Production and use of plant degrading materials

• US 10,676,751 B2
• Filed: 03/02/2009
• Issued: 06/09/2020
• Est. Priority Date: 02/29/2008
• Status: Active Grant

• Current Assignee: Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Original Assignee: Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Inventors: Daniell, Henry
• Primary Examiner(s): Visone, Lee A
• Application Number:
  US12/396,382
• Publication Number:
  US 20090325240A1
• Time in Patent Office: 4,117 Days
• CPC Class Codes: C12N 15/8214 Plastid transformation
• CPC Class Codes: C12N 15/8242 with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
• CPC Class Codes: C12N 15/8257 for the production of primary gene products, e.g. pharmaceutical products, interferon
• 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 9/2482 Endo-1,4-beta-xylanase (3.2.1.8)
• CPC Class Codes: C12P 7/12 substrate containing sulfite waste liquor or citrus waste
• 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: C12Y 302/01091 Cellulose 1,4-beta-cellobiosidase (3.2.1.91)

Abstract

Disclosed herein are materials useful for degrading plant biomass material. In exemplary embodiments, the plant material comprises one or more enzymes that are expressed in plants and/or bacteria. Specifically exemplified herein are plant degrading enzymes expressed in chloroplasts. The chloroplast expressed enzymes may be provided as cocktails for use in conjunction with conventional methods of converting biomass into biofuels, such as cellulosic ethanol.

Claims

1. A method of degrading a plant biomass sample so as to release fermentable sugars therein, the method comprising obtaining a plant degrading enzyme cocktail comprising plant cell extracts comprising active plant degrading enzymes recombinantly expressed in chloroplasts of plant cells, and admixing said plant degrading enzyme cocktail with said biomass sample, wherein said cocktail comprises recombinant CelD cellulase encoded by sequences obtained from Clostridium thermocellum, ligninase, recombinant beta-glucosidase encoded by sequences obtained from Trichoderma reesei, hemicellulase, xylanase encoded by sequences obtained from Trichoderma reesei, recombinant alpha amylase encoded by sequences obtained from Bacillus ssp., amyloglucosidase, recombinant PelB and PelD pectate lyases encoded by sequences obtained from Fusarium solani, lipase encoded by sequences obtained from Mycobacterium tuberculosis, maltogenic alpha-amylase encoded by sequences obtained from Bacillus ssp., acetyl xylan esterase encoded by sequences obtained from Trichoderma reesei, cutinase encoded by sequences obtained from Fusarium solani, and swollenin encoded by sequences obtained from Trichoderma reesei, wherein said CelD, PelB, and PelD exhibit elevated levels of enzymatic activity at higher temperatures and/or broader pH ranges relative to E. coli-expressed CelD, PelB, and PelD enzymes, respectively, said plant degrading enzyme cocktail acting synergistically to release fermentable sugars produced from said biomass sample.

2. The method of claim 1, comprising at least one bacterial cell extract comprising a second plant degrading enzyme.

3. The method of claim 1, wherein said plant degrading enzyme cocktail comprises at least one additional chloroplast-expressed recombinant cellulase selected from the group consisting of CelO exoglucanase encoded by sequences obtained from Clostridium thermocellum, egI endoglucanase, egII endoglucanase, each being encoded by sequences obtained from Trichoderma reesei.

4. The method of claim 1, wherein said plant biomass sample comprises grain and/or grain residues, sugar beet, sugar cane, grasses, wood-based biomass, fruits and/or fruit waste residues, or a combination thereof.

5. The method of claim 4, wherein said plant biomass is corn, wheat, barley, or citrus, or waste residues obtained therefrom.

6. The method of claim 4, wherein said plant biomass is switchgrass.

8. The method of claim 4, wherein said plant biomass is sugar cane.

10. The method of claim 4, wherein said plant biomass is sugar beet.

11. The method of claim 1, wherein said plant degrading cocktail further comprises rubisco.

7. A method of degrading a plant biomass sample so as to release fermentable sugars therein, the method comprising obtaining a plant degrading enzyme cocktail comprising plant cell extracts comprising active plant degrading enzyme recombinantly expressed in chloroplasts of plant cells, and admixing said plant degrading enzyme cocktail with said biomass sample, wherein said plant biomass is sawdust or a wood-based biomass and said plant degrading enzymes consist of CelD endoglucanase, CelO exoglucanase each being encoded by sequences obtained from Clostridium thermocellum;

beta-glucosidase, xylanase, acetyl xylan esterase each being encoded by sequences obtained from Trichoderma reesei;

PelA pectate lyase, PelB pectate lysase, PelD pectate lyase each being encoded by sequences obtained from Fusarium solani; and

swollenin encoded by sequences obtained from Trichoderma reesei, and said plant degrading enzyme cocktail is effective to release 100% fermentable sugars produced from said sawdust or wood-based biomass.

9. A method of degrading a plant biomass sample so as to release fermentable sugars therein, the method comprising obtaining a plant degrading enzyme cocktail comprising plant cell extracts comprising active plant degrading enzyme recombinantly expressed in chloroplasts of plant cells, and admixing said plant degrading enzyme cocktail with said biomass sample, wherein said plant biomass is citrus peel and said plant degrading cocktail contains enzymes consisting of CelD endoglucanase, CelO exoglucanase each being encoded by sequences obtained from Clostridium thermocellum;

beta-glucosidase, xylanase, acetyl xylan esterase each being encoded by sequences obtained from Trichoderma reesei;

lipase encoded by sequences obtained from Mycobacterium tuberculosis, PelA pectate lysase, PelB pectate lyase, PelD pectate lyase each being encoded by sequences obtained from Fusarium solani;

cutinase encoded by a sequence obtained from Fusarium solani, and swollenin encoded by sequences obtained from Trichoderma reesei, and said plant degrading enzyme cocktail is effective to release 100% fermentable sugars from said citrus peel biomass.

12. A plant material useful for degrading a processed wood biomass, said material comprising chloroplast-expressed CelD endoglucanase, CelO exoglucanase each being encoded by sequences obtained from Clostridium thermocellum;

beta-glucosidase, xylanase, acetyl xylan esterase each being encoded by sequences obtained from Trichoderma reesei;

PelA pectate lyase, PelB pectate lysase, PelD pectate lyase, each being encoded by sequences obtained from Fusarium solani; and

swollenin encoded by a sequence obtained from Trichoderma reesei, and said plant degrading enzyme cocktail being effective to release 100% fermentable sugars produced from said sawdust or wood-based biomass.

14. The homogenized plant material of claim 12, in powdered form.

13. A plant material for degrading a citrus peel biomass wherein said plant material comprises chloroplast expressed CelD endoglucanase, CelO exoglucanase each being encoded by sequences obtained from Clostridium thermocellum;

beta-glucosidase, xylanase, acetyl xylan esterase each being encoded by sequences obtained from Trichoderma reesei;

lipase encoded by sequences obtained from Mycobacterium tuberculosis, PelA pectate lysase, PelB pectate lyase, PelD pectate lyase each being encoded by sequences obtained from Fusarium solani;

cutinase encoded by sequences obtained from Fusarium solani, and swollenin encoded by sequences obtained from Trichoderma reesei, and said plant degrading enzyme cocktail is effective to release 100% fermentable sugars from said citrus peel biomass.

15. The method of claim 13, wherein said plant material is in powdered form.