Genetic tools for probing long evolutionary pathways

Directed evolution is a powerful tool that has been used for novel drug discovery, commodity chemical synthesis, biodegradation, and many other medical and industrial advancements. However, one challenge with traditional directed evolution experiments is the divide between ex vivo diversification and in vivo selection, as the two are kept separate both to take advantage of their respective settings and to avoid unintentional off-target effects. This results in labor and time-intensive evolution experiments, limiting the number of replicates, rounds of evolution, or both. To address this, previous work in our lab sought to develop OrthoRep, a plasmid system in yeast enabling continuous in vivo mutagenesis of genes at high rates, allowing for the coupling of diversification and selection. Expanding on this, we developed a panel of constructs allowing genes encoded on OrthoRep to be expressed at levels spanning a wide range comparable to genes encoded on nuclear promoters. This has expanded OrthoRep’s capabilities for more ambitious targets, including nanobodies and poorly functioning enzymes. Next, we paired OrthoRep with a continuous culture device to enable Automated Continuous Evolution, a hands-free evolution device that automatically adjusts culture conditions to maintain a programmable level of selection. We show that this pairing enables faster adaptation compared to manual passaging and prevents overly stringent conditions that can lead to extinction of evolving cultures. Finally, we used OrthoRep to evolve HisA from Thermotoga maritima (TmHisA) to catalyze the related Trp1 activity in yeast and demonstrated the ability to survey a broad fitness landscape and probe multiple selection conditions. We further reveal that after reaching a plateau for Trp1 activity, we can escape this plateau through an alternative selection for the original activity of TmHisA while continued strong selection for Trp1 activity is ineffective. These advancements have enabled OrthoRep to be a capable alternative to traditional mutagenesis, as well as offered insights into the design of selection schemes that may facilitate reaching higher catalytic peaks