Designing a Mutagenesis Circuit for the Directed Evolution of a Target Gene In Vivo

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Directed evolution has emerged as an important tool in the Biotechnology industry as it gravitates towards development of biocatalysts and utilising living organisms to produce consumer products. Biocatalysts and cell bases systems offer certain advantages over chemical methods for production1. They provide high efficiencies and substrate specificities, while operating at physiological pH and temperature as opposed to chemical methods that may require extreme reaction conditions. The use of biocatalysts at the moment is limited as one enzyme equals one function and many of the enzymes and metabolic pathways required to produce certain products are not found in nature or do not exist. Directed evolution allows us to modify the characteristics of existing enzymes and generate novel enzymes with function not found in nature. This is done by accelerating the natural process of Darwinian Evolution2,3. So far, in vitro methods like error-prone PCR have been the most popular for conducting Directed evolution. Error-prone PCR is extremely high-throughput and can generate a library of 1010 mutants of a target gene4. However, transforming all these mutants into the desired chassis for screening and characterisation can be cumbersome and requires a significant amount of time. Many of these mutants can be non-functional and deleterious to the chassis. In vivo directed evolution methods have been developed to overcome the transformation bottle-neck of screening such a large library of mutants5. The most popular method for single gene directed evolution is Phage-assisted Continuous Evolution (PACE), which separates the mutation and selection between two hosts; E. coli and a bacteriophage6 (Fig. 1). The E. coli host contains a mutator plasmid that leads to global mutation that ultimately affects its fitness. Selection via successful re-infection by the bacteriophage enables the E. coli host to be recycled and overcomes the loss of fitness due to continuous mutation. This was performed by mutating T7 RNA polymerase to be active from a T3 promoter sequence that produced the selection signal. The use of PACE is limited by the ability to link selection to transcriptional activation.Unless this is possible, the phage will be unable to infect the host cells, making screening impossible. Also, optimising the conditions for selection can be tricky. If the flow rate of the lagoon is too high, the phage get washed out in the outflow. In some cases, genetic drift might be required to generate a diverse gene pool for continuous evolution and selection7. We hope to design an in vivo continuous evolution system to target a single gene like PACE, but with a simpler selection strategy, whereby both mutation and selection can take place in a single host. The system would apply the concept of somatic hypermutation found in lymphocytes to generate variant antigen-binding regions8. During this process, specific genes are targeted for mutations, while maintaining the fidelity of the overall genome. We hope to apply this tightly regulated mutation to evolve the lasR gene in the E. coli chassis and select for functional mutants via a selectable marker and fluorescent output. The system will be designed to perform continuous evolution and select for desired mutants in a pool of replicating cells with minimal human intervention.