BIOFUEL PRODUCTION METHODS AND COMPOSITIONS

• US 20080274528A1
• Filed: 11/21/2007
• Published: 11/06/2008
• Est. Priority Date: 11/21/2006
• Status: Active Grant

• Current Assignee: Noble Research Institute, LLC
• Original Assignee: The Samuel Roberts Noble Foundation
• Inventors: Dixon, Richard A., Chen, Fang, Wang, Zengyu
• Granted Patent: US 9,309,528 B2
• Time in Patent Office: Days
• US Class Current: 435/161
• CPC Class Codes: C12N 15/8245 involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
• CPC Class Codes: C12N 15/8255 involving lignin biosynthesis
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The invention provides methods for increasing the level of fermentable carbohydrates in a biofuel crop plant such as alfalfa or switchgrass, by modification of the lignin biosynthetic pathway. Also provided are plants prepared by the methods of the invention. Methods for processing plant tissue and for producing ethanol by utilizing such plants are also provided.

Claims

1. A transgenic plant of a biofuel crop species comprising a selected DNA that down regulates lignin biosynthesis in the plant and wherein the plant exhibits an increase in fermentable carbohydrates relative to a plant of the same genotype lacking the selected DNA.

2. The plant of claim 1 wherein the selected DNA down regulates at least a first lignin biosynthesis gene selected from the group consisting of 4-coumarate 3-hydroxylase (C3H), phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), hydroxycinnamoyl transferase (HCT), caffeic acid O-methyltransferase (COMT), caffeoyl coA 3-O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), cinnamyl alcohol dehydrogenase (CAD), cinnamoyl CoA-reductase (CCR), 4-coumarate-CoA ligase (4CL), monolignol-lignin-specific glycosyltransferase, and aldehyde dehydrogenase (ALDH).

3. The transgenic plant of claim 1, wherein the selected DNA is an antisense or RNAi construct.

4. The transgenic plant of claim 1, defined as from a species selected from the group consisting of switchgrass (Panicum virgatum), giant reed (Arundo donax), reed canarygrass (Phalaris arundinacea), Miscanthus×

giganteus, Miscanthus sp., sericea lespedeza (Lespedeza cuneata), corn, sugarcane, sorghum, millet, ryegrass (Lolium multiflorum, Lolium sp.), timothy, Kochia (Kochia scoparia), forage soybeans, alfalfa, clover, sunn hemp, kenaf, bahiagrass, bermudagrass, dallisgrass, pangolagrass, big bluestem, indiangrass, fescue (Festuca sp.), Dactylis sp., Brachypodium distachyon, smooth bromegrass, orchardgrass, Kentucky bluegrass, and poplar.

5. The transgenic plant of claim 1, further defined as a monocot.

6. The transgenic plant of claim 5, further defined as switchgrass.

7. The transgenic plant of claim 1, further defined as a dicot.

8. The transgenic plant of claim 7, further defined as alfalfa.

9. The transgenic plant of claim 3, wherein the antisense or RNAi construct comprises a promoter selected from the group consisting of a developmentally-regulated, organelle-specific, inducible, tissue-specific, constitutive, cell-specific, seed specific, or germination-specific promoter.

10. The transgenic plant of claim 1, further defined as an R0 transgenic plant.

11. The transgenic plant of claim 1, further defined as a progeny plant of any generation of an R0 transgenic plant, wherein the transgenic plant has inherited the selected DNA from the R0 transgenic plant.

12. The transgenic plant of claim 2, wherein at least two of said lignin biosynthesis genes are down-regulated.

13. The transgenic plant of claim 12, wherein at least three of said lignin biosynthesis genes are down-regulated.

14. The transgenic plant of claim 13, wherein at least four of said lignin biosynthesis genes are down-regulated.

15. A seed of the transgenic plant of claim 1, wherein the seed comprises the selected DNA.

16. A transgenic cell of the plant of claim 1.

33. A method for producing lignocellulosic biomass, comprising:

(a) obtaining a plant according to claim 1; and

(b) harvesting lignocellulosic biomass from the plant.

35. The method of claim 34, wherein the plant is a plant according to claim 1.

17. A method of increasing the level of one or more fermentable carbohydrates in a biofuel crop species plant comprising down-regulating in the plant at least a first lignin biosynthesis gene.

18. The method of claim 17, wherein the down-regulating is accomplished by introduction of an isolated nucleic acid sequence that encodes all or part of a lignin biosynthesis gene or its complement.

19. The method of claim 18, wherein the isolated nucleic acid sequence is in sense orientation.

20. The method of claim 17, wherein the fermentable carbohydrate is selected from the group consisting of xylose, arabinose, mannose, glucose, galactose, xyloglucan, arabinoglucan, galacturonan, starch, and cellobiose.

21. The method of claim 17, wherein down-regulating a lignin biosynthesis gene comprises mutating the lignin biosynthesis gene.

22. The method of claim 18, wherein the isolated nucleic acid sequence is an antisense or RNAi construct.

23. The method of claim 17, wherein the isolated nucleic acid sequence encodes a ribozyme or zinc-finger protein that inhibits the expression of the lignin biosynthesis gene.

24. The method of claim 17, wherein down-regulating comprises introducing into the plant a selected DNA that down regulates at least a first enzyme activity selected from the group consisting of:

4-coumarate 3-hydroxylase (C3H), phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), hydroxycinnamoyl transferase (HCT) caffeic acid O-methyltransferase (COMT), caffeoyl coA 3-O-methyltransferase (CCoAOMT), and ferulate 5-hydroxylase (F5H), cinnamyl alcohol dehydrogenase (CAD), cinnamoyl CoA-reductase (CCR), 4-coumarate-CoA ligase (4CL), monolignol-lignin-specific glycosyltransferase, and aldehyde dehydrogenase (ALDH).

25. The method of claim 24, wherein down-regulating comprises introducing into the plant a selected DNA that down regulates 4-coumarate 3-hydroxylase (C3H) and/or hydroxycinnamoyl transferase (HCT).

26. The method of claim 17, wherein introducing the isolated nucleic acid comprises plant breeding.

27. The method of claim 17, wherein introducing the isolated nucleic acid comprises genetic transformation.

28. The method of claim 17, wherein the plant is from a species selected from the group consisting of:

switchgrass (Panicum virgatum), giant reed (Arundo donax), reed canarygrass (Phalaris arundinacea), Miscanthus×

giganteus, Miscanthus sp., sericea lespedeza (Lespedeza cuneata), corn, sugarcane, sorghum, millet, ryegrass (Lolium multiflorum, Lolium sp.), timothy, Kochia (Kochia scoparia), forage soybeans, alfalfa, clover, sunn hemp, kenaf, bahiagrass, bermudagrass, dallisgrass, pangolagrass, big bluestem, indiangrass, fescue (Festuca sp.), Dactylis sp., Brachypodium distachyon, smooth bromegrass, orchardgrass, Kentucky bluegrass, and poplar.

29. The method of claim 17, wherein the plant is a monocot.

30. The method of claim 17, wherein the plant is further defined as switchgrass.

31. The method of claim 17, wherein the plant is further defined as a dicot.

32. The method of claim 17, wherein the plant is alfalfa.

34. A method for producing ethanol comprising:

(a) obtaining lignocellulosic biomass from a plant of a biofuel crop species, wherein the biomass comprises reduced lignin content as a result of the presence of at least a first transgene or through a classical selection and breeding process for reduced lignin content, relative to a plant of the same biofuel crop species that lacks the transgene or has not undergone said selection and breeding process,

(b) treating the biomass to render carbohydrates in the biomass fermentable and wherein at least about 40% of the total carbohydrates present in the biomass are released and fermentable; and

(c) fermenting the carbohydrates to produce ethanol.

36. The method of claim 34, wherein the step of treating the biomass comprises contacting the biomass with a cellulolytic enzyme.

37. The method of claim 36, wherein the biomass is contacted with the cellulolytic enzyme without an acid pretreatment step.

38. The method of claim 36, further comprising pretreating the biomass with an acid prior to contacting the biomass with the cellulolytic enzyme.

39. The method of claim 34, wherein the plant comprises a selected DNA that down regulates at least a first enzyme activity selected from the group consisting of:

4-coumarate 3-hydroxylase (C3H), phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), hydroxycinnamoyl transferase (HCT) caffeic acid O-methyltransferase (COMT), caffeoyl coA 3-O-methyltransferase (CCoAOMT), and ferulate 5-hydroxylase (F5H), cinnamyl alcohol dehydrogenase (CAD), cinnamoyl CoA-reductase (CCR), 4-coumarate-CoA ligase (4CL), monolignol-lignin-specific glycosyltransferase, and aldehyde dehydrogenase (ALDH).

40. An isolated nucleic acid sequence selected from the group consisting of:

(a) an isolated nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO;

10, or a fragment thereof;

(b) an isolated nucleic acid sequence that hybridizes to the nucleic acid sequence of SEQ ID NO;

10 or a fragment thereof, under conditions of 5×

SSC, 50% formamide and 42°

C.;

(c) an isolated nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

10, or a fragment thereof; and

(d) the complement of any of (a)-(c).