Engineering a feedback-based synthetic gene circuit for targeted continuous evolution of a gene in E. coli

Abstract

Directed evolution is an invaluable technique for engineering proteins to possess desired physical and chemical properties when very little structural and functional information is known. It is divided into two sequential steps: generating a library of protein variants using mutagenic techniques; and applying a screening or selection strategy to scan the library for variants displaying desired properties. Library generation is performed using either in vitro or in vivo techniques, while screening or selection typically occurs in a suitable host cell. Currently, in vitro methods like error-prone PCR are popular for library generation. However, these techniques can be labour intensive, prone to mutation biases, and generate limited library sizes for screening. In vivo mutagenic techniques overcome these limitations by enabling simultaneous library generation and selection within cells. By generating random mutations in the gene-of-interest within one cell cycle, each cell in a batch culture potentially represents a library variant. Such a continuous evolution system can run for weeks with minimal human intervention, greatly expanding the genetic search space for protein engineering. The challenge lies in developing a mutator system that specifically generates mutations in the target gene, while maintaining the cell’s genomic fidelity. With this goal in mind, a mutator system was engineered in E. coli that introduces targeted cytidine deamination damage and subsequently performs error-prone DNA repair by hijacking the base excision repair pathway. The targeted damage occurs via activation induced cytidine deaminase fused to T7 RNA polymerase, while the error-prone DNA repair is performed by a three-protein fusion comprising a 5’-3’-exonuclease, an AP-endonuclease and an error-prone DNA polymerase. The mutagenic characteristics of this system was tested by knocking out GFP expression and analysing the mutant library using next generation sequencing techniques. The system was also experimentally shown to generate functionally active mutations that reverted inactivated β-lactamase gene variants to confer ampicillin resistance.