Consolidated pretreatment and hydrolysis of plant biomass expressing cell wall degrading enzymes

• US 10,006,038 B2
• Filed: 12/07/2015
• Issued: 06/26/2018
• Est. Priority Date: 11/06/2009
• Status: Active Grant

• Current Assignee: Agrivida, Inc. (Nippon Soda Company Limited)
• Original Assignee: Agrivida, Inc. (Nippon Soda Company Limited)
• Inventors: Raab, R. Michael, Zhang, Dongcheng, Bougri, Oleg
• Primary Examiner(s): Page, Brent T
• Application Number:
  US14/961,426
• Publication Number:
  US 20160138035A1
• Time in Patent Office: 932 Days
• Field of Search: None
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8246 Non-starch polysaccharides, e.g. cellulose, fructans, levans
• CPC Class Codes: C12N 9/2437 Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• CPC Class Codes: C12N 9/2445 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: C12N 9/248 Xylanases
• 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: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: C12Y 302/01008 Endo-1,4-beta-xylanase (3.2.1.8)
• CPC Class Codes: C12Y 302/01021 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02E 50/30 Fuel from waste, e.g. synthetic alcohol or diesel

Abstract

Methods for consolidated pretreatment and hydrolysis of genetically engineered plants expressing cell wall degrading enzymes are provided. Expression cassettes and vectors for making transgenic plants are described. Plants engineered to express one or more cell wall degrading enzymes using expression cassettes and vectors of the invention are also provided.

Claims

1. An engineered plant comprising a first polynucleotide sequence, a second polynucleotide sequence, and a third polynucleotide sequence, wherein the first polynucleotide sequence encodes a xylanase comprising an amino acid sequence of SEQ ID NO:

6, the second polynucleotide sequence encodes an endoglucanase comprising an amino acid sequence of SEQ ID NO;

4, and the third polynucleotide sequence encodes a cellobiohydrolase comprising an amino acid sequence of SEQ ID NO;

10 or 12.

2. The engineered plant of claim 1, wherein at least one of the first polynucleotide sequence, the second polynucleotide sequence, or the third polynucleotide sequence further comprises a targeting polynucleotide sequence encoding a targeting peptide selected from the group consisting of:

an amyloplast targeting signal, a cell wall targeting peptide, a vacuole targeting peptide, a mitochondrial targeting peptide, a cytosol localization signal, a chloroplast targeting signal, and a nuclear targeting peptide.

3. The engineered plant of claim 2, wherein the targeting polynucleotide sequence encodes a targeting sequence selected from the group consisting of:

SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

22, SEQ ID NO;

23, and SEQ ID NO;

24.

4. The engineered plant of claim 1, wherein the engineered plant is selected from the group consisting of:

corn, sugar cane, sugar beet, sorghum, switchgrass, miscanthus, eucalyptus, willow and poplar.

5. The engineered plant of claim 1, wherein the engineered plant produces more xylose under hydrolysis condition in the absence of exogenous enzymes compared to a non-engineered plant of the same genetic background lacking a xylanase, an endoglucanase and a cellobiohydrolase.

6. The engineered plant of claim 1, wherein the engineered plant produces more glucose under hydrolysis condition in the absence of exogenous enzymes compared to a non-engineered plant of the same genetic background lacking a xylanase, an endoglucanase and a cellobiohydrolase.

7. The engineered plant of claim 1, wherein the engineered plant produces at least 10% more xylose under hydrolysis condition in the absence of exogenous enzymes compared to a non-engineered plant of the same genetic background lacking a xylanase, an endoglucanase and a cellobiohydrolase.

8. The engineered plant of claim 1, wherein the engineered plant produces at least 10% more glucose under hydrolysis condition in the absence of exogenous enzymes compared to a non-engineered plant of the same genetic background lacking a xylanase, an endoglucanase and a cellobiohydrolase.