# US 2019/0169587 A1 · Variant enzymes

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2019/0169587 A1
- Document ID: US20190169587A1
- Status: Published application
- Kind: A1
- Application number: 16/185,619
- Filed: 2018-11-09
- Published: 2019-06-06
- Earliest claimed: 2013-07-29
- Assignees: Danisco US Inc.
- Applicants: Danisco US Inc
- Inventors: MARIA FOUKARAKI; RONALDUS WILHELMUS JOANNES HOMMES; THIJS KAPER; BRADLEY R. KELEMEN; SLAVKO KRALJ; SUZANNE E. LANTZ; IGOR NIKOLAEV; WILHELMUS ANTONIUS HENDRICUS VAN DER KLEIJ; JOHANNES FRANCISCUS THOMAS VAN LIESHOUT; SANDER VAN STIGT THANS
- Web page: https://patentgrep.com/patent/US20190169587A1

## CPC

- C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)}
- C12N 9/0083: {Miscellaneous (1.14.99)}
- C12N 9/2402: {hydrolysing O- and S- glycosyl compounds (3.2.1)}
- C12N 9/2434: {acting on beta-1,4-glucosidic bonds}
- C12P 19/02: Monosaccharides (2-ketogulonic acid C12P7/60)
- C12P 19/14: produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase}
- C12Y 302/01004: Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES

## 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.

## Claims

### Claim 1

A variant of a parent glycosyl hydrolase 61 (GH61) enzyme, wherein said variant has cellulase augmenting 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, (f) performance in a Dilute Ammonia Pretreated Corn Stover (daCS) hydrolysis assay.

### Claim 2 (depends on 1)

The variant of claim 1, wherein said variant comprises at least one amino acid substitution at a position selected from the group consisting of: T287, W13, L288, P19, T20, P29, A37, L39, D47, A48, S68, T107, S121, G122, T127, S164, G166, Q167, A168, N216, I218, I219, V224, T289, Y291, S297, T303, R304, A306, P308, S312, T313, N315, Y317, Y318, N5, I7, V12, Q15, T21, F22, S26, P28, V32, G33, T35, A36, N41, Q50, N51, K58, N59, V80, P89, T108, G116, L120, G123, V132, S135, N147, A161, A181, S183, S185, Q189, G195, T196, H200, A201, T202, I208, P222, T223, S226, S231, G235, L290, G295, G296, G298, Y299, S300, G301, P302, P307, A309, T310, P316, A319, L322, I4, I9, N10, P23, Y24, E25, N27, D38, F43, V44, Y49, P52, I54, T61, N62, K64, V69, K70, A71, I90, V91, N96, N98, D100, T103, E110, V117, L119, D124, P125, G126, V141, D146, A149, I160, H163, A171, N173, P175, I180, S187, L188, S191, L194, D197, P204, V206, L207, N209, Y211, S213, P214, Y217, T230, V232, A233, G292, Q293, Q320, and N323, wherein the position of each amino acid substitution corresponds to SEQ ID NO:3.

### Claim 3

The variant of any preceding claim, wherein said variant comprises one or more of:
an amino acid substitution at position T287 selected from the group consisting of: H, A, F, G, S, and I; an amino acid substitution at position W13 selected from the group consisting of: G, L, N, R, T, V, F, Q, Y, A, E, I, K, and M; an amino acid substitution at position L288 selected from the group consisting of: E, F, G, I, K, M, N, R, S, T, A, C, D, V, and W; an amino acid substitution at position P19 selected from the group consisting of: F, M, E, H, Q, T, K, and W; an amino acid substitution at position T20 selected from the group consisting of: K, P, G, M, R, Y, A, and N; an amino acid substitution at position P29 selected from the group consisting of: C, E, F, K, T, Y D, G, M, R, and S; an amino acid substitution at position A37 selected from the group consisting of: D, S, E, L, W, F, K, and Q; an amino acid substitution at position L39 selected from the group consisting of: A, D, E, M, T, Y, F, I, N, P, Q, S, and V; an amino acid substitution at position D47 selected from the group consisting of: A, K, T, H, R, P, and W; an amino acid substitution at position A48 selected from the group consisting of: C, E, G, K, L, N, T, Y, Q, W, H, I, and S; an amino acid substitution at position S68 selected from the group consisting of: E, I, L, M, N, T, V, and Y; an amino acid substitution at position T107 selected from the group consisting of: A, D, E, K, M, R, S, and G; an amino acid substitution at position S121 selected from the group consisting of: E, F, N, T, C, L, V, D, G, K, M, and Y; an amino acid substitution at position G122 selected from the group consisting of: E, N, S, V, A, M, T, F, and L; an amino acid substitution at position T127 selected from the group consisting of: A, E, F, I, L, M, V, W, and Y; an amino acid substitution at position S164 selected from the group consisting of: A, D, E,G, K, L, M, Q, R, V, and Y; an amino acid substitution at position G166 selected from the group consisting of: A, D, E, F, H, K, M, N, Q, and S; an amino acid substitution at position Q167 selected from the group consisting of: E, A, D, F, G, K, L, N, R, V, and Y; an amino acid substitution at position A168 selected from the group consisting of: E, M, D, F, I, L, N, P, Q, R, T, and Y; an amino acid substitution at position N216 selected from the group consisting of: D, Q, T, K, and P; an amino acid substitution at position 1218 selected from the group consisting of: T, A, D, E, K, M, N, P, S, and V; an amino acid substitution at position 1219 selected from the group consisting of: M, A, D, L, N, P, and Q; an amino acid substitution at position V224 selected from the group consisting of: Q, I, A, E, N, P, S, and T; an amino acid substitution at position T289 selected from the group consisting of: A, D, K, L, M, R, and S; an amino acid substitution at position Y291 selected from the group consisting of: A, F, G, I, K, L, M, N, R, S, V, W, D, and E; an amino acid substitution at position S297 selected from the group consisting of: D, E, K, N, Q, R, and T; an amino acid substitution at position T303 selected from the group consisting of: K, M, P, R, S, V, and Y; an amino acid substitution at position R304 selected from the group consisting of: A, C, D, E, N, Q, T, and V; an amino acid substitution at position A306 selected from the group consisting of: E, F, G, I, K, L, M, Q, T, and Y; an amino acid substitution at position P308 selected from the group consisting of: A, G, H, I, K, L, Q, R, S, T, V, W, and Y; an amino acid substitution at position S312 selected from the group consisting of: Q, F, G, I, K, L, M, N, T, V, D, R, and Y; an amino acid substitution at position T313 selected from the group consisting of: S, D, G, L, P, V, E, F, I, K, M, and Y; an amino acid substitution at position N315 selected from the group consisting of: S, H, L, Q, R, V, Y, E, K, and M; an amino acid substitution at position Y317 selected from the group consisting of: Q, S, V, R, E, G, L, M, P, and T; a Y318T amino acid substitution; an amino acid substitution at position N5 selected from the group consisting of: A, D, G, and S; an amino acid substitution at position I7 selected from the group consisting of: L, M, and V; an amino acid substitution at position V12 selected from the group consisting of: M and T; an amino acid substitution at position Q15 selected from the group consisting of: H, K, and D; an amino acid substitution at position T21 selected from the group consisting of: I, K, M, and S; an amino acid substitution at position F22 selected from the group consisting of: K, I, L, and R; an amino acid substitution at position S26 selected from the group consisting of: M, P, and T; an amino acid substitution at position P28 selected from the group consisting of: M, S, and V; an amino acid substitution at position V32 selected from the group consisting of: A and P; an amino acid substitution at position G33 selected from the group consisting of: Q, A, and S; an amino acid substitution at position T35 selected from the group consisting of: A, D, E, K, N, and S; an amino acid substitution at position A36 selected from the group consisting of: T, I, N, S, and Y; an amino acid substitution at position N41 selected from the group consisting of: D, E, L, and M; an amino acid substitution at position Q50 selected from the group consisting of: A, T, Y, D, E, and K; an amino acid substitution at position N51 selected from the group consisting of: S, E, H, K, L, and T; an amino acid substitution at position K58 selected from the group consisting of: I, R, and V; an amino acid substitution at position N59 selected from the group consisting of: G, E, Q, and S; an amino acid substitution at position V80 selected from the group consisting of: A, E, L, and M; an amino acid substitution at position P89 selected from the group consisting of: T, S, V, and L; an amino acid substitution at position T108 selected from the group consisting of: A, Q, R, and S; an amino acid substitution at position G116 selected from the group consisting of: H, Q, and S; an amino acid substitution at position L120 selected from the group consisting of: V, T, I, and S; an amino acid substitution at position G123 selected from the group consisting of: E, Q, and R; an amino acid substitution at position V132 selected from the group consisting of: D, E, M, and R; an amino acid substitution at position S135 selected from the group consisting of: A, E, G, H, M, and N; an amino acid substitution at position N147 selected from the group consisting of: E, M, S, and T; an amino acid substitution at position A161 selected from the group consisting of: E, G, L, and S; an amino acid substitution at position A181 selected from the group consisting of: K, N, and R; an amino acid substitution at position S183 selected from the group consisting of: E, K, N, and T; an amino acid substitution at position S185 selected from the group consisting of: D, G, and T; an amino acid substitution at position Q189 selected from the group consisting of: E, L, M, R, G, and T; an amino acid substitution at position G195 selected from the group consisting of: A, E, and S; an amino acid substitution at position T196 selected from the group consisting of: H, Y, and R; an amino acid substitution at position H200 selected from the group consisting of: R, A, Q, and S; an amino acid substitution at position A201 selected from the group consisting of: P, K, and Q; an amino acid substitution at position T202 selected from the group consisting of: S, E, and Q; an amino acid substitution at position 1208 selected from the group consisting of: F, L, and V; an amino acid substitution at position P222 selected from the group consisting of: D and G; an amino acid substitution at position T223 selected from the group consisting of: S, E, and Q; an amino acid substitution at position S226 selected from the group consisting of: D, G, K, M, P, and Q; an amino acid substitution at position S231 selected from the group consisting of: C and I; an amino acid substitution at position G235 selected from the group consisting of: A, F, I, M, N, Q, V; an amino acid substitution at position L290 selected from the group consisting of: K, M, and T; an amino acid substitution at position G295 selected from the group consisting of: A, F, H, Q, and R; an amino acid substitution at position G296 selected from the group consisting of: A and M; an amino acid substitution at position G298 selected from the group consisting of: K, A, I, N, and Q; an amino acid substitution at position Y299 selected from the group consisting of: N, S, F, and P; an amino acid substitution at position S300 selected from the group consisting of: F, L, N, T, and Q; an amino acid substitution at position G301 selected from the group consisting of: D, S, and Y; an amino acid substitution at position P302 selected from the group consisting of: G, L, T, V, Y, and F; an amino acid substitution at position P307 selected from the group consisting of: A, F, H, L, and V; an amino acid substitution at position A309 selected from the group consisting of: I, K, R, T, V, and Y; an amino acid substitution at position T310 selected from the group consisting of: L, M, Q, S, and W; an amino acid substitution at position P316 selected from the group consisting of: D, N, R, and T; an amino acid substitution at position A319 selected from the group consisting of: Q, W, D, F, and S; an amino acid substitution at position L322 selected from the group consisting of: A, E, S, T, and V; an amino acid substitution at position 14 selected from the group consisting of: V and T; an amino acid substitution at position 19 selected from the group consisting of: V and A; an N10D amino acid substitution; a P23G amino acid substitution; a Y24T amino acid substitution; an amino acid substitution at position E25 selected from the group consisting of: N and Q; an N27E amino acid substitution; an amino acid substitution at position D38 selected from the group consisting of: E and N; an F43Y amino acid substitution; a V441 amino acid substitution; a Y49W amino acid substitution; an amino acid substitution at position P52 selected from the group consisting of: A and N; an amino acid substitution at position 154 selected from the group consisting of: Q and E; an amino acid substitution at position T61 selected from the group consisting of: Q and S; an N62P amino acid substitution; an amino acid substitution at position K64 selected from the group consisting of: A and Q; a V69Y amino acid substitution; a K70R amino acid substitution; an amino acid substitution at position A71 selected from the group consisting of: Y and P; an I90V amino acid substitution; an amino acid substitution at position V91 selected from the group consisting of: L and I; an amino acid substitution at position N96 selected from the group consisting of: A and S; an N98H amino acid substitution; an amino acid substitution at position D100 selected from the group consisting of: P and S; a T103A amino acid substitution; an E110K amino acid substitution; a V117A amino acid substitution; an amino acid substitution at position L119 selected from the group consisting of: N and T; a D124N amino acid substitution; a P125D amino acid substitution; an amino acid substitution at position G126 selected from the group consisting of: M and Q; a V141T amino acid substitution; a D146E amino acid substitution; an A149E amino acid substitution; an amino acid substitution at position I160 selected from the group consisting of: F and M; an H163A amino acid substitution; an A171T amino acid substitution; an N173H amino acid substitution; a P175F amino acid substitution; an I180L amino acid substitution; an amino acid substitution at position S187 selected from the group consisting of: A and D; an amino acid substitution at position L188 selected from the group consisting of: A and D; an S191D amino acid substitution; an amino acid substitution at position L194 selected from the group consisting of: D and M; an amino acid substitution at position D197 selected from the group consisting of: A and V; a P204A amino acid substitution; a V206I amino acid substitution; an L207F amino acid substitution; an N209D amino acid substitution; a Y211A amino acid substitution; an S213T amino acid substitution; a P214S amino acid substitution; a Y217H amino acid substitution; a T230S amino acid substitution; an amino acid substitution at position V232 selected from the group consisting of: F and Y; comprises an amino acid substitution at position A233 selected from the group consisting of: F and S; an amino acid substitution at position G292 selected from the group consisting of: D and K; an amino acid substitution at position Q293 selected from the group consisting of: K and P; a Q320R amino acid substitution; and an N323V amino acid substitution.

### Claim 4

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

### Claim 5 (depends on 4)

The variant of claim 4, wherein said variant is a combinatorial variant.

### Claim 6

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

### Claim 7 (depends on 6)

The variant of claim 6, 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.

### Claim 8

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

### Claim 9

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

### Claim 10

A polynucleotide comprising a polynucleotide sequence encoding a variant of a parent GH61 polypeptide according to any preceding claim.

### Claim 11 (depends on 10)

A vector comprising the polynucleotide of claim 10.

### Claim 12 (depends on 11)

The vector of claim 11, wherein said vector is an expression vector.

### Claim 13 (depends on 10, 11, 12)

A host cell comprising the polynucleotide of claim 10, the vector of claim 11, or the expression vector of claim 12.

### Claim 14 (depends on 13)

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

### Claim 15 (depends on 14)

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 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.

### Claim 16 (depends on 13, 14, 15)

The host cell of claim 13, 14 or 15, wherein said host cell expresses the variant of a parent GH61 polypeptide encoded by said polynucleotide, vector, or expression vector.

### Claim 17 (depends on 16)

A method of producing a variant GH61 polypeptide comprising culturing a host cell according to claim 16 in a suitable culture medium under suitable conditions to produce said variant.

### Claim 18 (depends on 17)

The method of claim 17, further comprising isolating said produced variant.

### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

A composition comprising a GH61 variant according to any one of claims 1 to 9.

### Claim 20 (depends on 19)

The composition of claim 19, wherein said composition is selected from the group consisting of: a detergent, an animal feed, a feed additive, and a cell culture supernatant.

### Claim 21 (depends on 19, 20)

The composition of claim 19 or 20, wherein said composition is enriched for said GH61 variant.

### Claim 22 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

A method for hydrolyzing a cellulosic substrate, comprising contacting said substrate with a variant GH61 polypeptide according to any one of claims 1 to 9.