Use of Glycoside Hydrolase 61 Family Proteins in Processing of Cellulose

• US 20120083019A1
• Filed: 08/22/2011
• Published: 04/05/2012
• Est. Priority Date: 08/20/2010
• Status: Active Grant

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, Inc.
• Inventors: Baidyaroy, Dipnath, Clark, Louis, Campopiano, Onorato, Rao, Kripa, Szabo, Lorand, Torok, Janos
• Granted Patent: US 8,298,795 B2
• Time in Patent Office: Days
• US Class Current: 435/99
• CPC Class Codes: C12N 9/242 Fungal source
• CPC Class Codes: C12N 9/2434 acting on beta-1,4-glucosidic bonds
• CPC Class Codes: C12P 19/02 Monosaccharides 2-ketogulonic acid C12P7/60
• CPC Class Codes: C12P 19/14 produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase , e.g. by cellulase, hemicellulase
• CPC Class Codes: C12P 7/10 substrate containing cellulosic material
• CPC Class Codes: C12P 7/14 Multiple stages of fermentation; Multiple types of microorganisms or re-use of microorganisms
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

The invention provides recombinant GH61 proteins obtained from Myceliophtora thermophila, and nucleic acids that encode such proteins. The invention also provides protein fractions isolated from M. thermophila supernatant that have GH61 protein activity. These preparations can be used to increase yield of products from reactions in which a cellulose-containing substrate undergoes saccharification by one or more cellulase enzymes, such as endoglucanase, β-glucosidase, or cellobiohydrolase. Combinations of GH61 protein and cellulases can be used to break down cellulosic biomass into fermentable sugars in the production of ethanol.

Claims

1-70. -70. (canceled)

71. A composition comprising a cellulosic substrate, and a recombinantly produced GH61 polypeptide that comprises an amino acid sequence that is at least 90% identical to residues 11-323 of SEQ ID NO:

2, or to a biologically active fragment thereof.

72. The composition of claim 71, wherein the GH61 protein comprises an amino acid sequence that is at least 95% identical to residues 11-323 of SEQ ID NO:

2.

73. The composition of claim 71, wherein the GH61 protein comprises an amino acid sequence that is 100% identical to the secreted portion of SEQ ID NO:

2.

74. The composition of claim 71, further comprising at least one recombinant cellulase enzyme selected from an endoglucanase (EG), a β

-glucosidases (BGL), a Type 1 cellobiohydrolase (CBH1), and a Type 2 cellobiohydrolase (CBH2).

75. The composition of claim 71, comprising:

a) a recombinantly produced GH61 protein comprising an amino acid sequence that is at least about 90% identical to SEQ ID NO;

2; and

b) one or more recombinant cellulase enzymes selected from endoglucanases (EG), β

-glucosidases (BGL), Type 1 cellobiohydrolases (CBH1), and Type 2 cellobiohydrolases (CBH2);

wherein the yield of glucose in a reaction in which cellulose undergoes saccharification by an enzyme combination comprising EG, BGL, CBH1, and CBH2, is at least 20% greater in the presence of the GH61 protein compared with the yield of glucose under the same reaction conditions in the absence of the GH61 protein.

76. The composition of claim 75, wherein the cellulase enzyme(s) are fungal enzymes.

77. The composition of claim 76, wherein the cellulase enzyme(s) are from M. thermophila.

78. The composition of claim 77, comprising a host cell in which a recombinant gene encoding the GH61 protein and a recombinant gene encoding a cellulase enzyme are expressed.

79. The composition of claim 78, wherein the gene encoding the GH61 protein encodes a GH61 protein comprising a heterologous signal peptide.

80. The composition of claim 78, wherein the gene encoding the GH61 protein comprises a GH61 coding sequence operably linked to a heterologous promoter.

81. The composition of claim 75, wherein the GH61 protein comprises an amino acid sequence that is 100% identical to the secreted portion of SEQ ID NO:

2.

87. A method of producing an end product from a cellulosic substrate, comprising:

a) contacting the cellulosic substrate with a composition according to claim 75, whereby fermentable sugars are produced from the substrate; and

b) contacting the fermentable sugars with a microorganism in a fermentation to produce an end product.

88. The method of claim 87, wherein the end product is ethanol.

89. The method of claim 87, wherein the cellulase enzymes are obtained from a culture broth from M. thermophila cells.

82. A method for increasing yield of fermentable sugars in a reaction in which a cellulose-containing substrate undergoes saccharification by one or more cellulase enzymes, comprising conducting the reaction in the presence of a recombinant GH61 protein that comprises an amino acid sequence that is at least 80% identical to residues 11-323 of SEQ ID NO:

2 or to a biologically active fragment thereof.

83. The method of claim 82, wherein the GH61 protein comprises an amino acid sequence that is at least 95% identical to residues 11-323 of SEQ ID NO:

2, or to a fragment of SEQ ID NO;

2.

84. The method of claim 82, wherein the one or more cellulase enzymes comprise a combination of an endoglucanase (EG), a β

-glucosidase (BGL), a Type 1 cellobiohydrolase (CBH1), and a Type 2 cellobiohydrolase (CBH2).

85. The method of claim 82, wherein the GH61 protein comprises an amino acid sequence that is at least 98% identical to residues 11-323 of SEQ ID NO:

2.

86. The method of claim 82, wherein the reaction results in glucose yield that is at least about 20% higher than glucose yield of a saccharification reaction under the same conditions in the absence of said GH61 protein.