Glucoamylase variants and polynucleotides encoding same

Patent metadata

Field	Value
Publication number	US-10865399-B2
Application number	US-201816135407-A
Country	US
Kind code	B2
Filing date	Sep 19, 2018
Priority date	Oct 11, 2011
Publication date	Dec 15, 2020
Grant date	Dec 15, 2020

Abstract

The present invention relates to glucoamylase variants having improved thermostability. 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

The invention claimed is:

1. A glucoamylase variant of Penicillium having glucoamylase activity, the glucoamylase variant comprising:
a) a substitution or deletion at two or more positions corresponding to positions 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 18, 26, 31, 33, 34, 65, 72, 74, 79, 80, 103, 105, 112, 161, 172, 218, 220, 221, 245, 253, 255, 279, 325, 327, 359, 364, 370, 375, 377, 405, 445, 447, 460, 463, 465, 468, 477, 501, 502, 504, 516, 524, 526, 563, 564, 568, 571 of the polypeptide of SEQ ID NO: 3; and
b) an amino acid sequence that has at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 3.

2. The variant of claim 1 , further comprising a substitution or deletion in at least one position selected from the group consisting of position 65, 327, 501, and 504 of SEQ ID NO: 3.

3. The variant of claim 1 , wherein the variant further comprises or consists of one or more substitutions selected from the group consisting of T65A, Q327F, E501V, Y504T, and Y504*.

4. The variant of claim 1 , wherein the variant further comprises at least one of the following substitutions or combinations of substitutions:
T65A;
Q327F;
E501V;
Y504T;
Y504*;
T65A+Q327F;
T65A+E501V;
T65A+Y504T;
T65A+Y504*;
Q327F+E501V;
Q327F+Y504T;
Q327F+Y504*;
E501V+Y504T;
E501V+Y504*;
T65A+Q327F+E501V;
T65A+Q327F+Y504T;
T65A+E501V+Y504T;
Q327F+E501V+Y504T;
T65A+Q327F+Y504*;
T65A+E501V+Y504*;
Q327F+E501V+Y504*;
T65A+Q327F+E501V+Y504T; or
T65A+Q327F+E501V+Y504*.

5. The variant of claim 1 , wherein the variant comprises at least one of the following combinations of substitutions:
P11F+T65A+Q327F;
R1K+D3W+K5Q+G7V+N8S+T10K+P11S+T65A+Q327F;
P2N+P4S+P11F+T65A+Q327F;
P11F+D26C+K33C+T65A+Q327F;
P2N+P4S+P11F+T65A+Q327W+E501V+Y504T;
R1E+D3N+P4G+G6R+G7A+N8A+T10D+P11D+T65A+Q327F;
P11F+T65A+Q327W;
P2N+P4S+P11F+T65A+Q327F+E501V+Y504T;
P11F+T65A+Q327W+E501V+Y504T;
P2N+P4S+P11F+K34Y+T65A+Q327F;
P2N+P4S+P11F+T65A+Q327F+D445N+V447S;
P2N+P4S+P11F+T65A+I172V+Q327F;
P2N+P4S+P11F+T65A+Q327F+N502*;
P2N+P4S+P11F+T65A+Q327F+N502T+P563S+K571E;
P2N+P4S+P11F+R31S+K33V+T65A+Q327F+N564D+K571S;
P2N+P4S+P11F+T65A+Q327F+S377T;
P2N+P4S+P11F+T65A+V325T+Q327W;
P2N+P4S+P11F+T65A+Q327F+D445N+V447S+E501V+Y504T;
P2N+P4S+P11F+T65A+I172V+Q327F+E501V+Y504T;
P2N+P4S+P11F+T65A+Q327F+S377T+E501V+Y504T;
P2N+P4S+P11F+D26N+K34Y+T65A+Q327F;
P2N+P4S+P11F+T65A+Q327F+I375A+E501V+Y504T;
P2N+P4S+P11F+T65A+K218A+K221D+Q327F+E501V+Y504T;
P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T;
K5A+P11F+T65A+Q327F+E501V+Y504T;
P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T568N;
P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+K524T+G526A;
P2N+P4S+P11F+K34Y+T65A+Q327F+D445N+V447S+E501V+Y504T;
P2N+P4S+P11F+R31S+K33V+T65A+Q327F+D445N+V447S+E501V+Y504T;
P2N+P4S+P11F+D26N+K34Y+T65A+Q327F+E501V+Y504T;
P2N+P4S+P11F+T65A+F80*+Q327F+E501V+Y504T;
P2N+P4S+P11F+T65A+K112S+Q327F+E501V+Y504T;
P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A;
P2N+P4S+P11F+T65A+Q327F+E501V+N502T+Y504*;
P2N+P4S+P11F+T65A+Q327F+E501V+Y504T;
P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T;
K5A+P11F+T65A+Q327F+E501V+Y504T;
P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A;
P2N+P4S+P11F+T65A+V79A+Q327F+E501V+Y504T;
P2N+P4S+P11F+T65A+V79G+Q327F+E501V+Y504T;
P2N+P4S+P11F+T65A+V79I+Q327F+E501V+Y504T;
P2N+P4S+P11F+T65A+V79L+Q327F+E501V+Y504T;
P2N+P4S+P11F+T65A+V79S+Q327F+E501V+Y504T;
P2N+P4S+P11F+T65A+L72V+Q327F+E501V+Y504T;
P2N+P4S+P11F+T65A+E74N+V79K+Q327F+E501V+Y504T;
P2N+P4S+P11F+T65A+G220N+Q327F+E501V+Y504T;
P2N+P4S+P11F+T65A+Y245N+Q327F+E501V+Y504T;
P2N+P4S+P11F+T65A+Q253N+Q327F+E501V+Y504T;
P2N+P4S+P11F+T65A+D279N+Q327F+E501V+Y504T;
P2N+P4S+P11F+T65A+Q327F+S359N+E501V+Y504T;
P2N+P4S+P11F+T65A+Q327F+D370N+E501V+Y504T;
P2N+P4S+P11F+T65A+Q327F+V460S+E501V+Y504T;
P2N+P4S+P11F+T65A+Q327F+V460T+P468T+E501V+Y504T;
P2N+P4S+P11F+T65A+Q327F+T463N+E501V+Y504T;
P2N+P4S+P11F+T65A+Q327F+S465N+E501V+Y504T; or
P2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T.

6. The variant of claim 1 , which has reduced sensitivity to protease degradation compared to SEQ ID NO: 3.

7. The variant of claim 1 , which has improved thermostability compared to SEQ ID NO: 3.

8. A composition comprising the Penicillium glucoamylase variant of claim 1 .

9. The composition of claim 8 , comprising an alpha-amylase and Penicillium glucoamylase variant of claim 1 .

10. A process of producing a fermentation product 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 (a) and/or step (b) is carried out using at least the Penicillium glucoamylase variant of claim 1 .

11. The process of claim 10 , wherein the fermentation product is ethanol and the starch-containing material is corn.

12. A process of producing a fermentation product from starch-containing material, comprising the steps of:
(a) saccharifying starch-containing material at a temperature below the initial gelatinization temperature of said starch-containing material; and
(b) fermenting with a fermenting organism,

wherein step (a) is carried out using at least the Penicillium glucoamylase variant of claim 1 .

13. The process of claim 12 , wherein the fermentation product is ethanol and the starch-containing material is corn.

14. The variant of claim 1 , wherein the variant further comprises at least one of the following combinations of substitutions:
E501V+Y504T;
T65A+K161S;
T65A+Q405T;
T65A+Q327W;
T65A+Q327F;
T65A+Q327Y;
T65A+Q327F+E501V+Y504T;
T65A+S105P+Q327W;
T65A+S105P+Q327F;
T65A+Q327W+S364P;
T65A+Q327F+S364P;
T65A+S103N+Q327F;
P2N+P4S+T10D+T65A+Q327F+E501V+Y504T;
P2N+P4S+F12Y+T65A+Q327F+E501V+Y504T;
P2N+P4S+T10E+E18N+T65A+Q327F+E501V+Y504T;
P2N+T10E+E18N+T65A+Q327F+E501V+Y504T; or
S255N+Q327F+E501V+Y504T.

Assignees

• Novozymes As

Inventors

• Friis Esben Peter
• De Maria Leonardo
• Vind Jesper
• Poulsen Thomas A
• Svendsen Allan
• Danielsen Steffen
• Lenhard Rolf T
• Friis-Madsen Henrik
• Skov Lars K

Classifications

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title

• C12N9/2428 Primary

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

• C12Y302/01003

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

• C12P37/00

  Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin · CPC title

• C12N9/242

  Fungal source · CPC title