Excavation Of Phage Resistance Gene In Escherichia Coli And Its Phage Resistance Construction Of Strain

Escherichia coli has the advantages of clear genetic background,fast growth rate and sim ple molecular manipulation.As a mature tool and strategy for metabolic engineering and high cell density culture,E.coli has become a cell factory for biosynthesis of natural and unnatural products.Bacteriophage,as a bacterial virus,has the characteristics of rapid reproduction and ubiquitous.In the process of fermentation,once contaminated by phage,the fermentation yiel d and quality will decrease at least,and the fermentation process can not be carried out,which brings serious economic losses to the production.Traditional phage control methods include f ormaldehyde fumigation and bacterial rotation,but it cannot fundamentally solve the problem of phage contamination.With the development of synthetic biology technology,the constructi on of bacteriophage resistant E.coli chassis cells has become an effective means of bacterioph age contamination during fermentation.In this study,phage coevolution and genome resequen cing were used to explore potential anti-phage genes on the E.coli genome,and the resistance mechanism of anti-phage genes was initially analyzed by combining gene overexpression,CR ISPR/Cas9 knockout technology,dot plate experiment,adsorption rate determination and RT-PCR.A wide spectrum and efficient engineering strain against phage was constructed.The ma in research results are as follows:（1）24 strains of bacteriophage resistant mutant E.coli BL21A1-A24 were selected by co-evolution of bacteria and phage.Seven phage mutants with strong resistance were identified a nd renamed E.coli BL21 A1-A7.In order to explore the key genes of phage resistance,genom e resequencing was used to analyze the mutant genome.According to mutation frequency and gene function,dna E（encoding DNA polymerase III subunitα）,yhj H（encoding cyclic di GMP phosphodiesterase）,rzo D（encoding putative phage lysed lipoprotein）,tu F（extension factor）,in s B（IS1 family protein）were selected for further study.Overexpression of dna E^I171L,yhj H^A251Vand rzo D^A2V was used to construct site mutant strains,and knockout strain BL21/Δrzo D was constructed by CRISPR/Cas9 technique.（2）Strain sensitivity was verified by spot plate test.The strain containing dna E^I171L mutat ion was resistant to BL21 Virus 01,BL21 Virus 03 and T7.The recombinant strain containing yhj H^A251V site mutation was resistant to BL21 Virus 01,BL21 Virus 03 and T1.The strain co ntaining the rzo D^A2V mutation was resistant to BL21 Virus 01,while the strain BL21/Δrzo D k nockout was resistant to all the phages tested.（3）Determination of adsorption rate and RT-PCR to determine whether the expression of mutant genes affected the infection of phage.The results showed that there was no significant difference in the adsorption rate of the strain containing dna E^I171L and yhj H^A251V mutation co mpared with the control group,and there was no significant change in the DNA content of the phage.It was speculated that the expression of dna E and yhj H genes could affect the replicati on of the phage to resist the infection of the phage.Compared with the control group,the adso rption rate of rzo D^A2V mutant strain and rzo D knockout strain decreased significantly,and the DNA content of phage increased significantly.It is speculated that the expression of gene rzo D can resist phage infection by affecting the adsorption of phage.（4）The bacteriostatic curve was used to determine the resistance of the recombinant strai n to phage.The results showed that the engineered strain BC11 could resist multiple phages at the same time under the condition of MOI=10,and had a wide range of phage resistance.At t he same time,E.coli BL21/p ETDuet-lpgad producingγ-aminobutyric acid was used as chassi s cells to characterize the production of resistance genes.The results show that contains the ex pression of recombinant vector p ET28a-dna E^I171Lyhj H^A251Vrzo D^A2V does not affect product yi eld of gamma-aminobutyric acid output.