Mining of phage resistance genes and construction of phage-resistant strains in Escherichia coli

Abstract:

[Objective] Phages are specific viruses that infect chassis cells in industrial fermentation. Due to the widespread existence and difficult eradication, phages greatly affect the yield of fermentation. The mining and functional verification of phage resistance genes can significantly enhance the anti-phage ability of chassis cells, so as to prevent phage pollution at the source. This study aims to mine the genes conferring the resistance to phages and construct phage-resistant strains. [Methods] Co-evolutionary screening, resequencing, recombinant strain construction, and phage infection experiments were carried out for the screening of the strains with phage resistance. [Results] Seven strains domesticated for resistance to phages were obtained through co-evolutionary screening. The genome resequencing and Annovar analysis identified mutations in 12 genes. Three genes with high mutation frequency, dnaE (DNA polymerase III subunit α): yhjH (cyclic diGMP phosphodiesterase): and rzoD (putative phage-lysed lipoprotein), were selected, and then the strains overexpressing the genes and the strains with knockout of the genes were constructed. The strains overexpressing the selected genes demonstrated obvious resistance to BL21 Virus 01, BL21 Virus 02, BL21 Virus 06, T1, and T7. The adsorption rates showed that dnaE and yhjH affected phage replication, while rzoD affected phage adsorption. Quantitative PCR was employed to further analyze the resistance of the strains with mutations of dnaE and yhjH to phages. The results showed that the mutations of dnaE and yhjH affected the replication process of BL21 Virus 01, while that of rzoD affected the adsorption process of BL21 Virus 01. [Conclusion] The mutations of dnaE, yhjH, and rzoD can resist phage infections, and the engineered strain BC11 with the mutations of all the three genes demonstrates a wide range of phage resistance. The findings provide a basis for the mining of phage resistance genes and references for the construction of strains with phage resistance.

Key words:anti-phage;evolutionary screening;resequencing;engineered strain