Method and nucleotide sequence for transforming microorganisms

Abstract

An isolated nucleic acid molecule is provided which contains a sequence which encodes a protein which mediates the uptake of L-malate, succinate, and malonate, and expression vectors and host cells containing the nucleic acid molecules. The nucleic acid molecules are used to transform cells for use in mediating malate, succinic acid or malonate uptake in particular malate uptake during the fermentation of wines.

We claim:  
     
         1 . An isolated nucleic acid molecule comprising a sequence which encodes a protein which mediates the uptake of L-malate, succinate, and malonate.  
     
     
         2 . An isolated nucleic acid molecule as claimed in  claim 1  wherein the protein contains a PEST region, and a leucine zipper motif.  
     
     
         3 . An isolated nucleic acid molecule as claimed in  claim 1  comprising 
 (i) a nucleic acid sequence encoding a protein having the amino acid sequence shown in SEQ ID NO: 2 or FIG. 3;  
 (ii) nucleic acid sequences complementary to (i); and  
 (iii) a nucleic acid capable of hybridizing under stringent conditions to a nucleic acid of (i).  
 
     
     
         4 . An isolated nucleic acid molecule as claimed in  claim 1  comprising 
 (i) a nucleic acid sequence as shown in SEQ ID NO:1 or FIG. 3, wherein T can also be U;  
 (ii) nucleic acid sequences complementary to (i), preferably complementary to the full length nucleic acid sequence shown in SEQ ID NO: 1 or FIG. 3;  
 (iii) a nucleic acid capable of hybridizing under stringent conditions to a nucleic acid of (i); and  
 (iv) a nucleic acid molecule differing from any of the nucleic acids of (i) to (iii) in codon sequences due to the degeneracy of the genetic code.  
 
     
     
         5 . An isolated nucleic acid molecule as claimed in  claim 1  comprising a sequence encoding a truncation of a protein having the amino acid sequence shown in SEQ ID NO: 2 or FIG. 3; an analog, or a homolog of a protein having the amino acid sequence shown in SEQ ID NO: 2 and FIG. 3; or a truncation thereof.  
     
     
         6 . An isolated nucleic acid molecule encoding a fusion protein comprising a protein having the amino acid sequence shown in SEQ ID NO: 2 or FIG. 3 and a heterologous protein or peptide.  
     
     
         7 . An isolated nucleic add molecule as claimed in  claim 6 , wherein the heterologous protein is a selectable marker protein, or a protein involved in the metabolism of L-malate, succinate, or malonate.  
     
     
         8 . An isolated nucleic acid molecule as claimed in  claim 6 , wherein the heterologous protein is a malic enzyme, a malolactic enzyme, or an enzyme involved in the metabolism of malate in plants.  
     
     
         9 . An isolated nucleic acid molecule as claimed in  claim 8 , wherein the heterologous protein is a malic enzyme from  S. pombe.    
     
     
         10 . An isolated nucleic acid molecule as claimed in  claim 8 , wherein the heterologous protein is a malolactic enzyme from  S. cerevisiae.    
     
     
         11 . An isolated nucleic acid molecule as claimed in any one of  claims 1  to  11  containing regulatory sequences for the transcription and translation of the protein.  
     
     
         12 . A recombinant expression vector adapted for transformation of a host cell comprising a nucleic acid molecule as claimed in any one of  claims 1  to  11 .  
     
     
         13 . A host cell having integrated into its genome a nucleic acid molecule as claimed in any one of  claims 1  to  11 .  
     
     
         14 . A host cell as claimed in  claim 13  which is a yeast strain.  
     
     
         15 . A host cell as claimed in  claim 13  which is  S. cerevisiae.    
     
     
         16 . A method for preparing a protein which mediates the uptake of L-malate, succinate, and malonate comprising (a) transferring a recombinant expression vector as claimed in  claim 12  into a host cell; (b) selecting transformed host cells from untransformed host cells; (c) culturing a selected transformed host cell under conditions which allow expression of the protein; and (d) isolating the protein.  
     
     
         17 . An isolated protein characterized in that it mediates the uptake of L-malate, succinate, and malonate.  
     
     
         18 . An isolated protein as claimed in  claim 17  further characterized in that it has part or all of the primary structural conformation and the enzymatic activity of Mae1 from  S. pombe.    
     
     
         19 . An isolated protein as claimed in  claim 18  which has the amino acid sequence as shown in SEQ ID NO:2 or FIG. 3.  
     
     
         20 . An isolated protein as claimed in  claim 17 ,  18  or  19  which is a truncation of the protein, an analog, homolog, or isoform of the protein and truncations thereof, which has malate permease activity.  
     
     
         21 . A fusion protein comprising a protein as claimed in  claim 17 ,  18  or  19  and a heterologous protein or peptide.  
     
     
         22 . A fusion protein as claimed in  claim 21  wherein the heterologous protein is a malic enzyme or a malolactic enzyme.  
     
     
         23 . Antibodies having specificity against an epitope of a protein as claimed in  claim 17 ,  18  or  19 .  
     
     
         24 . A method of increasing uptake of L-malate, succinic acid or malonate in a cell comprising transforming the cell with a nucleic acid molecule as claimed in any one of  claims 1  to  11 .  
     
     
         25 . A method of providing a microorganism with the capability of degrading malate comprising transforming the microorganism with a nucleic acid molecule as claimed in any one of  claims 1  to  11 .  
     
     
         26 . A method as claimed in  claim 25  wherein the microorganism is a yeast strain.  
     
     
         27 . A method of degrading malate which comprises cultivating, in the presence of a supply of malate, a microorganism which has been transformed with a nucleic acid molecule as claimed in any one of  claims 1  to  11 .  
     
     
         28 . A method of degrading malate during fermentation of wine, which method comprises, cultivating, in grape musts which contain a supply of malate, a yeast strain as claimed in  claim 14  or  15 .  
     
     
         29 . A method of fermenting wine, which includes cultivating, in a wine fermentation medium which includes grape must containing a supply of malate, a yeast strain as claimed in  claim 14  or  15 .  
     
     
         30 . A method for identifying a substance that mediates transport of L-malate, succinate or malonate comprising incubating a protein as claimed in  claim 17 ,  18  or  19  with a substrate of the protein, and a test substance which is suspected of affecting the activity of the protein, and determining the effect of the substance by comparing to a control.