大肠杆菌抗噬菌体基因的挖掘及其抗噬菌体菌株的构建

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

摘要:

【目的】噬菌体是工业发酵侵染底盘细胞的专一性病毒，由于其广泛存在以及难以根除，极大影响了发酵生产，从基因水平进行抗噬菌体基因的挖掘以及功能验证可以显著增强底盘细胞的抗噬菌体的能力，从而达到从源头上抵御噬菌体污染的目的。为了选育具有噬菌体抗性的工程菌株，本研究旨在分析选取抗噬菌体的基因以及构建抗噬菌体的工程菌株。【方法】采用共进化筛选、重测序手段、重组菌株构建以及噬菌体侵染的敏感验证实验等方法，获得具有抗噬菌体属性的工程菌株。【结果】实验通过共进化共筛选得到7株抗噬菌体驯化菌株，基因组重测序以及Annovar软件分析，发现了12个基因发生位点突变，选取位点突变频率较高的dnaE (DNA聚合酶III亚基α)、yhjH (环状二GMP磷酸二酯酶)及rzoD (假定的噬菌体裂解脂蛋白) 3个基因分析对噬菌体的抗性，突变基因过表达菌株对噬菌体BL21 Virus 01、BL21 Virus 02、BL21 Virus 06、T1和T7具有明显的抗性；吸附率测定结果表明，基因dnaE和yhjH影响噬菌体复制，而基因rzoD突变影响噬菌体吸附过程。使用定量PCR进一步分析突变基因dnaE和yhjH对噬菌体的抗性作用，结果表明基因dnaE、yhjH突变影响噬菌体基因组BL21 Virus 01的复制过程，而rzoD突变影响噬菌体BL21 Virus 01的吸附过程。【结论】基因dnaE、yhjH、rzoD的位点突变能够有效抵御噬菌体的侵染，同时含有3个位点突变的工程菌株BC11具有较广范围内的噬菌体抗性，是潜在的抗噬菌体底盘细胞。本研究为抗噬菌体基因挖掘与初步解析以及开发抗噬菌体工程菌株提供借鉴。

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.