PULLULANASE VARIANTS AND POLYNUCLEOTIDES ENCODING SAME

• US 20180298364A1
• Filed: 06/25/2018
• Published: 10/18/2018
• Est. Priority Date: 01/22/2014
• Status: Active Grant

• Current Assignee: Novozymes A/S (Novozymes)
• Original Assignee: Novozymes A/S (Novozymes)
• Inventors: MATSUI, Tomoko, YAMAGISHI, Akihiko
• Granted Patent: US 10,640,755 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 9/2457 Pullulanase (3.2.1.41)
• CPC Class Codes: C12P 19/02 Monosaccharides 2-ketogulonic acid C12P7/60
• CPC Class Codes: C12P 19/16 produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin non-biological hydrolysis of starch C08B30/00
• CPC Class Codes: C12P 2203/00 Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source ethanol C12P7/10
• CPC Class Codes: C12P 7/06 Ethanol, i.e. non-beverage
• CPC Class Codes: C12Y 302/01041 Pullulanase (3.2.1.41)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The present invention relates to pullulanase variants comprising substitutions of the parent pullulanase at one or more positions corresponding to positions 393, 143, 150, 243, 244, 345, 346, 368, 370, 373, 381, 382, 385, 387, 402, 429, 430, 431, 432, 456, 486, 492, 610, 624, 631, 632, 665 and 699 of the polypeptide of SEQ ID NO: 3. 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.

Claims

1. A pullulanase variant comprising a substitution at one or more positions corresponding to positions 393, 143, 150, 243, 244, 346, 368, 370, 373, 381, 385, 387, 402, 429, 430, 456, 486, 492, 610, 631, 632, 665 and 699 of the polypeptide of SEQ ID NO:

3 wherein the variant has pullulanase activity, and;

a) wherein the variant has 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;

3 and wherein the variant has increased thermoactivity compared to the pullulanase of SEQ ID NO;

3, particularly the variant has at least 60% relative activity when measured at 70°

C. relative to activity at 65°

C., more particularly at least 65%, more particularly at least 70%, more particularly at least 60%, more particularly at least 75%, more particularly at least 80%, more particularly at least 90%, more particularly at least 100%;

or

b) wherein the variant has 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;

6 and wherein the variant has increased thermoactivity compared to the pullulanase of SEQ ID NO;

6, particularly the variant has at least 30% relative activity when measured at 70°

C. relative to activity at 65°

C., more particularly at least 40%, more particularly at least 50%, more particularly at least 60%, more particularly at least 70%, more particularly at least 80%, more particularly at least 90%, more particularly at least 100%;

or

c) wherein the variant has 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;

9 and wherein the variant has increased thermoactivity compared to the pullulanase of SEQ ID NO;

9, particularly the variant has at least 30% relative activity when measured at 70°

C. relative to activity at 65°

C., more particularly at least 40%, more particularly at least 50%, more particularly at least 60%, more particularly at least 70%, more particularly at least 80%, more particularly at least 90%, more particularly at least 100%;

or

d) wherein the variant has 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;

16 and wherein the variant has increased thermoactivity compared to the pullulanase of SEQ ID NO;

16, particularly the variant has at least 30% relative activity when measured at 70°

C. relative to activity at 65°

C., more particularly at least 40%, more particularly at least 50%, more particularly at least 60%, more particularly at least 70%, more particularly at least 80%, more particularly at least 90%, more particularly at least 100%;

or

e) wherein the variant has 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;

17 and wherein the variant has increased thermoactivity compared to the pullulanase of SEQ ID NO;

17, particularly the variant has at least 30% relative activity when measured at 70°

C. relative to activity at 65°

C., more particularly at least 40%, more particularly at least 50%, more particularly at least 60%, more particularly at least 70%, more particularly at least 80%, more particularly at least 90%, more particularly at least 100%.

2. The variant according to claim 1 wherein the variant comprises or consists of one or more substitutions selected from the group consisting of 393A, 143G, 150R, 243E, 244K, 345P, 346S, 368G, 370S, 373L, 381V, 382T, 385F, 387L, 402T, 429V, 430R, 431E, 432F, 456A, 486C, 492S,A, 610R,L, 624S, 631S, 632C, 665I and 699R.

3. The variant of claim 1, wherein the variant comprises at least one of the following substitutions or combinations of substitutions:

N368G;

E699R;

E150R;

N346S;

N243E;

S244K;

V143G;

N393A;

N610R;

N610L;

G624S;

F456A;

T492S,A;

V486C+T492S,A;

N368G+M402T;

T631S+S632C;

V486C+T492S,A+T631S+S632C;

N393A+T631S+S632C;

T631S+S632C+E699R;

N393A+V486C+T492S,A+T631S+S632C;

N393A+G624S+S632C;

N393A+N610R+T631S+S632C;

N393A+G624S+T631S+S632C;

N393A+N610R+G624S+T631S+S632C;

N393A+V486C+T492S,A+G624S+T631S+S632C;

N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;

N368G+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;

N393A+V486C+T492S,A+N610R+G624S+T631S+S632C+E699R;

N346S+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;

N393A+F456A+V486C+T492S,A+N610R+G624S+T631S+S632C;

N393A+T492S,A+N610R+G624S+T631S+S632C;

N368G+N393A+T492S,A+N610R+G624S+T631S+S632C;

A345P+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;

N368G+K370S+I373L+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;

I381V+Q385E+Q387L+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;

I381V+N382T+Q385E+Q387L+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;

A345P+N368G+N393A+T492S,A+N610R+G624S+T631S+S632C;

N368G+I381V+Q385E+Q387L+N393A+T492S,A+N610R+G624S+T631S+S632C;

A345P+N368G+I381V+Q385E+Q387L+N393A+T492S,A+N610R+G624S+T631S+S632C;

A345P+N368G+I381V+Q385E+Q387L+N393A+T492S,A+N610R+G624S+T631S+S632C+V665I;

N393A+T430R+Q431E+L432F+V486C+T492S,A+N610R+G624S+T631S+S632C;

N393A+Q431E+L432F+V486C+T492S,A+N610R+G624S+T631S+S632C;

N393A+I429V+Q431E+V486C+T492S,A+N610R+G624S+T631S+S632C;

N393A+I429V+T430R+Q431E+L432F+V486C+T492S,A+N610R+G624S+T631S+S632C;

N368G+N393A+A492S,A;

N368G+N393A;

N393A+N610R;

N368G+N393A+N610R;

N368G+N393A+T492S,A+N610R+G624S;

N368G+N393A+T492S,A+N610R+G624S+T631 S+S632C+Q431E+L432F;

N368G+N393A+N610R+G624S+T631 S+S632C;

N368G+N393A+T492S,A+N610R+G624S+T631S+S632C

N368G+N393A+N610R+G624S+T631 S+S632C+Q431E+L432F.

12. A composition comprising the variant pullulanase of claim 1.

13. The composition according to claim 12, further comprising one or more enzymes selected from the group consisting of:

glucoamylase, alpha-amylase, beta-amylase, and protease.

14. The composition according to claim 12, comprising a variant pullulanase of claim 1 and:

i) a glucoamylase, an alpha-amylase and a protease;

ii) an alpha-amylase and a protease;

iii) a glucoamylase and an alpha-amylase;

iv) a beta-amylase;

or v) a glucoamylase.

15. 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 in the presence of a glucoamylase; and

(c) fermenting with a fermenting organism;

wherein step (a) and/or step (b) is carried out in the presence of a variant pullulanase of claim 1.

16. 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 a glucoamylase, and a variant pullulanase of claim 1.

17. A polynucleotide encoding the variant pullulanase of claim 1.

18. A nucleic acid construct or expression vector comprising the polynucleotide of claim 17.

19. A host cell comprising the polynucleotide of claim 17.

20. A whole broth formulation or cell culture composition comprising a variant pullulanase of claim 1.

4. A variant catalytic domain, wherein the variant catalytic domain comprises a substitution at one or more positions corresponding to positions 393, 346, 368, 370, 373, 381, 385, 387, 402, 429, 430, 456, 486, 492, 531, 532, 610, 631, 632, 665 and 699 of the polypeptide of SEQ ID NO:

3; and

;

a) wherein the variant catalytic domain has 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 amino acids 330 to 828 of SEQ ID NO;

3;

or

b) wherein the variant catalytic domain has 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 amino acids 430 to 928 of SEQ ID NO;

6;

or

c) wherein the variant catalytic domain has 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 amino acids 330 to 829 of SEQ ID NO;

9;

or

d) wherein the variant catalytic domain has 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 amino acids 430 to 928 of SEQ ID NO;

16;

or

e) wherein the variant catalytic domain has 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 amino acids 430 to 928 of SEQ ID NO;

17.

10. A method for producing a variant pullulanase of a parent pullulanase comprising substitution of the parent pullulanase at one or more positions corresponding to positions 393, 143, 150, 243, 244, 345, 346, 368, 370, 373, 381, 382, 385, 387, 402, 429, 430, 431, 432, 456, 486, 492, 531, 532, 610, 624, 631, 632, 665 and 699 of the polypeptide of SEQ ID NO:

3 wherein the variant has pullulanase activity and increased thermoactivity compared to the parent; and

;

a) wherein the variant has 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;

3;

or

b) wherein the variant has 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;

6;

or

c) wherein the variant has 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;

9;

or

d) wherein the variant has 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;

16;

or

e) wherein the variant has 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;

17.

5. A pullulanase variant comprising the variant catalytic domain according to claim 10, wherein the pullulanase variant has pullulanase activity and increased thermoactivity compared to the parent pullulanase, and the variant has at least 60% relative activity when measured at 70°

C. relative to activity at 65°

C., more particularly at least 65%, more particularly at least 70%, more particularly at least 60%, more particularly at least 75%, more particularly at least 80%, more particularly at least 90%, more particularly at least 100%.

6. The pullulanase variant according to claim 5, wherein the variant comprises or consists of one or more substitutions selected from the group consisting of 393A, 345P, 346S, 368G, 370S, 373L, 381V, 382T, 385F, 387L, 402T, 429V, 430R, 431E, 432F, 456A, 486C, 492S,A, 610R,L, 624S, 631S, 632C, 665I and 699R.

7. The pullulanase variant according to claim 6, further comprising an N-terminal part comprising at least one of the domains selected from a CBM41 domain, an X45 domain and a CBM48 domain.

8. The pullulanase variant according to claim 7, comprising an N-terminal part comprising a CBM41 domain, an X45 domain and a CBM48 domain.

9. The pullulanase variant according to claim 8, further comprising an X25 domain.

11. A variant pullulanase produced by the method of claim 10.