BR-112013001671-B1 - FERULOIL-CoA: MONOLIGNOL TRANSFERASE

Abstract

FERULOYL-CoA:MONOLIGNOL TRANSFERASE. The invention relates to nucleic acids encoding a feruloyl-CoA:monolignol transferase and the feruloyl-CoA:monolignol transferase enzyme that enables the incorporation of ferulic monolignols, for example, including p-coumaryl ferulic acid, coniferyl ferulic acid, and sunapil ferulic acid, into plant lignin.

Inventors

• Curtis Wilkerson
• John Ralph
• Saunia Withers
• SHAWN MANSFIELD

Assignees

• BOARD OF TRUSTEES OF MICHIGAN STATE UNIVERSITY
• WISCONSIN ALUMNI RESEARCH FOUNDATION
• THE UNIVERSITY OF BRITISH COLUMBIA / UNIVERSITY - INDUSTRY LIAISON OFFICE

Dates

Publication Date

20260317

Application Date

20110722

Priority Date

20100723

Claims (15)

1. 1. Expression cassette characterized by: comprising an isolated nucleic acid having SEQ ID NO:1, or the sequence that is degenerate as a consequence of the genetic code from SEQ ID NO:1 and encoding a feruloyl-CoA: monolignol transferase with the sequence SEQ ID NO:2, wherein the nucleic acid is operatively linked to a functional promoter in a host cell and wherein the nucleic acid encodes a feruloyl-CoA: monolignol transferase that can catalyze the synthesis of monolignol ferulate(s) from monolignol(s) and feruloyl-CoA.
2. 2. Expression cassette, according to claim 1, characterized by: additionally comprising a selectable marker gene.
3. 3. Expression cassette, according to claim 1, characterized by: being inside an expression vector.
4. 4. Expression cassette, according to claim 1, characterized in that: the promoter is a functional promoter during plant development or growth.
5. 5. Expression cassette, according to claim 1, characterized in that: the promoter being a secondary xylem-specific poplar promoter of the cell wall-specific cellulose synthase 8, cauliflower mosaic virus promoter, Z10 promoter from the promoter of a gene encoding a 10 kD zein protein, Z27 promoter from a gene encoding a 27 kD zein protein, pea rbcS gene or rice actin promoter.
6. 6. Method for incorporating ferulic acids monolignols into plant lignin, characterized by: comprising the steps of: a) stably transforming plant cells with the expression cassette of claim 1 to generate transformed plant cells; b) regenerating the transformed plant cells into at least one transgenic plant wherein feruloyl-CoA: monolignol transferase is expressed in at least one transgenic plant, thereby incorporating ferulic acid monolignol into the lignin of the transgenic plant.
7. 7. Method for incorporating ferulic acids monolignols into plant lignin, according to claim 6, characterized in that: the plant is fertile.
8. 8. Method for incorporating ferulic acids monolignols into lignin from a plant, according to claim 6, characterized by: further comprising recovery from transgenic seeds of the transgenic plant, wherein the transgenic seeds comprise the nucleic acid encoding a feruloyl-CoA: monolignol transferase.
9. 9. Method for incorporating ferulic acids monolignols into lignin from a plant, according to claim 6, characterized in that: the plant is a dicotyledon.
10. 10. Method for incorporating ferulic acids monolignols into plant lignin, according to claim 6, characterized in that: the plant lignin comprises at least ferulated monolignol, or at least 5% ferulated monolignol, or at least 25% ferulated monolignol.
11. 11. Method for incorporating ferulic acids monolignols into plant lignin, according to claim 6, characterized by: further comprising creating fertile transgenic plants to produce a progeny plant that has an increased percentage of ferulic acids monolignols in the lignin of the progeny plant compared to the corresponding non-transformed plant.
12. 12. Method of manufacturing a product from a transgenic plant characterized by: including the steps of: a) providing a transgenic plant that includes an isolated nucleic acid encoding a feruloyl-CoA: monolignol transferase comprising the isolated nucleic acid of any of claims 1, 3-6, and b) processing tissues of the transgenic plant under conditions sufficient to digest the lignin, and thus generate the product from the transgenic plant, wherein the tissues of the transgenic plant comprise lignin with a higher percentage of ferulic acids monolignols compared to that of a corresponding untransformed plant.
13. 13. Method of manufacturing a product from a transgenic plant, according to claim 12, characterized by: the conditions sufficient to digest lignin comprising conditions sufficient to cleave ester linkages in lignin containing ferulic acids monolignols.
14. 14. A method for manufacturing a product from a transgenic plant, according to claim 12, characterized in that: the conditions sufficient to digest lignin would not be sufficient to substantially cleave any of the ether and carbon-carbon linkages in lignin from a corresponding plant that does not contain the isolated nucleic acid encoding feruloyl-CoA: monolignol transferase.
15. 15. Method according to claim 6, characterized in that: the plant is a eucalyptus plant.

Description

This application claims the benefit of priority to U.S. Patent Application Serial Number 61/366,977, filed July 23, 2010, the contents of which are specifically incorporated in full herein by reference. This application also reports to the publication of U.S. Patent Application Serial Number 12/830,905, filed July 6, 2010, and to U.S. Patent Application Serial Number 61/213,706, filed July 6, 2009, the contents of both of which are specifically incorporated in full herein by reference. This invention was made with government support from the U.S. Department of Energy, Office of Biological and Environmental Research (BER) Office of Science, Grant # DE-FC02-07ER64494. The government has certain rights in the invention. This invention was realized as a result of activities undertaken within the scope of a joint Research Agreement between Michigan State University and the Wisconsin Alumni Research Foundation. BACKGROUND OF THE INVENTION Lignin is an important cell wall component that provides structural support for plants and is necessary for the function of plant vascular tissue. It is one of the most abundant organic polymers in the world, constituting about 30% of non-fossil organic carbon and one-third to one-quarter of the dry mass of wood. Because the chemical structure of lignin is difficult to degrade by chemical and enzymatic means, lignin performs the task of producing paper and biofuels from plant cell walls. SUMMARY OF THE INVENTION The invention relates to the identification and isolation of novel acyltransferase nucleic acids and polypeptides. The acyltransferase enzyme is a feruloyl-CoA:monolignol transferase (FMT, also called a ferulic monolignol transferase) that produces ferulic monolignols, which can be used to create plants that readily contain cleavable lignin. The use of feruloyl-CoA:monolignol transferase nucleic acids and/or polypeptides in plants can simplify the process used to make biofuels and paper from these plants, because these plants have lignin that is more readily removed by chemical (pre)treatment. No other cloned or isolated enzymes with these beneficial properties are currently available. One aspect of the invention is an isolated nucleic acid encoding a feruloyl-CoA:monolignol transferase, characterized in that the nucleic acid can selectively hybridize to a DNA with a SEQ ID NO:1 sequence. For example, the nucleic acid can selectively hybridize to a DNA with a SEQ ID NO:1 sequence under strict hybridization conditions. In some embodiments, the strict hybridization conditions comprise washing in 0.1 x SSC, 0.1% SDS at 65 °C. Such an isolated nucleic acid, which selectively hybridizes to a DNA with a SEQ ID NO:1 sequence, can have at least about 90% sequence identity with SEQ ID NO:1. In some embodiments, the isolated nucleic acid encoding the feruloyl-CoA:monolignol transferase has the SEQ ID NO:1 sequence. Furthermore, the nucleic acid encoding a feruloyl-CoA:monolignol transferase, for example, can catalyze the synthesis of ferulic monolignol(s). For example, the monolignol can be coniferyl alcohol, p-coumaryl alcohol, sinapyl alcohol, or a combination thereof, and the feruloyl-CoA:monolignol transferase can, for example, synthesize coniferyl ferulic alcohol, p-coumaryl ferulic alcohol, sinapyl ferulic alcohol, or a combination thereof. As described in more detail in this document, feruloyl-CoA:monolignol transferase nucleic acids and polypeptides produce ferulic monolignols, which can be used to create plants containing readily cleavable lignin. In some embodiments, the feruloyl-CoA:monolignol transferase nucleic acid encodes a feruloyl-CoA:monolignol transferase polypeptide with a SEQ ID NO:2 sequence. In other embodiments, the nucleic acid may, for example, encode a feruloyl-CoA:monolignol transferase that can catalyze the synthesis of ferulic monolignol(s) from a monolignol(s) and feruloyl-CoA with at least about 50% of the activity of a feruloyl-CoA:monolignol transferase with SEQ ID NO:2. Another aspect of the invention is a transgenic plant cell comprising an isolated nucleic acid encoding a feruloyl-CoA:monolignol transferase. The nucleic acid may include any of the nucleic acids of feruloyl-CoA:monolignol transferase, as long as any of the nucleic acids described in this document may selectively hybridize to a DNA with a sequence SEQ ID NO:1. Another aspect of the invention is an expression cassette comprising one of the feruloyl-CoA:monolignol transferase nucleic acids described in this document that is operationally linked to the functional promoter in a host cell. Such nucleic acid includes a nucleic acid that can selectively hybridize to a DNA with the sequence SEQ ID NO: 1. An expression cassette may additionally comprise a selectable marker gene. In some embodiments, an expression cassette additionally comprises plasmid DNA. For example, the expression cassette may be within an expression vector. Promoters that may be used within such an expression cassett