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 carrying out the methods, resulting cells, and methods for producing a polypeptide of interest using the resulting cells.

1 . A method of constructing a recombinant cell comprising in its chromosome two 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 promoter, said method comprising the steps of:
 (a) providing a cell comprising in its chromosome two or more copies of a first recognition sequence (RS1) of a site specific recombinase, wherein each copy of RS1 is located downstream of the 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 promoter; and   (c) recombining RS2 with the two or more copies of RS1 in the presence of the site specific recombinase, wherein two or more copies of the ORF or operon are integrated into the chromosome and each placed:
 (i) either directly under the transcriptional control of the promoter, or 
 (ii) downstream of and in the same orientation as the 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 promoter. 
   
     
     
         2 . The method of  claim 1 , wherein the cell is a prokaryotic cell. 
     
     
         3 . The method of  claim 2 , wherein the prokaryotic cell is a  Bacillus  cell. 
     
     
         4 . The method of  claim 3 , wherein the prokaryotic cell is a  Bacillus subtilis  cell. 
     
     
         5 . The method of  claim 3 , wherein the prokaryotic cell is a  Bacillus licheniformis  cell. 
     
     
         6 . The method of  claim 1 , wherein the ORF or operon encodes at least one enzyme. 
     
     
         7 . The method of  claim 4 , wherein the ORF or operon encodes an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, an amylase or a ligase. 
     
     
         8 . The method of  claim 1 , wherein the site specific recombinase is a phage integrase. 
     
     
         9 . The method of  claim 8 , wherein the phage integrase is a tyrosine recombinase. 
     
     
         10 . The method of  claim 8 , wherein the phage integrase is a serine recombinase. 
     
     
         11 . The method of  claim 8 , wherein the phage integrase is a TP901-1 integrase. 
     
     
         12 . The method of  claim 1 , wherein RS1 comprises a nucleotide sequence at least 90% identical to attB 161  (SEQ ID NO: 21) or attBmin (SEQ ID NO: 22), RS2 comprises a nucleotide sequence at least 90% identical to attPmin (SEQ ID NO: 23), and the site specific recombinase is a TP901-1 integrase. 
     
     
         13 . The method of  claim 1 , wherein RS1 comprises a nucleotide sequence at least 90% identical to attPmin (SEQ ID NO: 23), RS2 comprises a nucleotide sequence at least 90% identical to attB 161  (SEQ ID NO: 21) or attBmin (SEQ ID NO: 22), and the site specific recombinase is a TP901-1 integrase. 
     
     
         14 . The method of  claim 1 , wherein RS1 comprises a nucleotide sequence at least 90% identical to attLmin (SEQ ID NO: 24), RS2 comprises a nucleotide sequence at least 90% identical to attRmin (SEQ ID NO: 25), the site specific recombinase is a TP901-1 integrase, and step (3) further comprises excisionase X is. 
     
     
         15 . The method of  claim 1 , wherein RS1 comprises a nucleotide sequence at least 90% identical to attRmin (SEQ ID NO: 25), RS2 comprises a nucleotide sequence at least 90% identical to attLmin (SEQ ID NO: 24), the site specific recombinase is a TP901-1 integrase, and step (3) further comprises excisionase X is. 
     
     
         16 . The method of  claim 1 , wherein each copy of RS1 is located downstream of the promoter is located downstream from a heterologous promoter. 
     
     
         17 . The method of  claim 1 , wherein each copy of RS1 is located downstream of the promoter is located downstream from an amyQ promoter, amyL promoter, cryIIIA promoter, or a consensus promoter comprising the nucleotide sequence TTGACA for the −35 region and the nucleotide sequence TATAAT for the −10 region. 
     
     
         18 . The method of  claim 1 , wherein the polynucleotide construct further comprises a region located upstream or downstream of the ORF or operon in the construct, said region being sufficiently homologous with a corresponding region located upstream or downstream, correspondingly, of RS1 in the chromosome of the cell to effectuate in vivo homologous recombination between the two homologous regions when both regions are present in the cell. 
     
     
         19 . The method of any of  claim 1 , wherein the polynucleotide construct further comprises at least one selectable marker, at least one counterselectable marker, or at least one screenable marker. 
     
     
         20 . A recombinant cell obtained by the method of  claim 1 .