Polynucleotide constructs and methods of expression in a host cell

Abstract

The invention includes polynucleotide constructs that contain a polynucleotide promoter sequence from the phosphoglycerate kinase 1 (pgk1) gene from Rhizopus oryzae . The invention also includes methods of expressing a polypeptide in a host cell using a polynucleotide construct containing a 3-phosphoglycerate kinase (pgk) gene promoter sequence operably linked to a polynucleotide encoding a polypeptide of interest. The invention also includes a system for producing a polypeptide. The system has a first polynucleotide sequence containing a 3-phosphoglycerate kinase gene promoter sequence isolated from a fungal species, and a second polynucleotide sequence which has been isolated from a second species and which encodes a polypeptide. A host cell contains the first polynucleotide sequence operably linked to the second polynucleotide sequence. The host cell is from a different species than the fungal species from which the first polynucleotide sequence is isolated.

1 . A polynucleotide construct comprising: 
 a promoter comprising at least nucleotides 1187-1254 of SEQ ID No.:7; and    a polynucleotide sequence that encodes a polypeptide of interest.    
     
     
         2 . A vector comprising the construct of  claim 1 .  
     
     
         3 . The vector of  claim 2  wherein the vector is a Ti plasmid.  
     
     
         4 . The vector of  claim 3  further comprising: 
 a T-DNA left border sequence; and  
 a T-DNA right border sequence.  
 
     
     
         5 . A cell comprising the construct of  claim 1 .  
     
     
         6 . A prokaryotic cell comprising the construct of  claim 1 .  
     
     
         7 . A bacterial cell comprising the construct of  claim 1 .  
     
     
         8 . A eukaryotic cell comprising the construct of  claim 1 .  
     
     
         9 . A plant cell comprising the construct of  claim 1 .  
     
     
         10 . A polynucleotide construct within a eukaryotic host cell, comprising: 
 a polynucleotide sequence encoding a protein other than a phosphoglycerate kinase; and    a phosphoglycerate kinase gene promoter sequence isolated from a first species, the first species belonging to a first phylogenetic kingdom, the promoter sequence being operably linked to the polynucleotide sequence, the eukaryotic host cell belonging to a second species within a second phylogenetic kingdom that is different from the first phylogenetic kingdom.    
     
     
         11 . The polynucleotide construct of  claim 10  wherein the first phylogenetic kingdom is fungi.  
     
     
         12 . The polynucleotide construct of  claim 10  wherein the second phylogenetic kingdom is plantae.  
     
     
         13 . The polynucleotide construct of  claim 10  wherein the promoter sequence comprises at least 100 nucleotides of SEQ ID NO.:7.  
     
     
         14 . The polynucleotide construct of  claim 10  wherein the protein confers a resistance to the host cell.  
     
     
         15 . The polynucleotide construct of  claim 14  wherein the resistance is a resistance to at least one of an antibiotic, a herbicide, a pathogen, a toxin and a parasite.  
     
     
         16 . The polynucleotide construct of  claim 10  wherein the polynucleotide construct is integrated into the genomic DNA of the eukaryotic host cell.  
     
     
         17 . A method of expressing a polypeptide in a host cell comprising: 
 providing a polynucleotide construct having a polynucleotide encoding a polypeptide of interest operably linked to a fungal phosphoglycerate kinase (pgk) gene promoter sequence; and    introducing the polynucleotide construct into a host cell.    
     
     
         18 . The method of  claim 17  wherein the pgk promoter sequence comprises at least 100 nucleotides as set forth in SEQ ID NO.7.  
     
     
         19 . The method of  claim 17  wherein the host cell is a yeast.  
     
     
         20 . The method of  claim 19  wherein the yeast is a species of the genus Saccharomyces.  
     
     
         21 . The method of  claim 17  wherein the host cell is a plant cell.  
     
     
         22 . The method of  claim 21  further comprising growing the plant cell in a cell culture.  
     
     
         23 . The method of  claim 21  further comprising growing the plant cell in plant tissue.  
     
     
         24 . The method of  claim 21  wherein the plant cell is an angiosperm plant cell.  
     
     
         25 . The method of  claim 24  wherein the plant cell is a monocot plant cell.  
     
     
         26 . The method of  claim 25  wherein the plant cell is a maize cell.  
     
     
         27 . The method of  claim 24  wherein the plant cell is a dicot plant cell.  
     
     
         28 . The method of  claim 27  wherein the plant cell is a tobacco plant cell.  
     
     
         29 . The method of  claim 17  wherein the introducing comprises transformation of the host cell utilizing at least one of electroporation, T-DNA mediated transformation, particle-bombardment mediated transformation, microinjection, freeze-thaw transformation, bacterial mediated transformation, virus-mediated transformation, chemical mediated transformation, whiskers mediated transformation, and plasmid transformation.  
     
     
         30 . A system for producing a polypeptide comprising: 
 a first polynucleotide sequence isolated from a fungal species; the first polynucleotide sequence comprising a promoter sequence of a phosphoglycerate kinase gene;    a second polynucleotide sequence isolated from a second species, the second polynucleotide sequence comprising a coding region that encodes the polypeptide;    a host cell containing the first polynucleotide sequence operably linked to the second polynucleotide sequence, the host cell being of a species that is different from the fungal species from which the first polynucleotide sequence is isolated.    
     
     
         31 . The system of  claim 30  wherein the fungal species is from the genus Rhizopus.  
     
     
         32 . The system of  claim 30  wherein the host cell is a plant cell.  
     
     
         33 . The system of  claim 30  wherein the host cell is a yeast cell.  
     
     
         34 . The system of  claim 30  wherein the polypeptide confers a resistance to the host cell.  
     
     
         35 . A method of producing a protein in a plant cell comprising: 
 creating a polynucleotide construct by operably linking an isolated fungal 3-phosphoglycerate kinase gene (pgk) promoter sequence to a polynucleotide sequence encoding at least one peptide of interest;    transforming the plant cell with the construct; and    utilizing the fungal pgk promoter to express the at least one peptide of interest within the plant cell.    
     
     
         36 . The method of  claim 35  wherein the polynucleotide sequence encodes a peptide foreign to the plant cell.  
     
     
         37 . The method of  claim 35  wherein the polynucleotide sequence encodes a peptide native to the plant cell.  
     
     
         38 . The method of  claim 37  wherein a non-transformed plant cell produces the peptide at a first level and the transforming the plant cell increases production of the peptide to a second level.  
     
     
         39 . A plant cell comprising a promoter sequence isolated from  Rhizopus Oryzae.    
     
     
         40 . The plant cell of  claim 39  wherein the promoter sequence comprises at least 100 nucleotides of SEQ ID. No 7.  
     
     
         41 . The plant cell of  claim 39  wherein the promoter sequence comprises the first 1254 nucleotides of SEQ ID No:7.  
     
     
         42 . The plant cell of  claim 39  wherein the promoter sequence is integrated into the plant cell genomic DNA.  
     
     
         43 . A yeast cell comprising at least one promoter sequence isolated from  Rhizopus Oryzae.    
     
     
         44 . The yeast cell of  claim 43  wherein the promoter sequence comprises at least 100 nucleotides of SEQ ID. No 7.  
     
     
         45 . The yeast cell of  claim 43  wherein the promoter sequence comprises the first 1254 nucleotides of SEQ ID No:7