Inducible Directed Evolution (IDE) of Complex Phenotypes in Bacteria

Many important microbial phenotypes are complex and difficult to engineer, as they emerge from interactions between many genes. Directed evolution is a method of engineering beneficial phenotypes in microbial organisms via an iterative process of mutagenesis and screening, improving function without the need for detailed biochemical models. Directed evolution often suggests novel or counterintuitive routes to improved function, making it ideal for engineering complex phenotypes and giving it diverse applications in natural product synthesis and microbial therapeutics. Recent methods for directed evolution have overcome the traditionally laborious and costly steps for generating error-prone libraries, which include molecular cloning and transformation. However, all methods for directed evolution developed to date are limited to small regions of DNA and/or allow the accumulation of off-target mutations. To enable directed evolution of complex phenotypes encoded by multigene pathways, we require large library sizes for DNA sequences >5-10 kb in length, elimination of off-target genomic mutations, and decoupling of diversification and screening steps.

To meet these challenges, we developed Inducible Directed Evolution (IDE), which uses a temperate bacteriophage to inducibly package large plasmids and transfer them to naive cells after intracellular mutagenesis. IDE combines (1) the P1 bacteriophage’s ability to deliver large plasmids efficiently to Escherichia coli cells and (2) inducible intracellular mutation machinery to introduce and propagate random mutations in the target pathway without the accumulation of off-target mutations. We demonstrate no reduction in library size with increasing pathway length, up to the largest pathway we have tested (35 kb). We also demonstrate IDE’s utility in evolving several complex multi-gene pathways, including a 5-gene tagatose catabolism pathway (5 kb) from Bacillus licheniformis, as well as a 10-gene melezitose catabolism pathway (15.4 kb) from Bifidobacterium breve.