Alcohol oxidase 1 regulatory nucleotide sequences for heterologous gene expression in yeast

Abstract

Polynucleotides that comprise regulatory regions of the yeast alcohol oxidase 1 promoter are provided. Isolated polynucleotides, recombinant polynucleotides, vectors, expression cassettes and transformed cells containing the regulatory regions are disclosed. Proteins produced by the transformed cells are also provided.

What is claimed is:  
     
         1 . An isolated polynucleotide comprising a regulatory region containing a nucleotide sequence less than about 1000 nucleotides long selected from the group consisting of: 
 (a) SEQ ID NO: 16, a fragment of SEQ ID NO: 16, the complement of SEQ ID NO: 16, or a fragment of the complement of SEQ ID NO: 16;    (b) a polynucleotide that hybridizes to the polynucleotide of (a) under conditions of high stringency; and    (c) a polynucleotide with at least 80% sequence homology to the polynucleotide of (a).    
     
     
         2 . An isolated polynucleotide comprising a regulatory region containing a nucleotide sequence less than about 1000 nucleotides long selected from the group consisting of: 
 (a) SEQ ID NO: 17, a fragment of SEQ ID NO: 17, the complement of SEQ ID NO: 17, or a fragment of the complement of SEQ ID NO: 17;    (b) a polynucleotide that hybridizes to the polynucleotide of (a) under conditions of high stringency; and    (c) a polynucleotide with at least 80% sequence homology to the polynucleotide of (a).    
     
     
         3 . An isolated polynucleotide comprising a regulatory region containing a nucleotide sequence less than about 1000 nucleotides long selected from the group consisting of: 
 (a) SEQ ID NO: 18, a fragment of SEQ ID NO: 18, the complement of SEQ ID NO: 18, or a fragment of the complement of SEQ ID NO: 18;    (b) a polynucleotide that hybridizes to the polynucleotide of (a) under conditions of high stringency; and    (c) a polynucleotide with at least 80% sequence homology to the polynucleotide of (a).    
     
     
         4 . An isolated polynucleotide comprising a regulatory region containing a nucleotide sequence less than about 1000 nucleotides long selected from the group consisting of: 
 (a) SEQ ID NO: 19,a fragment of SEQ ID NO: 19, the complement of SEQ ID NO: 19, or a fragment of the complement of SEQ ID NO: 19;    (b) a polynucleotide that hybridizes to the polynucleotide of (a) under conditions of high stringency; and    (c) a polynucleotide with at least 80% sequence homology to the polynucleotide of (a).    
     
     
         5 . An isolated-polynucleotide comprising a regulatory region containing a nucleotide sequence less than about 1000 nucleotides long selected from the group consisting of: 
 (a) SEQ ID NO: 20, a fragment of SEQ ID NO: 20, the complement of SEQ ID NO: 20, or a fragment of the complement of SEQ ID NO: 20;    (b) a polynucleotide that hybridizes to the polynucleotide of (a) under conditions of high stringency; and    (c) a polynucleotide with at least 80% sequence homology to the polynucleotide of (a).    
     
     
         6 . An isolated polynucleotide comprising a regulatory region containing a nucleotide sequence less than about 1000 nucleotides long selected from the group consisting of: 
 (a) SEQ ID NO: 21, a fragment of SEQ ID NO: 21, the complement of SEQ ID NO: 21, or a fragment of the complement of SEQ ID NO: 21;    (b) a polynucleotide that hybridizes to the polynucleotide of (a) under conditions of high stringency; and    (c) a polynucleotide with at least 80% sequence homology to the polynucleotide of (a).    
     
     
         7 . A recombinant vector comprising the isolated polynucleotide of any of claims  1 - 6  operably linked to a heterologous coding region.  
     
     
         8 . An expression cassette comprising operably linked in 5′ to 3′ order the isolated polynucleotide of any of claims  1 - 6 , a heterologous coding region, and a termination sequence.  
     
     
         9 . A host cell comprising the vector of  claim 7 .  
     
     
         10 . The host cell of  claim 9  wherein the host cell is a yeast cell.  
     
     
         11 . The yeast cell of  claim 10  wherein the yeast cell is a methylotrophic yeast cell.  
     
     
         12 . The methylotrophic yeast cell of  claim 11  wherein the yeast cell is selected from the group of genera consisting of Hansenula, Candida, Torulopsis, and Pichia.  
     
     
         13 . The yeast cell of  claim 12  wherein the yeast cell is from  Pichia pastoris.    
     
     
         14 . A host cell comprising the expression cassette of  claim 8 .  
     
     
         15 . The host cell of  claim 14  wherein the host cell is a yeast cell.  
     
     
         16 . The host cell of  claim 15  wherein the yeast cell is a methylotrophic yeast cell.  
     
     
         17 . The host cell of  claim 16  wherein the methylotrophic yeast cell is selected from the group of genera consisting of Hansenula, Candida, Torulopsis and Pichia.  
     
     
         18 . The host cell of  claim 17  wherein the yeast cell is from  Pichia pastoris.    
     
     
         19 . The host cell of  claim 9  wherein the host cell expresses a protein encoded by the vector.  
     
     
         20 . The host cell of  claim 14  wherein the host cell expresses a protein encoded by the expression cassette.  
     
     
         21 . A method for the production of a protein comprising growing the host cells of  claim 19  under conditions where the host cells express the protein encoded by the vector and isolating the expressed protein.  
     
     
         22 . A method for the production of a protein comprising growing the host cells of  claim 20  under conditions where the host cells express the protein encoded by the vector and isolating the expressed protein.