Cellobiohydrolase variants and polynucleotides encoding same

Patent metadata

Field	Value
Publication number	US-11965189-B2
Application number	US-201716088038-A
Country	US
Kind code	B2
Filing date	Mar 24, 2017
Priority date	Mar 24, 2016
Publication date	Apr 23, 2024
Grant date	Apr 23, 2024

Abstract

The present invention relates to cellobiohydrolase variants having increased thermal activity or thermostability, polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

First claim

What is claimed is:

1. A cellobiohydrolase variant, comprising a substitution relative to SEQ ID NO: 1 at one or more of positions 113, 157, 199, 240, 250, 274, 318, 325, 328, 347, 349, 358, 360, 391, 393, 394, 412, and 430 of SEQ ID NO: 1, wherein the variant has: (1) cellobiohydrolase activity, (2) at least 90%, but less than 100%, sequence identity to SEQ ID NO: 1, and (3) an increased thermal activity or increased thermostability relative to the cellobiohydrolase of SEQ ID NO: 1.

2. The cellobiohydrolase variant of claim 1 , which comprises one or more substitutions selected from the group consisting of: N113D; K157R; V199P; S250D; F274Y; G318A,P,S; T325P; T328P; T347P; D349V; G358A; G360S,T; N391D; S393D; S394P; T412A; and T430V.

3. The cellobiohydrolase variant of claim 1 , which further comprises an alteration at one or more positions corresponding to positions 4, 8, 17, 21, 26, 38, 39, 44, 45, 46, 51, 52, 53, 54, 72, 75, 87, 93, 94, 95, 100, 102, 108, 111, 114, 129, 130, 131, 137, 138, 139, 144, 150, 156, 157, 159, 183, 184, 187, 194, 195, 196, 197, 198, 199, 200, 201, 205, 206, 209, 211, 219, 237, 240, 241, 247, 253, 260, 264, 271, 280, 320, 322, 330, 332, 343, 345, 350, 357, 358, 360, 365, 371, 375, 379, 380, 381, 382, 385, 386, 389, 390, 392, 397, 400, 405, 407, 413, 426, 427, 430, 440, 444, 445, 446, 447, 450, 453, 455, 456, 458, 459, 462, 463, 464, 492, 493, 494, 496, 497, 498, 502, 503, 507, 510, 513, 515, 516, and 517 of SEQ ID NO: 1.

4. The cellobiohydrolase variant of claim 3 , wherein the alteration is selected from the group consisting of G4C, A21P, S26A, W38A, R39L, T44I, T44M, T44N, T44K, S45D, S45N, G46A, G46I, G46L, G46T, Y51I, T52R, T52W, G53A, G53M, G53R, G53W, N54S, N54I, N54D, A72C, G75S, S87T, L93V, N94S, N94A, N94R, N94Q, F95L, F95Y, S100T, S100V, S100W, S100L, S100G, K102S, K102R, L108I, L111T, D114E, F129S, D130N, D130E, V131A, P137S, C138S, G139E, G139M, G139Q, G139S, G139R, L144A, L144V, D150N, A156G, K157R, S159P, 1183N, D184S, A187L, P194*, P194Q, S195*, T196*, N197A, N197*, D198A, D198*, V199*, N200A, N200G, N200W, N200F, N2000, N200*, S201*, N205R, H206Y, C209S, A211T, N219S, M237T, D241L, D241R, D241V, Y247C, A253D, A253R, G260D, N264Y, T271I, V280I, P320A, S322V, N330D, I332F, A343V, G345D, F350L, A357S, G358R, G360M, D365S, M371V, D375A, D375G, A379T, D380H, D380Y, D380N, M381V, L382A, D385E, S386C, S386E, P389L, P389Q, P389I, T390A, T390S, T390A, A392G, A392I, A392L, P397A, P397G, P397K, P397W, P397G, P397L, K400A, D405P, S407G, T413P, S426F, N427D, T430Y, T440L, T440R, T440G, T444S, T445D, S446T, S447L, T450D, S453D, T455A, T456S, S458E, K459E, K459S, S462I, T463I, T464A, T464A, H492L, Y493S, Y493W, A494D, A494S, C496S, C496Y, G497C, G498D, G498S, T502N, G503D, 0507Y, P510V, P510S, 0513R, 0513W, K515D, Q516P, and N517D.

5. The cellobiohydrolase variant of claim 3 , which has at least 95%, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 1.

6. The cellobiohydrolase variant of claim 1 , which has at least 95%, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 1.

7. The cellobiohydrolase variant of claim 6 , which comprises one or more substitutions selected from the group consisting of: N113D; K157R; V199P; S250D; F274Y; G318A,P,S; T325P; T328P; T347P; D349V; G358A; G360S,T; N391D; S393D; S394P; T412A; and T430V.

8. The cellobiohydrolase variant of claim 7 , which further comprises an alteration at one or more positions corresponding to positions 4, 8, 17, 21, 26, 38, 39, 44, 45, 46, 51, 52, 53, 54, 72, 75, 87, 93, 94, 95, 100, 102, 108, 111, 114, 129, 130, 131, 137, 138, 139, 144, 150, 156, 157, 159, 183, 184, 187, 194, 195, 196, 197, 198, 199, 200, 201, 205, 206, 209, 211, 219, 237, 240, 241, 247, 253, 260, 264, 271, 280, 320, 322, 330, 332, 343, 345, 350, 357, 358, 360, 365, 371, 375, 379, 380, 381, 382, 385, 386, 389, 390, 392, 397, 400, 405, 407, 413, 426, 427, 430, 440, 444, 445, 446, 447, 450, 453, 455, 456, 458, 459, 462, 463, 464, 492, 493, 494, 496, 497, 498, 502, 503, 507, 510, 513, 515, 516, and 517 of SEQ ID NO: 1.

9. The cellobiohydrolase variant of claim 8 , wherein the alteration is selected from the group consisting of G4C, A21P, S26A, W38A, R39L, T44I, T44M, T44N, T44K, S45D, S45N, G46A, G46I, G46L, G46T, Y51I, T52R, T52W, G53A, G53M, G53R, G53W, N54S, N54I, N54D, A72C, G75S, S87T, L93V, N94S, N94A, N94R, N94Q, F95L, F95Y, S100T, S100V, S100W, S100L, S100G, K102S, K102R, L108I, L111T, D114E, F129S, D130N, D130E, V131A, P137S, 0138S, G139E, G139M, G139Q, G139S, G139R, L144A, L144V, D150N, A156G, K157R, S159P, 1183N, D184S, A187L, P194*, P194Q, S195*, T196*, N197A, N197*, D198A, D198*, V199*, N200A, N200G, N200W, N200F, N2000, N200*, S201*, N205R, H206Y, C209S, A211T, N219S, M237T, D241L, D241R, D241V, Y247C, A253D, A253R, G260D, N264Y, T271I, V280I, P320A, S322V, N330D, 1332F, A343V, G345D, F350L, A357S, G358R, G360M, D365S, M371V, D375A, D375G, A379T, D380H, D380Y, D380N, M381V, L382A, D385E, S386C, S386E, P389L, P389Q, P389I, T390A, T390S, T390A, A392G, A392I, A392L, P397A, P397G, P397K, P397W, P397G, P397L, K400A, D405P, S407G, T413P, S426F, N427D, T430Y, T440L, T440R, T440G, T444S, T445D, S446T, S447L, T450D, S453D, T455A, T456S, S458E, K459E, K459S, S462I, T463I, T464A, T464A, H492L, Y493S, Y493W, A494D, A494S, C496S, C496Y, G497C, G498D, G498S, T502N, G503D, C507Y, P510V, P510S, C513R, C513W, K515D, Q516P, and N517D.

10. The cellobiohydrolase variant of claim 1 , which comprises a N113D substitution.

11. The cellobiohydrolase variant of claim 1 , which comprises a K157R substitution.

12. The cellobiohydrolase variant of claim 11 , which has at least 95%, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 1.

13. The cellobiohydrolase variant of claim 1 , which comprises a V199P substitution.

14. The cellobiohydrolase variant of claim 13 , which has at least 95%, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 1.

15. The cellobiohydrolase variant of claim 1 , which comprises a S250D substitution.

16. The cellobiohydrolase variant of claim 15 , which has at least 95%, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 1.

17. The cellobiohydrolase variant of claim 1 , which comprises a F274Y substitution.

18. The cellobiohydrolase variant of claim 17 , which has at least 95%, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 1.

19. The cellobiohydrolase variant of claim 1 , which comprises a G318A, P, or S substitution.

20. The cellobiohydrolase variant of claim 19 , which has at least 95%, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 1.

21. The cellobiohydrolase variant of claim 1 , which comprises a T325P substitution.

22. The cellobiohydrolase variant of claim 21 , which has at least 95%, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 1.

23. The cellobiohydrolase variant of claim 1 , which comprises a T328P substitution.

24. The cellobiohydrolase variant of claim 23 , which has at least 95%, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 1.

25. The cellobiohydrolase variant of claim 1 , which comprises a T347P substitution.

26. The cellobiohydrolase variant of claim 25 , which has at least 95%, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 1.

27. The cellobiohydrolase variant of claim 1 , which comprises a D349V subs

Assignees

• Novozymes As

Inventors

• Wogulis Mark
• Demars Leslie
• Osborn David

Classifications

• C12N9/2437 Primary

  Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150) · CPC title

• C12P7/10

  substrate containing cellulosic material · CPC title

• C12P19/02

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

• C12P19/14

  produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase} · CPC title

• C12Y302/01

  Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1) · CPC title