Study On The Knockout Methodology Of Corynebacterium Glutamicum ATCC 13032

Gene deletion is an essential strategy to eludate gene function and constructions of highly efficient microbial cell factories.Corynebacterium glutamicum strains are important producers of amino acid and derivatives.C.glutamicum ATCC 13032 is the model strain,modification of the strain would be helpful for the study of the functions of genes and proteins,improvement of the yied of amicno acid,and inductrial process of amino acid production.R6K replicon needs a special Pir protein to replicate;Pir is devoid in many bacteria.Therefore R6K replicon plasmids are widely used in the construction of suicide plasmids.When deliveried into the host cells,suicide vector will integrate into the chromosome under the selective pressure.The none-replicative property makes it possible convinent to construct the precise gene deletion plasmid via the splicing of homologous arms.Current C.glutamicum ATCC 13032 genome modication methods suffer the shortcomings of tedious procedure and low efficiency.To investigate the problem,our research group established a suicide plasmid-based C.glutamicum ATCC 13032 genome engineering strategy and obtained a series of strains with suicide plasmid integration.The first part of the thesis is the elimination of the suicide plasmid from the integratve strains followed by the deletions of prohages CGP2 and CGP3 with the strategy.Suicide plasmids pLS3730 and pLS3732 were constructed and transferred into C.glutamicum ATCC 13032,KmR clones were obtained with suicide plasmid integration.Then 1 mM IPTG was added to the cultue medium for the induction of homing endonuclease I-SceI,which cleaved on its recognition sites brought by the suicide plasmid and created double strand break.The inherent recombination machinery of the strain catalyzed the recombination of homologous fragments and generated the scarless gene deletion strains.Results showed that the gene deletion strains of crtI2 gene,crt operon,CGP1,CGP2 were obtained with average deletion efficiency of 50%,and the highest efficiency reached 90%.However,prophage CGP3 deletion was failed,possibly due to the extremly large size of CGP3(more than 180 kb)which makes homogous recombination between such a long distrance difficult.To establish an efficient procedure to delete CGP3,We applied recombineering and I-SceI tools.The tools were used for the CGP3 deletion through both the plasmid-based and chromosome-based I-SceI expression models.Recombineering(recombination-mediated genetic engineering),also called?-Red/ET,is a genetic engineering technology which based on the recombinase catalyzed homologous recombination between DNA molecules.The recombinases are mainly originate from the ? phage red?? genes or Escherichia coli prohage Rac recET genes.The distinguished advantages of recombineering include simplicity,no DNA moleculre size and no restriction site limitations,thus tedious cloning steps are circumvented and only PCR or OE-PCR is required to generate targeting DNA used for precise genome engineering.We constructed CGP3 gene deletion targeting plasmid pLS4121 and plasmid-based recombinase and I-SceI expression plasmids pLS3635,pLS4132,pLS4133,pLS4136,pLS4137.Each plasmid was transformeal into C.glutamicum ATCC 13032.Chromosome-based recombinase and I-SceI expression system was obtained by pLS4141 into the chromosome and eliminated the plasmid backbone via the suicide-based strategy.Targeting DNA was transferred into the recombination-proficient cells;and replacement strain was obtained under the kanamycin selection.Then I-SceI was induced for double-strand break repair leading to the CGP3 scarles deletion.Results showed that plasmid pLS4132 with L-arabinose-induced recET gene,codon optimized I-Scel gene showed the highest deletion efficiency.Nevertheless,the efficiency is still too low to have practical applications.Further investigation will focus the optimization of various parameters to establish an efficienct manipulation system.