Variant enzymes

Patent metadata

Field	Value
Publication number	US-2016186155-A1
Application number	US-201414909041-A
Country	US
Kind code	A1
Filing date	Jul 24, 2014
Priority date	Jul 29, 2013
Publication date	Jun 30, 2016
Grant date	—

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

1 . A variant of a parent glycoside hydrolase family 61 (GH61) enzyme, wherein said variant has cellulase activity, has at least 80% sequence identity to SEQ ID NO:3, and has at least one improved property over said parent GH61 enzyme selected from: (a) expression, (b) thermostability and/or Tm, (c) performance in a Whole Hydrolysate Dilute Acid Pretreated Corn Stover (whPCS) hydrolysis assay, and (d) performance in a Dilute Ammonia Pretreated Corn Stover (daCS) hydrolysis assay, wherein said variant comprises at least one amino acid substitution at one or more position corresponding to amino acids 111 to 114 and 144 to 163 of SEQ ID NO:3.

2 . The variant of claim 1 , wherein said variant has improved thermostability and/or Tm and wherein said at least one amino acid substitution is selected from the group consisting of: I144G, D146F, L148P, A149P, G151Q, G151V, N152F, N152G, N152Q, N152S, N152Y, L155M, R156S, H157W, E158W, E158Y, I159Q, I160D, I160F, A161E, A161L, A161Y, L162A, L162F, H163L, H163R, H163Y, and combinations thereof.

3 . The variant of claim 1 , wherein said variant has improved performance in a whPCS hydrolysis assay and wherein said at least one amino acid substitution is selected from the group consisting of: I144K, I144R, I144S, P145G, P145H, P145I, P145K, P145R, P145S, P145V, D146A, D146C, D146E, D146F, D146K, D146M, D146Q, D146T, D146Y, N147F, N147M, L148K, L148V, A149C, A149D, A149F, A149G, A149I, A149N, A149V, P150A, P150C, P150D, P150E, P150F, P150G, P150H, P150I, P150K, P150L, P150Q, G151E, G151F, G151H, G151I, G151K, G151M, G151P, G151Q, G151S, G151V, G151W, G151Y, N152E, N152F, N152G, N152H, N152K, N152P, Y153F, Y153K, Y153L, Y153M, Y153P, Y153Q, Y153R, Y153S, Y153V, V154A, V154C, V154D, V154E, V154G, V154H, V154K, V154N, V154P, V154Q, V154R, V154T, L155C, L155F, L155K, L155M, L155N, R156A, R156C, R156D, R156E, R156F, R156H, R156I, R156K, R156L, R156M, R156P, R156Q, H157D, E158A, E158C, E158D, E158F, E158H, E158I, E158L, E158M, E158N, E158P, E158S, E158T, E158W, A161C, L162I, L162N, H163A, H163C, H163D, H163E, H163F, H163G, H163I, H163K, H163M, H163P, H163R, H163T, H163V, H163W, H163Y, and combinations thereof.

4 . The variant of claim 1 , wherein said variant has improved performance in a daCS hydrolysis assay and wherein said at least one amino acid substitution is selected from the group consisting of: F112V, K113L, K113M, K113N, K113R, K113S, I114F, I114V, I144F, I144V, P145A, Y153F, V154T, H163D, H163Y, and combinations thereof.

5 . The variant of claim 1 , wherein said variant further comprises at least one second amino acid substitution selected from the group consisting of: F112M, F112W, K113P, K113T, I114L, I114M, I114T, I144A, I144C, I144D, I144E, I144H, I144N, I144P, I144Q, I144T, I144W, I144Y, P145C, P145D, P145E, P145F, P145L, P145M, P145N, P145Q, P145T, P145W, P145Y, D146G, D146H, D1461, D146L, D146N, D146P, D146R, D146S, D146V, D146W, N147A, N147C, N147D, N147E, N147G, N147H, N147I, N147K, N147L, N147P, N147Q, N147R, N147S, N147T, N147V, N147W, N147Y, L148A, L148C, L148D, L148E, L148F, L148G, L148H, L148I, L148M, L148N, L148Q, L148R, L148S, L148T, L148W, L148Y, A149E, A149H, A149K, A149L, A149M, A149Q, A149R, A149S, A149T, A149W, A149Y, P150M, P150N, P150R, P150S, P150T, P150V, P150W, P150Y, G151A, G151C, G151D, G151L, G151N, G151R, G151T, N152A, N152C, N152D, N152I, N152L, N152M, N152R, N152T, N152V, N152W, Y153A, Y153C, Y153D, Y153E, Y153G, Y153H, Y153I, Y153N, Y153T, Y153W, V154F, V154I, V154L, V154M, V154S, V154W, V154Y, L155A, L155D, L155E, L155G, L155H, L1551, L155P, L155Q, L155R, L155S, L155T, L155V, L155W, L155Y, R156G, R156N, R156T, R156V, R156W, R156Y, H157A, H157C, H157E, H157F, H157G, H1571, H157K, H157L, H157M, H157N, H157P, H157Q, H157R, H157S, H157T, H157V, H157Y, E158G, E158K, E158R, E158V, I159A, I159C, I159D, I159E, I159F, I159G, I159H, I159K, I159L, I159M, I159N, I159P, I159R, I159S, I159T, I159V, I159W, I159Y, I160A, I160C, I160E, I160G, I160H, I160K, I160L, I160M, I160N, I160P, I160Q, I160R, I160S, I160T, I160V, I160W, I160Y, A161D, A161F, A161G, A161H, A161I, A161K, A161M, A161N, A161P, A161Q, A161R, A161S, A161T, A161V, A161W, L162C, L162D, L162E, L162G, L162H, L162K, L162M, L162P, L162Q, L162R, L162S, L162T, L162V, L162W, L162Y, H163Q, H163S, and combinations thereof.

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

7 . The variant of claim 6 , wherein said variant is a combinatorial variant.

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

9 . The variant of claim 8 , 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.

10 . The variant of any preceding claim, wherein said variant has at least 90% sequence identity to SEQ ID NO:3.

11 - 14 . (canceled)

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

16 . The host cell of claim 15 , wherein said host cell is a fungal cell or a bacterial cell.

17 . The host cell of claim 16 , wherein said fungal cell is a filamentous fungal cell selected from the group consisting of: Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma viride, Trichoderma koningii, 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.

18 . The host cell of claim 15 , wherein said host cell expresses the variant of a parent GH61 polypeptide encoded by said polynucleotide, vector, or expression vector.

19 - 23 . (canceled)

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

25 . The method of claim 31 wherein the cellulosic substrate is wheat straw, corn stover or bagasse.

26 . The method of claim 32 wherein the cellulosic substrate is pretreated.

Assignees

• Danisco Us Inc

Inventors

• Douglas Nicholai
• Foukaraki Maria
• Hommes Ronaldus Wilhelmus
• Kaper Thijs
• Kelemen Bradley R
• Kralj Slavko
• Lantz Suzanne E
• Lassila Jonathan K
• Nikolaev Igor
• Torres-Pazmino Daniel Esteban
• Van Der Kley Wilhelmus
• Van Lieshout Johannes Franciscus Thomas
• Van Stigt Thans Sander

Classifications

• C12N9/0083

  Miscellaneous (1.14.99) · CPC title

• C12Y302/01004

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

• C12N9/2437 Primary

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

• C12P19/02

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

• C12N9/2402

  hydrolysing O- and S- glycosyl compounds (3.2.1) · CPC title