Is-targeting system for gene insertion and genetic engineering in deinococcus bacteria

Abstract

The present invention relates to methods and compositions for chromosome integration of nucleic acids into Deinococcus bacteria. The invention more particularly relates to IS-mediated multicopy gene insertion or chromosome engineering in Deinococcus bacteria, the resulting bacteria, and the uses thereof.

We claim: 
     
         1 . A  Deinococcus  bacterium comprising one or several copies of a gene of interest inserted into an insertion sequence (IS) element. 
     
     
         2 . The  Deinococcus  bacterium of  claim 1 , wherein said bacterium is produced by a method comprising introducing in a targeted manner said gene of interest into an IS present in the genome of said bacterium and, optionally, amplifying the copy number by subjecting said bacterium or a descendant thereof to a gene amplification treatment. 
     
     
         3 . The  Deinococcus  bacterium of  claim 1 , wherein the insertion sequence is present in several copies in the genome of said bacterium. 
     
     
         4 . The  Deinococcus  bacterium of  claim 1 , wherein the IS is selected from IS200/IS605; IS630; IS701; IS607; IS982; IS3; IS1; IS6; IS5; IS4; or IS66. 
     
     
         5 . The  Deinococcus  bacterium of  claim 1 , wherein the gene of interest is flanked, on one or both sides, by an insertion-mediating sequence. 
     
     
         6 . The  Deinococcus  bacterium of  claim 5 , wherein the insertion-mediating sequence comprises a sequence homologous to a sequence of the IS, allowing insertion of the gene of interest into the IS element by homologous recombination. 
     
     
         7 . The  Deinococcus  bacterium of  claim 5 , wherein the insertion-mediating sequence comprises an Inverted Repeat element of an IS, allowing insertion of the gene of interest into the IS element by transposition or an intron sequence, allowing insertion of the gene of interest into the IS element by retro-homing. 
     
     
         8 . The  Deinococcus  bacterium of  claim 1 , wherein the gene of interest comprises an open reading frame encoding a biologically active polypeptide or RNA. 
     
     
         9 . The  Deinococcus  bacterium of  claim 1 , wherein the gene of interest comprises an open reading frame encoding a polypeptide having pharmaceutical and/or agro-chemical interest or which is an enzyme. 
     
     
         10 . The  Deinococcus  bacterium of  claim 9 , wherein the polypeptide is a pharmaceutical compound. 
     
     
         11 . The  Deinococcus  bacterium of  claim 1 , wherein the  Deinococcus  bacterium is selected from  D. deserti; D. geothermalis; D. maricopensis; D. proteolyticus; D. radiodurans; D. murrayi; D. cellulolysiticus; D. guilhemensis; D. aquaticus; D. ficus; D. gobiensis; D. grandis; D. radiopugnans ; or  D. roseus , or any thermophile  Deinococcus  bacterium. 
     
     
         12 . A method of producing a pharmaceutical and/or agro-chemical polypeptide of interest or an enzyme comprising culturing a bacterium according to  claim 9  under conditions that permit the expression of a polypeptide encoded by a gene of interest encoding said pharmaceutical and/or agro-chemical polypeptide or said enzyme.