Mitigating genetic instability caused by the excision activity of the C31 integrase in .

Over the past three decades, the integrase (Int) from phage C31 has become a valuable genome engineering tool across various species. C31 Int was thought to mediate unidirectional site-specific integration ( × to and ) in the absence of the phage-encoded recombination directionality factor (RDF). However, we have shown in this study that Int can also catalyze reverse excision ( × to and ) at low frequencies in and , causing genetic instability in engineered strains. To address this issue, we developed a two-plasmid co-conjugation (TPC) system. This system consists of an -containing integration vector and an Int expression suicide plasmid, both carrying to facilitate efficient conjugation transfer from to . Using the TPC system, genetically stable integrants free of Int can be generated quickly and easily. The indigoidine-producing strains generated by the TPC system exhibited higher genetic stability and production efficiency compared to the indigoidine-producing strain generated by the conventional integration system, further demonstrating the utility of the TPC system in the field of biotechnology. We anticipate that the strategies presented here will be widely adopted for stable genetic engineering of industrial microbes using phage integrase-based integration systems.IMPORTANCELarge serine recombinases (LSRs), including the bacteriophage C31 integrase, were previously thought to allow only unidirectional site-specific integration ( × to and attR). Our study is the first to show that the C31 integrase can also catalyze a low-efficiency reverse excision reaction in and without the involvement of the phage-encoded recombination directionality factor (RDF). The genetic instability caused by the low excisionase activity of the C31 integrase is a major challenge for biotechnological applications. Our study addresses this issue by developing a two-plasmid co-conjugation (TPC) system that facilitates the construction of Int-deficient genomic engineering strains. The Int-deficient integrants produced by this TPC system exhibit strong genetic stability for introduced genes and maintain stable production traits even in the absence of selection pressure, making them highly valuable for industrial applications.