Constitutive Yeast LLP Promotor-Based Expression Systems

Abstract

The present invention belongs to the field of biotechnology, recombinant protein production, molecular biology, microbiology and microbial genetics. It provides a modified eukaryotic cell that is modified to the effect that the modified eukaryotic cell is not able to provide an SSN6-like related protein or an SSN6-like protein that exerts its wildtype function and/or wildtype activity, the amount of SSN6-like related protein or of SSN6-like protein being present in the modified eukaryotic cell differs from the amount of SSN6-like related protein or of SSN6-like protein being present in its wildtype form, and/or essentially no SSN6-like related protein or SSN6-like protein is present in the modified cell. Additionally, the present invention provides a polynucleotide sequence comprising a modified ssn6-like related gene or modified ssn6-like gene, and a vector comprising said polynucleoptide. Additionally provided is an expression vector comprising a promoter that is repressed in the presence of SSN6-like protein or SSN6-like related protein, and a host cell comprising said vectors. The present invention further refers to a method for determining the purity of a composition by using the modified eukaryotic cell, to a method of expressing gene(s) of interest, and eukaryotic cells comprising modified ssn6-like related gene or ssn6-like gene.

1 - 27 . (canceled) 
     
     
         28 . A polynucleotide sequence comprising a modified ssn6-like gene,
 wherein either (A) or (B) applies:   (A):
 the ssn6-like gene is defined as comprising SEQ ID NO: 1, 
 the nucleotide sequence of the ssn6-like gene is modified by introduction of a point mutation, or a partial deletion, 
   
       wherein, if said polynucleotide sequence is 
       (a) introduced into a suitable expression system, and tried to be expressed, essentially no SSN6-like protein is expressed, or 
       (b) expressed in a suitable expression system, SSN6-like protein is expressed that does not exert its wildtype function and/or wildtype activity, and/or 
       (c) expressed in a suitable expression system, an amount of SSN6-like protein is expressed that differs from the amount of wildtype SSN6-like protein;
 (B):
 the polynucleotide sequence comprises SEQ ID NO: 7. 
 
 
     
     
         29 . The polynucleotide sequence according to  claim 28 , wherein the polynucleotide comprises modifications of SEQ ID NO: 1. 
     
     
         30 . A The polynucleotide according to  claim 28 , wherein the polynucleotide is a vector. 
     
     
         31 . An expression vector comprising a promoter, wherein said promoter is characterized in that it is repressed in the presence of SSN6-like protein if said expression vector is introduced into a suitable expression system, wherein the ssn6-like protein is defined as comprising the amino acid sequence as depicted in SEQ ID NO: 8, and wherein said promoter is an LLP promoter comprising SEQ ID NO: 133 or modified versions thereof, said modified versions being characterized in that they still exhibit the promoter function, wherein an LLP protein, which is encoded by the nucleotide sequence depicted in SEQ ID NO: 16, is not encoded by the polynucleotide sequence of this expression vector. 
     
     
         32 - 33 . (canceled) 
     
     
         34 . The expression vector of  claim 31 , which further comprises one or more gene(s) of interest, wherein this one or more gene(s) of interest is under control of said LLP promoter. 
     
     
         35 . (canceled) 
     
     
         36 . A host cell, comprising the vector according to  claim 30 . 
     
     
         37 - 45 . (canceled) 
     
     
         46 . The polynucleotide sequence according to  claim 28 , wherein, if said polynucleotide sequence is expressed in a suitable expression system, or is introduced in a suitable expression system and tried to be expressed, the amount of SSN6-like protein is reduced, or there is no SSN6-like protein at all, compared to the amount of SSN6-like protein that is expressed when the corresponding wildtype polynucleotide sequence is expressed under comparable or the same conditions. 
     
     
         47 . The polynucleotide sequence according to  claim 28 , comprising SEQ ID NO: 7. 
     
     
         48 . The expression vector of  claim 34 , wherein the one or more gene(s) of interest is (are) selected from the group consisting of genes encoding enzymes, antibodies or fragments thereof, hormones, structural proteins, and protein-antigens being present in vaccines. 
     
     
         49 . A host cell, comprising an expression vector according to  claim 31 .