PRODUCTION AND USE OF PLANT DEGRADING MATERIALS

• US 20090325240A1
• Filed: 03/02/2009
• Published: 12/31/2009
• 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
• Granted Patent: US 10,676,751 B2
• Time in Patent Office: Days
• US Class Current: 435/101
• 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 cocktail comprising at least two cell extracts, each cell extract comprising an active plant degrading compound recombinantly expressed in cells from which each said cell extract is derived, said at least two cell extracts being plant extracts or bacterial extracts, or a combination of both;

and admixing said plant degrading cocktail with said biomass sample.

2. The method of claim 1, wherein said at least two cell extracts comprise at least one plant cell extract comprising a first plant degrading compound and at least one bacterial cell extract comprising a second plant degrading compound.

3. The method of claim 1, wherein said plant degrading cocktail comprises at least three cell extracts, each of said at least three cell extracts comprising cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase, maltogenic alpha-amylase, pectolyase or expansin.

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.

7. The method of claim 4, wherein said plant biomass is sawdust or otherwise processed wood.

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

9. The method of claim 4, wherein said plant biomass is citrus peel.

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.

24. The method of claim 1 optimized for digesting a citrus biomass sample wherein said plant degrading cocktail comprises cellulase, beta-glucosidase, xylanase, alpha amylase, amyloglucosidase, pectin lyase or pectate lyase, or a combination thereof;

and said biomass sample is a citrus biomass sample.

49. The method of claim 1, wherein said plant material is in powdered form.

74. The method of claim 1, wherein said plant degrading cocktail includes at least two of the following:

beta-glucosidase, xylanase, alpha amylase, amyloglucosidase, pectin lyase or pectate lyase

12. A method of producing a plant biomass degrading material comprising

producing a first plant comprising chloroplasts that express a first plant degrading enzyme and a second plant comprising chloroplasts that express a second plant degrading enzyme;

harvesting said first and second plants; and

processing said first and second plants to produce an enzyme source comprising a combination of enzymes suitable for mixing with and degrading a biomass sample.

13. The method of claim 12, wherein said first and/or second plant is tobacco.

14. The method of claim 12 wherein said processing comprises homogenizing said first and/or second plant or a portion thereof.

15. The method of claim 12, wherein said producing step comprises at least two of the following:

producing a first plant comprising chloroplasts that express cellulase, producing a second plant comprising chloroplasts that express lignanse, producing a third plant comprising chloroplasts that express beta-glucosidase;

producing a fourth plant comprising chloroplasts that express hemicellulase;

producing a fifth plant comprising chloroplasts that express xylanase;

producing a sixth plant comprising chloroplasts that express alpha-amylase;

producing a seventh plant comprising chloroplasts that express amyloglucosidase;

producing an eighth plant comprising chloroplasts that express pectate lyase;

producing a ninth plant comprising chloroplasts that express cutinase;

producing a tenth plant comprising chloroplasts that express lipase;

producing an eleventh plant comprising chloroplasts that express maltogenic alpha amylase, producing a twelfth plant comprising chloroplasts that express pectolyase or a thirteenth plant comprising chloroplasts that express expansin (e.g. swollenin).

25. The method of claim 12, wherein said first and second plants are monocots, dicots or algae or a combination thereof.

50. The method of claim 12, wherein said first or second plant is homoplasmic or heteroplasmic with respect to chloroplasts expressing first or second enzyme, respectively.

16-19. -19. (canceled)

20. A plant material useful for degrading a plant biomass, said material comprising a first chloroplast genome or genome segment having a heterologous gene that encodes a first plant degrading enzyme and a second chloroplast genome or genome segment having a heterologous gene that encodes a second plant degrading enzyme.

21. The plant material of claim 20 wherein said plant material comprises at least two of the following:

a first chloroplast genome or genome segment having a heterologous gene that encodes cellulase, a second chloroplast genome or genome segment having a heterologous gene that encodes lignanase, a third chloroplast genome or genome segment having a heterologous gene that encodes beta-glucosidase;

a fourth chloroplast genome or genome segment having a heterologous gene that encodes hemicellulase;

a fifth chloroplast genome or genome segment having a heterologous gene that encodes xylanase;

a sixth chloroplast genome or genome segment having a heterologous gene that encodes alpha-amylase;

a seventh chloroplast genome or genome segment having a heterologous gene that encodes amyloglucosidase;

an eighth chloroplast genome or genome segment having a heterologous gene that encodes pectate lyase;

a ninth plant chloroplast genome or genome segment having a heterologous gene that encodes cutinase;

a tenth chloroplast genome or genome segment having a heterologous gene that encodes lipase;

an eleventh chloroplast genome or genome segment having a heterologous gene that encodes pectolyase;

a twelfth chloroplast genome or genome segment having a heterologous gene that encodes maltogenic alpha-amylase;

or a thirteenth chloroplast genome or genome segment have a heterologous gene that encodes expansin.

22. A plant comprising a plant cell having recombinantly expressing cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase pectolyase, maltogenic alpha-amylase, or expansin, said plant a commercial cultivar of tobacco.

23. The plant of claim 22, wherein said enzyme is in a chloroplast in said cell.

26. A homogenized plant material comprising at least two of the following active proteins:

cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase or pectolyase, or a combination thereof; and

wherein said plant material comprises cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase, pectolyase or maltogenic alpha-amylase;

said plant material optionally comprising rubisco and/or swollenin.

27. The homogenized plant material of claim 26, where the enzyme comprises more than 0.1 percent of total protein in said powdered plant material.

28. The homogenized plant material of claim 26, wherein said plant material comprises rubisco.

29. The homogenized plant material of claim 26 wherein said plant material comprises cellulase, beta-glucosidase, xylanase, alpha amylase, amyloglucosidase, pectin lyase, swollenin or pectate lyase, or a combination thereof expressed in a plant, optionally with an amount of rubisco.

30-48. -48. (canceled)

51-73. -73. (canceled)