(67d) Retron-Based Targeted Mutagenesis Enabling in vivo Continuous Evolution in E. coli

Envisioned as an accelerated imitation of Darwinian evolution, in vivo continuous evolution, when compared with traditional in vitro methods, is supposed to be a highly effective approach of engineering enzymes or pathways with tailor-made properties under few human intervention. A typical in vivo continuous evolution process contains iterative rounds of global mutagenesis and fitness-coupled selection. Generally used global mutagenesis methods, however, usually lead to error catastrophe or evolutionary escape. Targeted mutagenesis, which can largely improve the mutation rate of a target region against the relative steady genetic background, has raised much attention. Existing targeted mutagenesis tools mainly rely on mutagenic protein anchoring or orthogonal DNA polymerase-plasmid pairs, while lacking stringent targeting property and general applicability. Recently, a novel targeted mutagenesis tool based on yeast native retrotransposons, where diversity is generated in transcription level and recorded by reverse transcription and recombination, has opened a new direction.

In this work, we proposed a novel targeted mutagenesis tool based on an E. coli native retron cassette, which is able to produce ssDNAs encoded within it in vivo through transcription and reverse transcription processes. By inserting the target region sequence into the wild-type retron cassette and applying the error-prone T7 RNA polymerase, an ssDNA mutant library can be generated, and integrated chromosomally into the target area through single-stranded DNA recombination. As a proof of concept, the gene sacB, encoding levansucrase, was integrated into E. coli DH10B genome as a counter-selection marker for mutants. When the ssDNA mutant library targeting the sacB region was generated in this strain, a considerable mutation rate improvement over its genetic background as well as the wild-type strain can be observed through sucrose lethal selection. Details of this work will be given in the conference. 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 be developed into a practical method in rapid and robust protein and metabolic engineering.