Method of producing recombinant Aspergillus niger beta-glucosidase and an aroma spreading plant

Abstract

A polypeptide having β-glucosidase enzymatic activity, a polynucleotide encoding the polypeptide, a nucleic acid constructs carrying the polynucleotide, transformed or infected cells, such as yeast cells, and transgenic organisms expressing the polynucleotide and various uses of the polypeptide, the polynucleotide, cells and/or organisms, including, producing a recombinant polypeptide having the β-glucosidase enzymatic activity, increasing the level of aroma compounds in alcoholic beverages, as well as other fermentation products of plant material, hydrolyzing cellobiose and thus increasing the level of fermentable glucose, increasing the production of alcohol, such as ethanol from plant material, increasing the aroma released from a plant or a plant product, and hydrolysis or transglycosylation of glycosides.

1 . A method of increasing a level of at least one fermentation substance in a fermentation product, the method comprising the step of fermenting a plant derived glucose containing fermentation starting material by a yeast cell, said plant expressing a nucleic acid construct comprising a polynucleotide encoding a polypeptide having a β-glucosidase catalytic activity and further encoding a signal peptide being in frame with said polypeptide, said plant having greater β-glucosidase catalytic activity as compared to β-glucosidase catalytic activity of a plant not expressing said nucleic acid construct, thereby increasing the level of the at least one fermentation substance in the fermentation product.  
     
     
         2 . The method of  claim 1 , wherein said polynucleotide further encodes an endoplasmic reticulum retaining peptide being in frame with said polypeptide.  
     
     
         3 . The method of  claim 1 , wherein said signal peptide is an apoplast and/or vacuole targeting signal peptide.  
     
     
         4 . The method of  claim 1 , wherein said signal peptide is Cel1.  
     
     
         5 . A method of increasing a level of at least one aroma substance in a plant derived product, the method comprising the step of incubating a glucose containing plant starting material with a yeast cell, said plant expressing a nucleic acid construct comprising a polynucleotide encoding a polypeptide having a β-glucosidase catalytic activity and further encoding a signal peptide being in frame with said polypeptide, said plant having greater β-glucosidase catalytic activity as compared to β-glucosidase catalytic activity of a plant not expressing said nucleic acid construct, thereby increasing the level of the at least one aroma substance in the plant derived product.  
     
     
         6 . The method of  claim 5 , wherein said polynucleotide further encodes an endoplasmic reticulum retaining peptide being in frame with said polypeptide.  
     
     
         7 . The method of  claim 5 , wherein said signal peptide is an apoplast and/or vacuole targeting signal peptide.  
     
     
         8 . The method of  claim 5 , wherein said signal peptide is Cel1.  
     
     
         9 . The method of  claim 5 , wherein said plant derived product is a fermentation product.  
     
     
         10 . A method of increasing a level of free glucose in a glucose containing fermentation starting material, the method comprising the step of fermenting the glucose containing fermentation starting material by a cell expressing a nucleic acid construct comprising a polynucleotide encoding a polypeptide having a β-glucosidase catalytic activity and further encoding a signal peptide being in frame with said polypeptide, said cell having greater β-glucosidase catalytic activity as compared to β-glucosidase catalytic activity of a cell not expressing said nucleic acid construct, thereby increasing the level of the free glucose in the glucose containing fermentation starting material.  
     
     
         11 . The method of  claim 10 , wherein said polynucleotide further encodes an endoplasmic reticulum retaining peptide being in frame with said polypeptide.  
     
     
         12 . The method of  claim 10 , wherein said signal peptide is an apoplast and/or vacuole targeting signal peptide.  
     
     
         13 . The method of  claim 10 , wherein said signal peptide is Cel1.  
     
     
         14 . A method of increasing a level of free glucose in a plant derived glucose containing fermentation starting material, the method comprising the step of fermenting the plant derived glucose containing fermentation starting material by a cell, said plant expressing a nucleic acid construct comprising a polynucleotide encoding a polypeptide having a β-glucosidase catalytic activity and further encoding a signal peptide being in frame with said polypeptide, said plant having greater β-glucosidase catalytic activity as compared to β-glucosidase catalytic activity of a plant not expressing said nucleic acid construct, thereby increasing the level of the free glucose in the plant.  
     
     
         15 . The method of  claim 14 , wherein said polynucleotide further encodes an endoplasmic reticulum retaining peptide being in frame with said polypeptide.  
     
     
         16 . The method of  claim 14 , wherein said signal peptide is an apoplast and/or vacuole targeting signal peptide.  
     
     
         17 . The method of  claim 14 , wherein said signal peptide is Cel1.  
     
     
         18 . A method of producing an alcohol, the method comprising the step of fermenting a glucose containing fermentation starting material by a cell expressing a nucleic acid construct comprising a polynucleotide encoding a polypeptide having a β-glucosidase catalytic activity and further encoding a signal peptide being in frame with said polypeptide, said cell having greater β-glucosidase catalytic activity as compared to β-glucosidase catalytic activity of a cell not expressing said nucleic acid construct and extracting the alcohol therefrom.  
     
     
         19 . The method of  claim 18 , wherein said polynucleotide further encodes an endoplasmic reticulum retaining peptide being in frame with said polypeptide.  
     
     
         20 . The method of  claim 18 , wherein said signal peptide is an apoplast and/or vacuole targeting signal peptide.  
     
     
         21 . The method of  claim 18 , wherein said signal peptide is Cel1.  
     
     
         22 . A method of producing an alcohol, the method comprising the step of fermenting a plant derived glucose containing fermentation starting material by a cell, said plant expressing a nucleic acid construct comprising a polynucleotide encoding a polypeptide having a β-glucosidase catalytic activity and further encoding a signal peptide being in frame with said polypeptide, said plant having greater β-glucosidase catalytic activity as compared to β-glucosidase catalytic activity of a plant not expressing said nucleic acid construct, and extracting the alcohol therefrom.  
     
     
         23 . The method of  claim 22 , wherein said polynucleotide further encodes an endoplasmic reticulum retaining peptide being in frame with said polypeptide.  
     
     
         24 . The method of  claim 22 , wherein said signal peptide is an apoplast and/or vacuole targeting signal peptide.  
     
     
         25 . The method of  claim 22 , wherein said signal peptide is Cel1.  
     
     
         26 . A method of producing a plant having increased release of flavor and/or aroma compounds in-vivo, the method comprising the step of expressing in the plant a nucleic acid construct comprising a polynucleotide encoding a polypeptide having a β-glucosidase catalytic activity and further encoding an apoplast and/or vacuole targeting signal peptide being in frame with said polypeptide and wherein said polypeptide is secreted into the apoplast and/or vacuole, said plant having greater β-glucosidase catalytic activity in the apoplast and/or vacuole as compared to β-glucosidase catalytic activity of the apoplast and/or vacuole of a plant not expressing said nucleic acid construct, thereby increasing in-vivo release of flavor and/or aroma compounds from the plant.  
     
     
         27 . The method of  claim 26 , wherein said signal peptide is Cel1.  
     
     
         28 . A method of producing a plant having increased release of flavor and/or aroma compounds upon processing of the plant or portion thereof, the method comprising the step of expressing in the plant a nucleic acid construct comprising a polynucleotide encoding a polypeptide having a β-glucosidase catalytic activity and further encoding a signal peptide and an endoplasmic retention peptide being in frame with said polypeptide and wherein said polypeptide accumulates in the endoplasmic reticulum, said plant having greater β-glucosidase catalytic activity in said endoplasmic reticulum as compared to β-glucosidase catalytic activity of said endoplamic reticulum of a plant not expressing said nucleic acid construct, thereby increasing release of flavor and/or aroma compounds from the processing of said plant or portion thereof, and wherein said processing comprises cell disruption and decompartmentalization.  
     
     
         29 . The method of  claim 28 , wherein said signal peptide is an apoplast and/or vacuole targeting signal peptide.  
     
     
         30 . The method of  claim 28 , wherein said signal peptide is Cel1.  
     
     
         31 . The method of  claim 28 , wherein said endoplasmic retention peptide is selected from the group consisting of KDEL and HDEL.