[Construction of an anti-phage network system based on synergistic mechanisms and its applications in synthetic...

In natural ecosystems, bacteriophages play a crucial role in bacterial lysis. Bacteriophage contamination frequently leads to fermentation failure, resulting in compromised product quality and purity, as well as substantial economic loss. Consequently, effective strategies are required in the fermentation industry to mitigate bacteriophage contamination. The use of a single anti-phage strategy is readily evaded by spontaneous phage mutations, resulting in limited and poorly applicable protection against phage infection. Synergistic defense mechanisms involving multiple systems suppress the spontaneous mutations that enable phage escape, thereby broadening phage resistance. This study is designed to tackle the bottleneck associated with single anti-phage strategies, which are susceptible to escape via spontaneous phage mutations, exhibit limited protective efficacy, and suffer from poor applicability. By integrating receptor engineering with synergistic defense mechanisms, a highly efficient, broad-spectrum, and non-toxic phage defense network system is constructed. This system is subsequently applied to industrial fermentation strains, with the aim of achieving effective resistance against phage contamination and improving the stability of fermentation processes. Initially, a synergistic resistance mechanism between Shedu and Septu was identified through laboratory screening of defense mechanisms. Further investigation revealed that the endonuclease activity of Shedu and the ATPase and HNH nuclease activities of Septu jointly contribute to synergistic resistance. Optimization of promoters was employed to adapt the Shedu and Septu mechanisms, thereby conferring resistance to phages T4, λ, P251, and P255. Subsequently, receptor engineering was applied to modify fhuA, a key adsorption site for phages T1 and T7, to achieve resistance against these phages and complete the construction of the anti-phage network system. Growth curve analysis demonstrated that the anti-phage network system exhibits minimal toxicity and high resistance efficiency, highlighting its strong industrial application potential. Finally, phage resistance was introduced into riboflavin- and ʟ-leucine-producing strains, enabling them to effectively counter phage contamination during fermentation. In a 5 L bioreactor, riboflavin production reached 11.2 g/L and ʟ-leucine production reached 64.0 g/L, thereby confirming the industrial potential of the anti-phage system. The resistance strategy reported herein facilitates the development of engineered Escherichia coli strains with robust and broad-spectrum phage resistance for using in the industrial fermentation of natural amino acids and high-value compounds.