Retron-Based Targeted Mutagenesis Enabling In Vivo Continuous Evolution in E. coli

By mimicking and accelerating Darwinian evolution, in vivo continuous evolution, when compared with the conventional in vitro methods, is supposed to be a highly effective approach to engineering tailor-made functions of enzymes or pathways with few human intervention. Generally used global mutagenesis methods, however, usually lead to high evolutionary escape ratio. Targeted mutation, which can largely improve the mutation rate of a target region against the relative steady genetic background, has raised much attention. However, existing targeted mutagenesis tools rely on mutagenic protein anchoring or bacteriophage assisting, which are limited in stringent targeting property and general applicability. Recently, two novel targeted mutagenesis tools based on orthogonal DNA replication system or error-prone reverse transcription process have been established in yeast. Effective targeted mutagenesis tools in prokaryote E. coli still remains underdeveloped.

In this work, we proposed a novel targeted mutagenesis tool based on E. coli native retron cassette. By inserting the target region sequence into the wild-type retron cassette and utilizing the error-prone T7 RNA polymerase, corresponding mutated ssDNAs can be generated via low-fidelity transcription and reverse transcription processes, and then integrated chromosomally into the target area through bet-guided single-stranded DNA recombination. As a proof of concept, the gene dbla, containing a premature stop codon in bla and encoding the inactivated β-lactamase, was integrated into E. coli DH1 genome as a selection marker for mutants. When the ssDNAs targeting the 100-bp stop-codon-containing region were generated in this strain, there was a chance of 10^-7 to obtain mutants acquiring ampicillin resistance, corresponding to an increase in targeted mutation rate of around 100 fold. In the light of its strong ability to construct mutant library of an artificially designated region with high throughput and low workload, we believe that the retron-based targeted mutagenesis, when integrated into in vivo continuous evolution, will become a promising method in protein and metabolic engineering.