Genetic tool for the transformation of clostridium bacteria

Abstract

The present invention relates to a genetic tool comprising at least two different nucleic acids allowing the transformation, by homologous recombination, of a bacterium of the genus Clostridium , typically of a solventogenic bacterium.

We claim: 
     
         1 . A process for transforming by homologous recombination a solventogenic bacterium of the genus  Clostridium , characterized in that it comprises a step of introduction into the bacterium of a genetic tool allowing the transformation by homologous recombination of a solventogenic bacterium of the genus  Clostridium , characterized in that it comprises:
 a first nucleic acid encoding at least Cas9, wherein the Cas9 coding sequence is placed under the control of a promoter, and   at least a second nucleic acid containing a repair template allowing, by a homologous recombination mechanism, the replacement of a portion of the Cas9-targeted bacterial DNA by a sequence of interest,   and in that i) at least one of said nucleic acids further encodes one or more guide RNAs (gRNAs), or ii) the genetic tool further comprises one or more guide RNAs, each guide RNA comprising a Cas9-enzyme-binding RNA structure and a sequence complementary to the targeted portion of the bacterial DNA.   
     
     
         2 . The process according to  claim 1 , characterized in that it comprises the following steps:
 a) introduction into the bacterium of said genetic tool, and   b) induction of the expression of the inducible promoter for genetically modifying the bacterium.   
     
     
         3 . The process according to  claim 1 , characterized in that it comprises one or more additional steps, subsequent to step b) when it is present, of introduction of an nth nucleic acid encoding i) a repair template different from that or those already introduced and ii) one or more guide RNAs allowing their integration into a targeted zone of the genome of the bacterium, each additional step being preceded by a step of removal of the nucleic acid encoding the repair template previously introduced, and preferably of removal of the one or more guide RNAs or sequences encoding the one or more guide RNAs previously introduced. 
     
     
         4 . A solventogenic bacterium of the genus  Clostridium  transformed using the process according to  claim 1 . 
     
     
         5 . A method of producing a solvent or a mixture of solvents comprising culturing a solventogenic bacterium according to  claim 4  and producing said solvent or mixture of solvents. 
     
     
         6 . The method according to  claim 5 , wherein said solvent or mixture of solvents is acetone, butanol, ethanol, isopropanol or a mixture thereof.