Stable Genomic Integration of Multiple Polynucleotide Copies

Abstract

Methods of constructing a cell comprising in its chromosome one or more copies of an open reading frame (ORF) or operon encoding at least one polypeptide of interest, each copy being under the transcriptional control of a heterologous promoter using a site specific recombinase and in vivo integration by recombination; means for Promoter carrying out the methods, resulting cells, and methods for producing a polypeptide of interest using the resulting cells.

1 - 79 . (canceled) 
     
     
         80 . A method of constructing a cell comprising in its chromosome one or more copies of an open reading frame (ORF) or operon encoding at least one polypeptide of interest, each copy being under the transcriptional control of a heterologous promoter, said method comprising the steps of:
 (a) providing a cell comprising in its chromosome one or more copies of a first recognition sequence (RS1) of a site specific recombinase, wherein each copy of RS1 is located downstream of a copy of said heterologous promoter;   (b) introducing into said cell a polynucleotide construct comprising the ORF or operon and a second recognition sequence (RS2) of the site specific recombinase, where RS2 is located and oriented with respect to the ORF or operon so that an in vivo recombination of RS2 with a copy of RS1 in the chromosome of the cell will integrate the construct into the chromosome and place the ORF or operon downstream of and in the same orientation as the heterologous promoter; and   (c) recombining RS2 with the one or more copies of RS1 in the presence of the site specific recombinase, whereby one or more copies of the ORF or operon are integrated into the chromosome and placed:
 (i) either directly under the transcriptional control of the heterologous promoter, or 
 (ii) downstream of and in the same orientation as the heterologous promoter but separated from it by a region, which can be excised after one or more optional recombination events, whereby the ORF or operon of interest is placed under the transcriptional control of the heterologous promoter. 
   
     
     
         81 . The method of  claim 80 , wherein the cell is a prokaryotic cell. 
     
     
         82 . The method of  claim 81 , wherein the prokaryotic cell is a  Bacillus  cell. 
     
     
         83 . The method of  claim 80 , wherein the ORF or operon encodes at least one enzyme. 
     
     
         84 . The method of  claim 83 , wherein the ORF or operon encodes an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, or a ligase. 
     
     
         85 . The method of  claim 80 , wherein the site specific recombinase comprises a phage integrase. 
     
     
         86 . The method of  claim 80 , wherein the site specific recombinase comprises the TP901-1 integrase. 
     
     
         87 . The method of  claim 80 , wherein RS1 comprises a nucleotide sequence at least 70% identical to attB 161  (SEQ ID NO: 21) or attBmin (SEQ ID NO: 22), RS2 comprises a nucleotide sequence at least 70% identical to attPmin (SEQ ID NO: 23), and the site specific recombinase comprises the phage TP901-1 integrase. 
     
     
         88 . The method of  claim 80 , wherein RS1 comprises a nucleotide sequence at least 70% identical to attPmin (SEQ ID NO: 23), RS2 comprises a nucleotide sequence at least 70% identical to attB 161  (SEQ ID NO: 21) or attBmin (SEQ ID NO: 22), and the site specific recombinase comprises the phage TP901-1 integrase. 
     
     
         89 . The method of  claim 80 , wherein RS1 comprises a nucleotide sequence at least 70% identical to attLmin (SEQ ID NO: 24), RS2 comprises a nucleotide sequence at least 70% identical to attRmin (SEQ ID NO: 25), and the site specific recombinase comprises the phage TP901-1 integrase and excisionase Xis. 
     
     
         90 . The method of  claim 80 , wherein RS1 comprises a nucleotide sequence at least 70% identical to attRmin (SEQ ID NO: 25), RS2 comprises a nucleotide sequence at least 70% identical to attLmin (SEQ ID NO: 24), and the site specific recombinase comprises the phage TP901-1 integrase and excisionase Xis. 
     
     
         91 . A cell comprising in its chromosome one or more copies of an open reading frame (ORF) or operon of interest, wherein each copy is under the transcriptional control of a heterologous promoter, and (i) wherein each copy of the ORF or operon is located in the chromosome upstream of a recognition sequence (RS) of a site specific recombinase, or (ii) wherein each copy of the ORF or operon is located in the chromosome downstream of a recognition sequence (RS) of a site specific recombinase. 
     
     
         92 . The cell of  claim 91 , wherein the cell is a prokaryotic cell. 
     
     
         93 . The cell of  claim 92 , wherein the prokaryotic cell is a  Bacillus  cell. 
     
     
         94 . The cell of  claim 91 , wherein the site specific recombinase comprises a phage integrase. 
     
     
         95 . The cell of  claim 91 , wherein the site specific recombinase comprises the TP901-1 integrase. 
     
     
         96 . The cell of  claim 91 , wherein the RS comprises a nucleotide sequence at least 70% identical to attB 161  (SEQ ID NO: 21), attBmin (SEQ ID NO: 22), or attPmin (SEQ ID NO: 23), and the site specific recombinase comprises the phage TP901-1 integrase. 
     
     
         97 . The cell of  claim 91 , wherein the RS comprises a nucleotide sequence at least 70% identical to attLmin (SEQ ID NO: 24) or attRmin (SEQ ID NO: 25), and the site specific recombinase comprises the phage TP901-1 integrase and excisionase Xis. 
     
     
         98 . The cell of  claim 91 , wherein the ORF or operon encodes at least one enzyme. 
     
     
         99 . The cell of  claim 98 , wherein the ORF or operon encodes an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, or a ligase. 
     
     
         100 . A method of producing a polypeptide of interest, said method comprising:
 (a) cultivating a cell as defined in  claim 91 ; and   (b) isolating the polypeptide of interest.