Cloning and Sequencing of the Ferulate Esterase Gene From Lactobacillus Buchneri LN4017

Abstract

Embodiments of the present invention include polypeptides having ferulate esterase activity and the nucleic acid sequences that encode them. Methods of the embodiments utilize these ferulate esterase polypeptides and nucleic acid sequences to enhance the digestibility of plant cell walls and the accessibility of carbohydrates in plants. The invention provides for transgenic plants and bacteria transformed with expression vectors containing a DNA sequence encoding ferulate esterase from Lactobacillus buchneri . Methods of using same to enhance plant fiber digestion in animals, are disclosed. Uses of this invention include, but are not limited to, forage and silage with improved digestibility for livestock, and enhanced biomass conversion.

1 . A DNA construct comprising a promoter that drives expression in a plant or plant cell operably linked to a nucleotide sequence that encodes a polypeptide having ferulate esterase activity, wherein the nucleotide sequence is selected from the group consisting of:
 a) a polynucleotide comprising SEQ ID NO: 1 or 4;   b) a polynucleotide having at least about 85% sequence identity to SEQ ID NO: 1 or 4;   c) a polynucleotide encoding SEQ ID NO: 5; and   d) a polynucleotide encoding a polypeptide having at least 85% sequence identity to SEQ ID NO: 5.   
     
     
         2 . A transformed plant cell comprising at least one DNA construct according to  claim 1 . 
     
     
         3 . The plant cell of  claim 2 , wherein said plant cell is from a monocot. 
     
     
         4 . The plant cell of  claim 3 , wherein said monocot is a member of a genus selected from the group consisting of  Festuca, Lolium, Sorghum, Zea, Triticum, Avena , and  Poa.    
     
     
         5 . A transgenic plant comprising at least one DNA construct according to  claim 1 . 
     
     
         6 . The transgenic plant of  claim 5 , wherein said plant displays increased digestibility. 
     
     
         7 . The transgenic plant of  claim 6 , wherein said plant is a monocot. 
     
     
         8 . The transgenic plant of  claim 7 , wherein said monocot is a member of a genus selected from the group consisting of  Festuca, Lolium, Sorghum, Zea, Triticum, Avena , and  Poa.    
     
     
         9 . A transformed seed of the transgenic plant of  claim 5 , wherein the seed comprises the construct. 
     
     
         10 . The transgenic plant of  claim 5 , further comprising introduction into the plant a second DNA construct comprising a promoter operably linked to a xylanase encoding polynucleotide. 
     
     
         11 . A method for increasing digestibility of a plant or plant part fed to an animal, the method comprising introducing into a plant a DNA construct comprising a promoter operably linked to a polynucleotide encoding ferulate esterase, wherein said polynucleotide has a sequence selected from the group consisting of:
 a) a polynucleotide comprising SEQ ID NO: 1 or 4;   b) a polynucleotide having at least about 85% sequence identity to SEQ ID NO: 1 or 4;   c) a polynucleotide encoding SEQ ID NO: 5; and   d) a polynucleotide encoding a polypeptide having at least 85% sequence identity to SEQ ID NO: 5.   
     
     
         12 . The method of  claim 11 , wherein said plant is a monocot. 
     
     
         13 . The method of  claim 12 , wherein said monocot is a member of a genus selected from the group consisting of  Festuca, Lolium, Sorghum, Zea, Triticum, Avena , and  Poa.    
     
     
         14 . The method of  claim 11 , further comprising introduction into the plant a second DNA construct comprising a promoter operably linked to a xylanase encoding polynucleotide. 
     
     
         15 . An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
 a) a polynucleotide comprising the sequence set forth in SEQ ID NOs: 1 or 4;   b) a polynucleotide having at least about 85% sequence identity to SEQ ID NOs: 1 or 4;   c) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 5; and   d) a polynucleotide encoding the amino acid sequence of a polypeptide having at least 85% sequence identity to SEQ ID NOs:5, wherein said polypeptide has ferulate esterase activity.   
     
     
         16 . The nucleic acid molecule of  claim 15 , wherein said nucleotide sequence is optimized for expression in a plant. 
     
     
         17 . A DNA construct that expresses in a bacterial cell, comprising a polynucleotide sequence that encodes a polypeptide having ferulate esterase activity, wherein the nucleotide sequence is selected from the group consisting of:
 a) a polynucleotide comprising SEQ ID NO: 1, 2 or 4;   b) a polynucleotide having at least about 85% sequence identity to SEQ ID NO: 1, 2 or 4;   c) a polynucleotide encoding SEQ ID NO: 3 or 5; and   d) a polynucleotide encoding a polypeptide having at least 85% sequence identity to SEQ ID NO: 3 or 5.   
     
     
         18 . A transformed bacterial cell comprising at least one DNA construct according to  claim 17 . 
     
     
         19 . The transformed bacterial cell of  claim 18 , wherein the bacteria is from a  Lactobacillus  species. 
     
     
         20 . The transformed bacterial cell of  claim 18 , where the bacteria is used as a silage incoculant.