Application And Improvement Of The Novel Counter-selection System Kil And Preliminary Study On Molecular Directed Evolution Of The Red Recombination System

Microbial pharmaceutical technology is closely related to microbial metabolites.Metabolic engineering is the method of DNA recombination to increase the content of metabolites or to generate new metabolites by changing metabolic fluxes or changing metabolic pathways to screen out excellent engineered strains or cells.At present,the DNA recombination technology was commonly used as the Red homologous recombination system.This system can be used to seamlessly modify the gene by using the counter-selection system(point mutation,substitution,deletion).However,excellent counter-selection genes are limited.This paper has a significantly help to genetic engineering editing technology through the directed evolution of the selection counter-selection system and Red recombination system,which has great theoretical research significance and practical application value.Objective To analyze the selection stringency and recombination efficiency of kil gene and compare it with the most widely used counter-selection gene sac B,we construct a new kil counter-selection system.The p Sim6 plasmid containing the highly efficient Red recombination system will then be used to enhance the efficiency of seamlessly modifying the gene by fusion with the novel tet/kil counter-selection system.In addition,directed evolution of the Red gene can improved MAGE(Multiplex Automated Genome Engineering)technology and accelerated the pace of cell evolution.Methods and Results According to Khetrapal’s method,the selection strigency of kil and sac B counter selection was performed.The result shows that the selection stringency of the kil gene was analyzed at three sites(lac I,ack and dbpa)of different Escherichia coli(E.coli)strains,and compared with the most widely used counter-selection gene sac B,it was found that the selection stringency was relatively Higher.Among the genetic loci of all these test strains,the selection stringency of kil was 2 to 28 times higher than that of sac B.When different lengths of exogenous ds DNA fragments are used for gene recombination,the recombination efficiency of the counter selection system was higher than that of the neo/sac B counter selection system.However,the kil system cannot be used for host strains containing the temperature sensitive kil gene.A Red system using a plasmid containing no kil gene was recommended for use in conjunction with this system.Then,we found that the neo/kil selection counter-selection system combined with the p Sim6 plasmid had a higher selectivity selection and a lower background than that of the kil selection counter-selection system combined with the p KD46 plasmid.Furthermore,four different non-essential gene loci(lac I,dbpa,ack,glk)in three different E.coli strains W3110,MG1655,DH10 B were tested,These loci were genetically replaced by exogenous gene fragments of different lengths(500 bp,1000 bp,2000 bp).The results showed that compared with the neo/kil system,the recombination efficiency increased significantly,and with the increase of exogenous fragments,the more obvious the recombination efficiency was.When the exogenous fragment was 1000 bp,which was 1.2-2times of neo/kil;when the exogenous fragment was 2000 bp,which was neo/kil 2.2-5times.In summary,the combination of the two makes the seamless modification operation time-saving,simple and recombination effect,and provides more choices for E.coli recombination engineering.In addition,we conducted a directed evolution analysis of the Red gene.First,a resistance gene(kanamycin resistance gene neo)containing a nonsense mutation was inserted into the chromosome of E.coli,and the bacteria do not have the resistance phenotype due to the presence of point mutation.Then,the promoter libraries of different intensities were constructed using E.coli lac promoter as template,and the promoter activity analysis of 35 different gradient intensities and SDS-PAGE protein gel electrophoresis analysis of the promoter library were performed.The promoters that were strongest(1283times the activity of the weakest promoter)and weaker(100 times the activity of the weakest promoter)were selected and compared with the wild lac promoter(500 times the activity of the weakest promoter).Three plasmids containing p RO38 a,p RO38 b and p RO38 c from the promoter to the weak recombinant system were constructed.The Red gene of the three plasmids was mutated,and the number of bacteria in the repaired neoplasm was analyzed.The results showed that the plasmid after mutating the Red gene has a higher number of colonies for repairing the kanamycin resistance gene than for the unmutated Red gene.The plasmid with the strongest promoter promoter,the number of colonies of the mutant Red gene repairing the neo resistance gene was about twice that of the unmutated;the plasmid initiated by the wild type promoter,the number of colonies of the mutant Red gene repairing the neo resistance gene was about no mutation 3 times;the plasmid with the weakest promoter promoter,the number of colonies of the mutant Red gene repairing the neo resistance gene was about 50 times that of the unmutated gene.Conclusion In this paper the selection stringency of the new counter-selection system kil was analyzed,which is equivalent to excellent tet-sac B counter-selection system.By combining the kil gene with different plasmids,the gene position of the E.coli strain The point of gene replacement confirmed the high recombination efficiency of kil.In addition,excellent mutants were screened by random mutation of the Red gene.These results are of great help to genetic engineering editing techniques and have great theoretical research significance and practical application value.