| Bacillus licheniformis has many excellent characteristics,such as strong protein secretion ability,high extracellular enzyme production,and simple fermentation conditions.Therefore,it is one of the most promising industrial microorganisms.Currently,the main methods of strain selection are natural screening and traditional mutagenesis,but low endogenous recombination efficiency of B.licheniformis greatly limits its genetic modification.Moreover,due to lack of efficient gene editing tools,the further expansion of B.licheniformis application is restricted.In recent years,bacteriophage recombination systems have been used to improve the recombination efficiency of many bacteria,but no effective bacteriophage recombination system for B.licheniformis has been reported.In this study,a phage derived recombinase Rec T was introduced into B.licheniformis.Using the rhamnose promoter as an expression element,a bacteriophage recombination system for conditional expression was constructed in B.licheniformis.The system was validated for the knockout of amylase genes,and the efficiency of gene editing in the system was improved to some extent through optimization of operating conditions.The main content and results are as follows:（1）Find bacteriophage recombinases and their expression elements.Select recombinase coding genes from Bacillus and its phages,and through amino acid homology comparison and phylogenetic tree analysis,identify five recombinases of the Rec T family that have both homology and diversity.Based on the metabolic pathway analysis,the rhamnose operon was found to be composed of three characteristic genes,rhamnose almutase gene yux G,rhamnose kinase gene yul C and its transcription regulator yul B.The promoter Prha in the rhamnose operon was obtained,and the transcription and expression characteristics of Prha promoter under different conditions were explored by using green fluorescent protein as a reporter gene.It was found that the endogenous rhamnose promoter of B.licheniformis was an inducible promoter strictly controlled by rhamnose,and the transcription and expression of the gene was enhanced with the increase of rhamnose concentration.（2）Construction and functional verification of the recombination system.A recombination system was constructed using rhamnose expression element Prha and five heterologous bacteriophage recombinases.The gene editing method based on this recombination system was established as a knockout plasmid using p HY300-PLK as the vector,carrying the recombinase expression cassette and the target gene knockout cassette.The knockout plasmid was introduced into Bacillus licheniformis by electroporation to induce the expression of the recombinase for homologous double exchange to achieve the knockout of the target gene,and then the knockout plasmid was lost in an antibiotic-free manner.The recombinase Rec T from Bacillus phage049ML001 showed high recombination activity in Bacillus licheniformis.The recombination system constructed with recombinase Rec T from Bacillus phage 049ML001 increased the recombination efficiency of Bacillus licheniformis to 5.56%.（3）Optimization of the operating conditions of the recombinant system.The operating conditions of rhamnose addition time,concentration,strain culture time,and passage times were optimized.Concluded that when theα-amylase gene amy L was knocked out,1.5%rhamnose was added after 8 h of growth to induce the expression of recombinase Rec T,and the gene editing efficiency was the highest after continuous culture for 24 h and passage 3 times,reaching 16.67%.At the same time,the recombination substrate is knocked in in the form of fragments,and found that random integration events occur in B.licheniformus in the process.（4）Application of recombination system.The recombination system was used to knock out the alkaline protease apr E gene and the key gene in the phosphotransferase system of B.licheniformis,the alkaline protease apr E gene was successfully knocked out and the gene editing efficiency was 12.5%,but the knockout of the key gene in the phosphotransferase system was still difficult. |
