invention relates to glucoamylase variants. 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.

First claim

1 . A glucoamylase variant comprising a substitution at a position corresponding to position 295 of the polypeptide of SEQ ID NO: 2, wherein the variant has glucoamylase activity and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 2.

2 . The variant of claim 1 , further comprises a substitution at least at one or more position corresponding to position 32, 83, 163, 169, 219, 224, 303 or 410 of the polypeptide of SEQ ID NO: 2, wherein the variant has glucoamylase activity and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 2.

3 . The variant of claim 1 , wherein the number of substitutions is 1-20.

4 . The variant of claim 1 , wherein the variant comprises at least one or more of the substitutions selected from 295F, 295W, 224A, 224I, 224T, 32V, 83D, 163A, 163W, 169I, 219R, 303N or 410K.

5 . The variant of claim 1 , wherein the variant comprises at least one of the following substitutions or combinations of substitutions:
295W; 295W+410K; 224I+295F; 224T+295W+318V; 295F; 224A+295F; 295W+83D+410K; 163A+295W+410K; 163W+295W+410K; 303N+295W; 169I+295W; or 32V+219R+295W; wherein the variant has glucoamylase activity and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 2.

6 . The variant according to claim 1 , wherein the variant comprises at least one of the following substitutions or combinations of substitutions:
295W+410K; or 163A+295W+410K; wherein the variant has glucoamylase activity and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 2.

7 . The variant according to claim 1 , wherein the substitutions are selected from Y295F, Y295W, L224A, L224I, L224T, A32V, S83D, N163A, N163W, V169I, T219R, S303NR410K, and Q318V.

8 . The variant of claim 1 , which has an improved property relative to the parent, wherein the improved property is reduced glucose inhibition.

9 . A glucoamylase variant, comprising a substitution at one or more positions corresponding to positions 271, 410, 72, 77, 145, 219, 303, 224, 318 of the polypeptide of SEQ ID NO: 2, wherein the variant has glucoamylase activity and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 2.

10 . The variant of claim 9 , wherein the number of substitutions is 1-20, e.g., 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions.

11 . The variant of claim 9 , wherein the variant comprises at least one or more of the substitutions selected from 72V, 77A, 145A, 219D, 271Q, 271N, 271A, 271S, 271V, 224Q, 303E, 318W, 318F, 410A, 410Q or 410H.

12 . The variant according to claim 9 , wherein the variant comprises at least one of the following substitutions or combinations of substitutions:
72V+145A; 271Q; 224Q+271N+410A; 77A; 77A+271A; 271S+318W+410Q; 271V+318F+410H; 77A+219D; 77A+303E; wherein the variant has glucoamylase activity and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 2.

13 . The variant according to claim 9 , wherein the substitutions are selected from D72V, L77A, L145A, T219D, L224Q, T271Q, T271N, T271A, T271S, T271V, S303E, Q318W, Q318F, R410Q, R410H and R410A.

14 . The variant according to claim 9 , which has an improved property relative to the parent, wherein the improved property is increased specific activity.

15 . A glucoamylase variant, comprising a substitution at one or more positions corresponding to positions 271, or 295 or 271 and 295 of the polypeptide of SEQ ID NO: 2, wherein the variant has glucoamylase activity and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 2.

16 . The variant of claim 15 , wherein the number of substitutions is 1-20, e.g., 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions.

17 . The variant of claim 15 , wherein the variant comprises at least one or more of the substitutions selected from 271Q, 271A, 271V, 295W, 60L, 73A, 77A, 77V, and 318Y.

18 . The variant according to claim 15 , wherein the variant comprises at least one of the following substitutions or combinations of substitutions:
F60L+S73A+T271Q; 271Q; 77V+271V+410A; 77A+271A; 271V+318Y; 295W; 271Q+295W; and wherein the variant has glucoamylase activity and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 2.

19 . The variant according to claim 15 , which has an improved property relative to the parent, wherein the improved property is increased ethanol yield when used in SSF.

20 . The variants according to claim 18 , wherein the substitutions are selected from F60L, 573A, T271A, T271V, T271Q, L77A, L77V, R410A, Y295W, and Q318Y.

21 . The variant according to claim 1 , wherein the amino acid at position 95 and 121 is a proline.

22 . A composition comprising the polypeptide of claim 1 .

23 . The composition according to claim 22 , further comprising a pullulanase.

24 . The composition according to claim 22 , further comprising an alpha-amylase, particularly a fungal alpha-amylase, more particularly an alpha-amylase derived from Rhizomucor pusillus or Aspergillus terreus , even more particularly an alpha-amylase selected from the alpha-amylase disclosed as SEQ ID NO: 4, preferably having one or more of the following substitutions: G128D, D143N, preferably G128D+D143N (using SEQ ID NO: 4 for numbering), and wherein the alpha-amylase variant has at least 75% identity preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the polypeptide of SEQ ID NO: 4

25 . (canceled)

26 . A process of producing a fermentation product, particularly ethanol, from starch-containing material comprising the steps of: (a) liquefying starch-containing material in the presence of an alpha amylase; (b) saccharifying the liquefied material; and (c) fermenting with a fermenting organism; wherein step (b) is carried out using at least a variant glucoamylase of claim 1 .

27 . The process

Assignees

• Novozymes As

Inventors

• Fukuyama Shiro
• Tsutsumi Noriko
• Ayabe Keiichi

Classifications

• C12P7/06

  Ethanol, i.e. non-beverage · CPC title

• C12Y302/01003

  Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase · CPC title

• C12P19/02

  Monosaccharides (2-ketogulonic acid C12P7/60) · CPC title

• C12Y302/01001

  Alpha-amylase (3.2.1.1) · CPC title

• C12N9/2457

  Pullulanase (3.2.1.41) · CPC title