Variants of cellobiohydrolases

Patent metadata

Field	Value
Publication number	US-9938551-B2
Application number	US-201314650285-A
Country	US
Kind code	B2
Filing date	Dec 10, 2013
Priority date	Dec 12, 2012
Publication date	Apr 10, 2018
Grant date	Apr 10, 2018

Abstract

Disclosed are a number of homologs and variants of Hypocrea jecorina Ce17A (formerly Trichoderma reesei cellobiohydrolase I or CBH1), nucleic acids encoding the same and methods for producing the same. The homologs and variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted and/or deleted.

First claim

What is claimed is:

1. A variant of a parent cellobiohydrolase 1 (CBH1) enzyme, wherein said variant has cellulase activity, comprises at least one amino acid substitution, has at least 90% amino acid sequence identity to SEQ ID NO:3, and has at least one improved property over said parent CBH1 enzyme selected from: (a) expression (protein content determination), (b) phosphoric acid swollen cellulose (PASC) hydrolysis assay activity, (c) PASC hydrolysis assay activity in the presence of endoglucanase 2 (EG2), (d) PASC hydrolysis assay activity after heat incubation, (e) whole hydrolysate acid-pretreated corn stover (whPCS) assay activity, (f) dilute ammonia corn cob (daCC) assay activity, and (g) dilute ammonia corn stover (daCS) assay activity; and wherein the at least one amino acid substitution is with an alanine, glutamine, or serine at a position that corresponds to position D249 of SEQ ID NO:3.

2. The variant of claim 1 , wherein said at least one amino acid substitution is with an alanine.

3. The variant of claim 1 , wherein said variant comprises an additional amino acid substitution at one or both amino acid positions corresponding to S92 and T41 of SEQ ID NO:3.

4. The variant of claim 3 , wherein said additional amino acid substitution is selected from the group consisting of: S92T, T41I, and both S92T and T41I.

5. A host cell comprising an expression vector comprising a polynucleotide sequence encoding the variant according to claim 1 .

6. The host cell of claim 5 , wherein said host cell is selected from the group consisting of:
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;
a yeast cell selected from the group consisting of: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Kluyveromyces lactis, Candida utilis, Candida albicans, Pichia stipitis, Pichia pastoris, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Arxula adeninivorans, Debaryomyces hansenii , and Debaryomyces polymorphus ; and
a Zymomonas mobilis cell.

7. The host cell of claim 5 , wherein said host cell expresses the variant.

8. A method for hydrolyzing a cellulosic substrate, comprising:
contacting said substrate with a composition comprising the variant according to claim 1 ,
and optionally producing ethanol by culturing an ethanologen in a medium comprising glucose and/or glucose-containing oligosaccharide products of the hydrolysis.

9. The method of claim 8 , wherein said cellulosic substrate is selected from the group consisting of grass, switch grass, cord grass, rye grass, reed canary grass, miscanthus , sugar-processing residues, sugarcane bagasse, agricultural wastes, rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, corn fiber, stover, soybean stover, corn stover, forestry wastes, wood pulp, recycled wood pulp fiber, paper sludge, sawdust, hardwood, softwood, and combinations thereof.

10. The method of claim 8 , wherein the composition further comprises one or more additional cellulases or hemicellulases.

11. The variant of claim 1 , wherein the variant has at least 95% amino acid sequence identity to SEQ ID NO:3.

12. The variant of claim 11 , wherein the variant has at least 97% amino acid sequence identity to SEQ ID NO:3.

13. The variant of claim 11 , wherein the variant has at least 98% amino acid sequence identity to SEQ ID NO:3.

Assignees

• Danisco Us Inc
• Danisco Us Inc

Inventors

• Bott Richard R
• Foukaraki Maria
• Hommes Ronaldus Wilhelmus
• Kaper Thijs
• Kelemen Bradley R
• Kralj Slavko
• Nikolaev Igor
• Sandgren Mats
• Van Lieshout Johannes Franciscus Thomas
• Van Stigt Thans Sander

Classifications

• C12Y302/01091

  Cellulose 1,4-beta-cellobiosidase (3.2.1.91) · CPC title

• C12P19/02

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

• C12N9/2437 Primary

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

• C12N9/2434

  acting on beta-1,4-glucosidic bonds · CPC title

• C12P19/14 Primary

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