An in vivo target mutagenesis system for multiple hosts

In vivo target mutagenesis is a powerful approach to accelerate protein evolution. However, current approaches have been primarily developed in conventional organisms, limiting their capacity to evolve proteins with subtle variations across non-conventional host species. Here, we design an in vivo target mutagenesis system for multiple hosts (ITMU) utilizing the broad host-range plasmid RSF1010 replication element. The ITMU, which is based on a deaminase-helicase fusion and a primase error-prone DNA polymerase I fusion, induces all types of mutation in the target plasmid harboring the RSF1010 replicon, at a mutation rate 1.18 × 10⁵-fold higher than that of the host genome. We show that ITMU-based in vivo continuous evolution is effective in Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, and Yarrowia lipolytica. This demonstrates that the ITMU is applicable to multiple microbial chassis and provides a viable alternative to in vivo continuous evolution systems.