Variant enzymes

Patent metadata

Field	Value
Publication number	US-10167460-B2
Application number	US-201414909031-A
Country	US
Kind code	B2
Filing date	Jul 24, 2014
Priority date	Jul 29, 2013
Publication date	Jan 1, 2019
Grant date	Jan 1, 2019

Abstract

Disclosed are glycosyl hydrolase enzyme variants, particularly variants of certain oxidoreductases of glycosyl hydrolase family 61. Nucleic acids encoding the glycosyl hydrolyase variants, compositions including the glycosyl hydrolase variants, methods of producing the variants, and methods of using the variants are also described.

First claim

What is claimed is:

1. A variant of a parent glycosyl hydrolase 61 (GH61) enzyme, wherein said variant has cellulase augmenting activity, and has at least 90% sequence identity to SEQ ID NO:3, wherein said variant comprises at least one amino acid substitution that is at position I4, wherein the position of each amino acid substitution corresponds to SEQ ID NO:3.

2. The variant of claim 1 , wherein said variant comprises from 1 to 10 amino acid substitutions.

3. The variant of claim 2 , wherein said variant is a combinatorial variant.

4. The variant of claim 1 , wherein said parent GH61 polypeptide is a fungal glycosyl hydrolase 61a (GH61A).

5. The variant of claim 4 , wherein said fungal GH61A is from Hypocrea jecorina, Hypocrea rufa, Hypocrea orientalis, Hypocrea atroviridis, Hypocrea virens, Emericella nidulans, Aspergillus terreus, Aspergillus oryzae, Aspergillus niger, Aspergillus kawachii, Aspergillus flavus, Aspergillus clavatus, Gaeumannomyces graminis, Trichoderma saturnisporum, Neurospora tetrasperma, Neurospora crassa, Neosartorya fumigate, Neosartorya fumigate, Neosartorya fischeri, Thielavia terrestris, Talaromyces sp., Sporotricum sp, and Thielavia heterothallica.

6. The variant of claim 1 , wherein said variant has at least 95% sequence identity to SEQ ID NO:3.

7. The variant of claim 1 , wherein the amino acid substitution at position I4 is selected from V or T.

8. The variant of claim 7 wherein said variant further comprises at least one amino acid substitution at a position selected from: N5, I7, I9, N10, V12, W13, Q15, A16, P19, T20, T21, F22, P23, Y24, E25, S26, N27, P28, P29, V32, G33, T35, A36, A37, D38, L39, N41, F43, V44, D47, A48, Y49, Q50, N51, P52, I54, K58, N59, T61, N62, K64, S68, V69, K70, A71, V80, P89, I90, V91, N96, N98, D100, T103, T107, T108, E110, G116, V117, L119, L120, S121, G122, G123, D124, P125, G126, T127, V132, S135, V141, D146, N147, A149, I160, A161, H163, S164, G166, C167, A168, A171, N173, P175, I180, A181, S183, S185, S187, L188, Q189, S191, L194, G195, T196, D197, H200, A201, T202, P204, V206, L207, I208, N209, Y211, S213, P214, N216, Y217, I218, I219, P222, T223, V224, S226, T230, S231, V232, A233, G235, T287, Q288, T289, L290, Y291, G292, Q293, G295, G296, S297, G298, Y299, S300, G301, P302, T303, R304, C305, A306, P307, P308, A309, T310, S312, T313, N315, P316, Y317, Y318, A319, Q320, L322, or N323, wherein the position of each amino acid substitution corresponds to SEQ ID NO:3.

9. The variant of claim 8 wherein the at least one amino acid substitution is selected from one or more of: I9V, V12T, A16F, A16G, A16H, A16K, A16L, A16P, A16R, A16W, T21I, F22R, S26P, S26T, A37W, D38N, L39F, L39I, L39V, L39Y, 154E, T61S, N62P, D100A, D100F, D100G, D100I, D100L, D100N, D100P, D100Q, D100S, D100W, D100Y, T103A, T103C, T103D, T103F, T103M, T103N, T103Q, T103S, T103V, T103Y, T127F, T127L, T127M, T127V, T127W, T127Y, S164A, S164L, S164M, S164Q, S164R, G166A, G166F, G166M, G166S, Q167K, Q167V, A168F, A168I, A168L, A168M, A168P, A168R, A168T, A168Y, S183N, S185D, S185G, S185T, S187A, S187D, Q189E, Q189R, L194M, V206I, S213T, N216T, I219M, S226Q, A233S, T287A, T287H, Q288A, Q288C, Q288D, Q288E, Q288F, Q2881, Q288M, Q288R, Q288S, Q288T, Q288W, L290K, L290M, Y291A, Y291I, Y291M, Y291N, Y291S, Y291V, Y291W, G298I, Y299F, Y299N, Y299S, S300F, S300L, S300T, G301Y, P302T, P302Y, A306L, A306M, P308G, P308H, P308K, P308L, P308Q, P308R, P308S, P308T, P308V, P308W, P308Y, T310L, T310M, T310S, T310W, S312F, S312I, S312L, S312M, S312N, S312Q, S312T, S312V, T313F, T313M, T313P, T313Y, N315S, Y317Q, Y317S, A319F, A319Q, A319S, Q320R, L322S, L322T, or L322V, wherein the position of each amino acid substitution corresponds to SEQ ID NO:3.

10. The variant of claim 1 wherein said variant further comprises at least one amino acid substitution at a position selected from: N5, I7, I9, N10, V12, W13, Q15, A16, P19, T20, T21, F22, P23, Y24, E25, S26, N27, P28, P29, V32, G33, T35, A36, A37, D38, L39, N41, F43, V44, D47, A48, Y49, Q50, N51, P52, I54, K58, N59, T61, N62, K64, S68, V69, K70, A71, V80, P89, I90, V91, N96, N98, D100, T103, T107, T108, E110, G116, V117, L119, L120, S121, G122, G123, D124, P125, G126, T127, V132, S135, V141, D146, N147, A149, I160, A161, H163, S164, G166, C167, A168, A171, N173, P175, I180, A181, S183, S185, S187, L188, Q189, S191, L194, G195, T196, D197, H200, A201, T202, P204, V206, L207, I208, N209, Y211, S213, P214, N216, Y217, I218, I219, P222, T223, V224, S226, T230, S231, V232, A233, G235, T287, Q288, T289, L290, Y291, G292, Q293, G295, G296, S297, G298, Y299, S300, G301, P302, T303, R304, C305, A306, P307, P308, A309, T310, S312, T313, N315, P316, Y317, Y318, A319, Q320, L322, or N323, wherein the position of each amino acid substitution corresponds to SEQ ID NO:3.

11. The variant of claim 10 wherein the at least one amino acid substitution is selected from one or more of: I9V, V12T, A16F, A16G, A16H, A16K, A16L, A16P, A16R, A16W, T21I, F22R, S26P, S26T, A37W, D38N, L39F, L39I, L39V, L39Y, I54E, T61S, N62P, D100A, D100F, D100G, D100I, D100L, D100N, D100P, D100Q, D100S, D100W, D100Y, T103A, T103C, T103D, T103F, T103M, T103N, T103Q, T103S, T103V, T103Y, T127F, T127L, T127M, T127V, T127W, T127Y, S164A, S164L, S164M, S164Q, S164R, G166A, G166F, G166M, G166S, Q167K, Q167V, A168F, A168I, A168L, A168M, A168P, A168R, A168T, A168Y, S183N, S185D, S185G, S185T, S187A, S187D, Q189E, Q189R, L194M, V206I, S213T, N216T, 1219M, S226Q, A233S, T287A, T287H, Q288A, Q288C, Q288D, Q288E, Q288F, Q288I, Q288M, Q288R, Q288S, Q288T, Q288W, L290K, L290M, Y291A, Y291I, Y291M, Y291N, Y291S, Y291V, Y291W, G298I, Y299F, Y299N, Y299S, S300F, S300L, S300T, G301Y, P302T, P302Y, A306L, A306M, P308G, P308H, P308K, P308L, P308Q, P308R, P308S, P308T, P308V, P308W, P308Y, T310L, T310M, T310S, T310W, S312F, S312I, S312L, S312M, S312N, S312Q, S312T, S312V, T313F, T313M, T313P, T313Y, N315S, Y317Q, Y317S, A319F, A319Q, A319S, Q320R, L322S, L322T, or L322V, wherein the position of each amino acid substitution corresponds to SEQ ID NO:3.

12. The variant of claim 1 , wherein the amino acid substitution at position I4 is selected from A, C, D, F, G, K, M, N, P, Q, V, T or Y.

13. A host cell comprising a polynucleotide encoding the variant of claim 1 .

14. The host cell of claim 13 , wherein said host cell is a fungal cell or a bacterial cell.

15. The host cell of claim 14 , wherein said fungal cell is a filamentous fungal cell selected from the group consisting of: Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma viride, Trichoderma Trichoderma harzianum, Penicillium, Humicola, Humicola insolens, Humicola grisea, Chrysosporium, Chrysosporium lucknowense, Myceliophthora thermophilia, Gliocladium, Aspergillus, Fusarium, Neurospora, Hypocrea, Emericella, Aspergillus niger, Aspergillus awamori, Aspergillus aculeatus , and Aspergillus nidulans.

16. The host cell of claim 13 , wherein said host cell expresses the variant.

17. A method for hydrolyzing a cellulosic substrate, comprising contacting said substrate with a variant GH61 polypeptide according to claim 1 .

18. The method of claim 17 wherein the cellulosic substrate is wheat straw, corn stover or bagasse.

19. The method of claim 18 wherein the cellulosic substrate is pretreated.

Assignees

• Danisco Us Inc

Inventors

• Foukaraki Maria
• Hommes Ronaldus Wilhelmus
• Kaper Thijs
• Kelemen Bradley R
• Kralj Slavko
• Lantz Suzanne E
• Nikolaev Igor
• Van Der Kley Wilhelmus
• Van Lieshout Johan Franciscus Thomas
• Van Stigt Thans Sander

Classifications

• C12Y302/01004

  Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase · CPC title

• C12N9/2434

  acting on beta-1,4-glucosidic bonds · 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

• C12N9/2437 Primary

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

• C12N9/0083

  Miscellaneous (1.14.99) · CPC title