Ura5 gene and methods for stable genetic integration in yeast

Abstract

A novel gene encoding P. pastoris orotate-phosphoribosyl transferase (URA5) is disclosed. Methods for producing and selecting yeast strains capable of stable genetic integration of heterologous sequences into the host genome are also provided.

1 - 26 . (canceled) 
     
     
         27 . An isolated antibody or antigen-binding fragment or derivative thereof which binds selectively to the isolated polypeptide of claim  12 ,  14  or  15 . 
     
     
         28 . A method for the genetic integration of a heterologous nucleic acid sequence in a host cell comprising the step of disrupting a host gene encoding orotate-phosphoribosyl transferase by introduction of a disrupted, deleted or mutated nucleic acid sequence derived from a sequence selected from the group consisting of:
 (a) SEQ ID NO:2;   (b) a nucleic acid sequence that is a degenerate variant of SEQ ID NO:2;   (c) a nucleic acid sequence at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.9% identical to SEQ ID NO:2;   (d) a nucleic acid sequence that encodes a polypeptide having the amino acid sequence of SEQ ID NO:3;   (e) a nucleic acid sequence that encodes a polypeptide at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.9% identical to SEQ ID NO:3;   (f) a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO:2; and   (g) a nucleic acid sequence comprising a fragment of any one of (a)-(f) that is at least 60 contiguous nucleotides in length.   
     
     
         29 . A method for the genetic integration of a heterologous nucleic acid sequence in a host cell lacking orotate-phosphoribosyl transferase activity comprising the step of introducing a sequence of interest into the host cell in linkage with a sequence encoding orotate-phosphoribosyl transferase activity selected from the group consisting of:
 (a) SEQ ID NO:2;   (b) a nucleic acid sequence that is a degenerate variant of SEQ ID NO:2;   (c) a nucleic acid sequence at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.9% identical to SEQ ID NO:2;   (d) a nucleic acid sequence that encodes a polypeptide having the amino acid sequence of SEQ ID NO:3;   (e) a nucleic acid sequence that encodes a polypeptide at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.9% identical to SEQ ID NO:3;   (f) a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO:2; and   (g) a nucleic acid sequence comprising a fragment of any one of (a)-(f) that is at least 60 contiguous nucleotides in length.   
     
     
         30 . The method of  claim 29 , wherein the heterologous nucleic acid sequence is stably integrated. 
     
     
         31 . The method of  claim 29 , wherein the sequence encoding orotate-phosphoribosyl transferase activity is flanked by direct repeat sequences. 
     
     
         32 . The method of  claim 29 , wherein the sequence of interest encodes a polypeptide. 
     
     
         33 . The method of  claim 32 , wherein the polypeptide is a glycosylation enzyme. 
     
     
         34 . The method of  claim 33 , wherein the glycosylation enzyme is selected from the group consisting of glycosidases, mannosidases, phosphomannosidases, phosphatases, nucleotide sugar transporters, mannosyltransferases, the N-acetylglucosaminyltransferases, the UDP-N-acetylglucosamine transporters, the galactosyltransferases, the sialyltransferases and the protein mannosyltransferases. 
     
     
         35 . The method of  claim 32 , wherein the polypeptide is a therapeutic protein. 
     
     
         36 . The method of  claim 35 , wherein the therapeutic protein is selected from the group consisting of kringle domains of the human plasminogen, erythropoietin, cytokines, coagulation factors, soluble IgE receptor α-chain, IgG, IgG fragments, IgM, urokinase, chymase, urea trypsin inhibitor, IGF-binding protein, epidermal growth factor, growth hormone-releasing factor, annexin V fusion protein, angiostatin, vascular endothelial growth factor-2, myeloid progenitor inhibitory factor-1, osteoprotegerin, α-1 antitrypsin, DNase II and α-feto proteins. 
     
     
         37 . The method of  claim 31 , further comprising the step of counterselecting for loss of orotate-phosphoribosyl transferase activity. 
     
     
         38 . The method of  claim 37 , wherein the counterselection is for growth on 5-fluoroorotic acid. 
     
     
         39 . The method of  claim 29 , wherein the heterologous nucleic acid sequence is introduced into an OCH1 locus of the host cell.