Self-processing plants and plant parts

• US 7,919,681 B2
• Filed: 10/18/2007
• Issued: 04/05/2011
• Est. Priority Date: 08/27/2001
• Status: Expired due to Term

• Current Assignee: Syngenta Participations AG (China National Chemical Corporation)
• Original Assignee: Syngenta Participations AG (China National Chemical Corporation)
• Inventors: Chen, Wen, Kinkema, Mark, Lanahan, Michael B., Craig, Joyce, Basu, Shib Sankar, Batie, Christopher J.
• Primary Examiner(s): Fox; David T
• Application Number:
  US11/874,560
• Publication Number:
  US 20080045702A1
• Time in Patent Office: 1,265 Days
• Field of Search: None
• US Class Current: 800/284
• CPC Class Codes: C12N 15/8245 involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
• CPC Class Codes: C12N 9/2408 acting on alpha -1,4-glucosidic bonds
• CPC Class Codes: C12N 9/2411 Amylases
• CPC Class Codes: C12N 9/2414 Alpha-amylase (3.2.1.1.)
• CPC Class Codes: C12N 9/2422 from plant source
• CPC Class Codes: C12N 9/2428 Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
• CPC Class Codes: C12N 9/2451 acting on alpha-1,6-glucosidic bonds
• CPC Class Codes: C12N 9/2457 Pullulanase (3.2.1.41)
• CPC Class Codes: C12N 9/246 Isoamylase (3.2.1.68)
• 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 7/06 Ethanol, i.e. non-beverage
• CPC Class Codes: C12Y 302/01001 Alpha-amylase (3.2.1.1)
• CPC Class Codes: C12Y 302/01003 Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
• CPC Class Codes: C12Y 302/0102 Alpha-glucosidase (3.2.1.20)
• CPC Class Codes: C12Y 302/01041 Pullulanase (3.2.1.41)
• CPC Class Codes: C12Y 302/01068 Isoamylase (3.2.1.68)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The invention provides polynucleotides, preferably synthetic polynucleotides, which encode processing enzymes that are optimized for expression in plants. The polynucleotides encode mesophilic, thermophilic, or hyperthermophilic processing enzymes, which are activated under suitable activating conditions to act upon the desired substrate. Also provided are “self-processing” transgenic plants, and plant parts, e.g., grain, which express one or more of these enzymes and have an altered composition that facilitates plant and grain processing. Methods for making and using these plants, e.g., to produce food products having improved taste and to produce fermentable substrates for the production of ethanol and fermented beverages are also provided.

Claims

1. A method to prepare ethanol comprising the steps of:

a) obtaining a plant part comprising a polynucleotide encoding an alpha-amylase operably linked to a promoter and a signal sequence, wherein the plant part comprises grain or seed, wherein the signal sequence directs the alpha-amylase to a specific compartment allowing the alpha-amylase to be localized in a manner that it will not come into contact with its substrate; and

b) milling or heating or means for disrupting the physical integrity of the plant part in a manner which allows the alpha-amylase to come into contact with its substrate and produce oligosaccharide or fermentable sugar; and

c) incubating the oligosaccharide or fermentable sugar under conditions that promote the conversion of the fermentable sugar or oligosaccharide into ethanol.

2. The method of claim 1, further comprising combining the transgenic plant part with a second plant part wherein the second plant part is transgenic or non-transgenic.

3. The method of claim 1, wherein the plant part comprises a polynucleotide encoding an alpha-amylase in combination with a polynucleotide encoding a non-starch processing enzyme.

4. The method of claim 3, wherein the non-starch processing enzyme is a phytase.

5. The method of claim 1, wherein the plant part is obtained from a corn plant.

6. The method of claim 1, wherein the promoter is a tissue-specific promoter.

7. The method of claim 1, wherein the promoter is a seed-specific promoter.

8. The method of claim 1, wherein the promoter is an endosperm specific promoter.

9. The method of claim 1, wherein the promoter is a maize gamma-zein promoter.

10. The method of claim 1, wherein the signal sequence is selected from the group consisting of an ER signal sequence, an ER retention sequence, an ER signal sequence and an additional ER retention sequence, and the combination of an ER signal sequence and an additional ER retention sequence encoding the amino acids SEKDEL (SEQ ID NO:

60).

11. The method of claim 1, wherein the promoter is a maize gamma-zein promoter and the ER signal sequence is a maize gamma-zein signal sequence and an ER retention sequence encoding the amino acid sequence SEKDEL (SEQ ID NO:

60).

12. A method of making fermentable sugars comprising the steps of:

a) obtaining a plant part comprising a polynucleotide encoding an alpha-amylase operably linked to a promoter and a signal sequence, wherein the plant part comprises a grain or seed, wherein the signal sequence directs the alpha-amylase to a specific compartment allowing the alpha-amylase to be localized in a manner that it will not come into contact with its substrate; and

b) milling or heating or means for disrupting the physical integrity of the plant part in a manner which allows the alpha-amylase to come into contact with its substrate and produce oligosaccharide or fermentable sugar.

13. The method of claim 12, further comprising combining the transgenic plant part with a second plant part wherein the second plant part is transgenic or non-transgenic.

14. The method of claim 12, wherein the plant part comprises a polynucleotide encoding an alpha-amylase in combination with a polynucleotide encoding a non-starch processing enzyme.

15. The method of claim 14, wherein the non-starch processing enzyme is a phytase.

16. The method of claim 12, wherein the plant part is obtained from a corn plant.

17. The method of claim 12, wherein the promoter is a tissue-specific promoter.

18. The method of claim 12, wherein the promoter is a seed-specific promoter.

19. The method of claim 12, wherein the promoter is an endosperm specific promoter.

20. The method of claim 12, wherein the promoter is a maize gamma-zein promoter.

21. The method of claim 12, wherein the signal sequence is selected from the group consisting of an ER signal sequence, an ER retention sequence, an ER signal sequence and an additional ER retention sequence, and the combination of an ER signal sequence and an additional ER retention sequence encoding the amino acids SEKDEL (SEQ ID NO:

60).

22. The method of claim 13, wherein the promoter is a maize gamma-zein promoter and the ER signal sequence is the maize gamma-zein signal sequence and the ER retention sequence encoding the amino acid sequence SEKDEL (SEQ ID NO:

60).

23. A method of making fermentable sugars comprising the steps of:

a) obtaining a plant part comprising a polynucleotide encoding a starch processing enzyme operably linked to a promoter and a signal sequence, wherein the plant part comprises grain or seed, wherein the signal sequence directs the starch processing enzyme to a specific compartment allowing the starch processing enzyme to be localized in a manner that it will not come into contact with its substrate;

b) milling or heating or means for disrupting the physical integrity of the plant part in a manner which allows the starch processing enzyme to come into contact with its substrate; and

c) liquefaction of the physically disrupted plant part at or below pH 6.0 to produce oligosaccharide or fermentable sugar.

24. The method of claim 23, further comprising the step of incubating the oligosaccharide or fermentable sugar under conditions that promote the conversion of the fermentable sugar or oligosaccharide into ethanol.

25. The method of claim 23, wherein the starch processing enzyme is selected from the group consisting of alpha-amylase and glucoamylase.

26. The method of claim 23, wherein the liquefaction of the physically disrupted plant part is at pH of about 5.2 to about 6.0.

27. The method of claim 24, wherein the liquefaction of the physically disrupted plant part is at pH of about 5.2 to about 6.0.