Research On The Application Of CglI Gene Complex To Construct Corynebacterium Crenatum Phage-resistance Strains

Corynebacterium crenatum is the most important producer of amino acid. Because of the large-scale and consistent of fermentation processes, the problem of phage infection has not been completely solved today, which was regarded as one of the most important restriction factors in the fermentation industry. Traditional methods used to solve the phage infection problem in fermentation industry include ameliorating sanitation condition and strengthening production management, using phage inhibitor and screening resistant mutants. Naturally occurring phage resistance mechanisms are widespread among bacterium, based on which a wide variety of engineered phage-resistance systems that target different stages in the lytic life cycle have been constructed. These systems include phage-encoded resistance, anti-sense RNA, suicide trap and the application of restriction and modification system, and the application of restriction and modification system is very effective.Bacterial restriction-modification (R-M) systems function as prokaryotic immune systems that attack foreign DNA, are widespread among prokaryotic cell. This defense mechanism is achieved by cleaving incoming DNA that is recognized as foreign by the absence of a characteristic modification at defined sites within the recognition sequence. The host DNA is resistant to cleavage as these sites are modified. The cglI gene complex, encoding a 5-cytosine methyltransferase enzyme and a restriction endonuclease, was first located in the genome of C. glutamicum.In order to efficiently solve the phage infection problem in fermentation industry, this work studied the feasibility of applying cglI gene complex to construct engineered phage-resistant strains using the principle of microbial immune. The cglI gene complex was amplified by PCR from Corynebacterium glutamicum, and then recombinant plasmid pJL23-cglI was constructed and transformed into E. coli. Based on exploring the available approach of cloning cglI gene complex into E.coli, gene engineering technique were used to study the role of cglI gene complex in constructing phage-resistant strains further and the influence of cglI gene complex on the recombinant strain's growth and metabolism were preliminary evaluated.The result showed that, first, selection of vector and host is the key factor when cglI gene complex was cloned into E. coli, and cglI gene complex was successfully cloned into McrBC deficiency E. coli strain HB101 at last. Second, the recombinant strains containing cglI gene complex had high phage-resistance activity in both qualitative and quantitative experiment. Third, the recombinant plasmid pJL23-cglI carrying cglI gene complex had little impact on the strain's growth and metabolism. The continued culture study result showed that the existence of cglI gene complex could enhance the plasmid's stability.In conclusion, this study verified the feasibility of using cglI gene complex to construct phage-resistance recombinant strains and provided an effective way to solve the phage infection problem in fermentation industry. At the same time, this study created an advantageous condition for constructing engineered recombinant E.coli. Also, it offered a new way to the application research on cglI gene complex.