Automated Continuous Evolution of Proteins in Vivo

Abstract

We present automated continuous evolution (ACE), a platform for the hands-free directed evolution of biomolecules. ACE pairs OrthoRep, a genetic system for continuous targeted mutagenesis of user-selected genes in vivo, with eVOLVER, a scalable and automated continuous culture device for precise, multi-parameter regulation of growth conditions. By implementing real-time feedback-controlled tuning of selection stringency with eVOLVER, genes of interest encoded on OrthoRep autonomously traversed multimutation adaptive pathways to reach desired functions, including drug resistance and improved enzyme activity. The durability, scalability, and speed of biomolecular evolution with ACE should be broadly applicable to protein engineering as well as prospective studies on how selection parameters and schedules shape adaptation.Continuous evolution has emerged as a powerful paradigm for the evolution of proteins and enzymes 1-3 towards challenging functions 4,5 . In contrast to classical directed evolution approaches that rely on stepwise rounds of ex vivo mutagenesis, transformation into cells, and selection 6 , continuous evolution systems achieve rapid diversification and functional selection autonomously, often through in vivo targeted mutagenesis systems (Fig. 1a). The result is a mode of directed evolution that requires only the basic culturing of cells, in theory, enabling extensive speed, scale, and depth in evolutionary search 3 . In practice, however, developing a continuous evolution method that realizes all three properties has been challenging.Recently, our groups made two independent advances that can pair to achieve continuous evolution at significant speed, scale, and depth. These advances are OrthoRep and eVOLVER. First, OrthoRep. OrthoRep is an engineered genetic system for continuous in vivo targeted mutagenesis of genes of interest (GOIs) 2,7 . OrthoRep uses a highly error-prone, orthogonal DNA polymerase-plasmid pair in yeast that replicates GOIs at a mutation rate of 10 -5 substitutions per base (spb) without increasing the genomic mutation rate of 10 -10 spb (Fig. 1a). This ~100,000-fold increase in the mutation rate of GOIs drives their accelerated evolution (speed). Because the OrthoRep system functions entirely in vivo and culturing yeast is straightforward, independent GOI evolution experiments can be carried out in high-throughput (scale). In addition, long multimutation pathways can be traversed using OrthoRep, owing to the durability of mutagenesis over many generations (depth). However, to practically realize depth in evolutionary search, in vivo

Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study

“…A particular advantage of continuous directed evolution is its scalability, which allows exploration of many trajectories through a fitness landscape simultaneously (which is challenging with classical directed evolution, see above). Some continuous directed evolution platforms are also compatible with automation, which furthers scalability and industrialization [30].…”

“…The~10 5 -fold mutational acceleration, combined with serial passaging, enables rapid, continuous evolution of the target gene entirely in vivo. OrthoRep is compatible with high-throughput, automated systems, including some that can impose diverse and adjustable selection pressures to help cross fitness valleys [30]. Fortunately, continuous directed evolution systems suitable for metabolic enzyme improvement have now been introduced.…”

“…To date, OrthoRep and EvolvR have only been used to evolve microbial enzymes, and for just two types of applications (antibiotic resistance and thermal adaptation) [28,30,33]. Furthermore, particularly for OrthoRep, the target gene plasmid construct is tailor-made and cannot be easily repurposed for different genes of interest.…”

Scalable, continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities

“…40 Multiple 24 hour periods were required for each experiment, but empty controls were included in each individual 96-well plate to ensure validity of growth in other cultures. Raw OD600 measurements were fed into a custom MATLAB script, 18 which carries out a logarithmic transformation to linearize the exponential growth phase, identifies this growth phase, and uses this to calculate the doubling time (T). Doubling time was then converted to growth rate by the equation ln(2)/T.…”

“…By reducing the manual stages of classical directed evolution down to a continuous process where cycles of diversification and selection occur autonomously in vivo, OrthoRep readily accesses depth and scale in evolutionary search. 16,18 Here, we apply OrthoRep to the evolution of the Thermotoga maritima tryptophan synthase β-subunit (TmTrpB) in multiple independent continuous evolution experiments, each carried out for at least 100 generations. While we only pressured TmTrpB to improve its primary activity of coupling indole and serine to produce tryptophan, the large number of independent evolution experiments we ran (scale) and the high degree of adaptation in each experiment (depth) resulted in a panel of variants encompassing expanded promiscuous activity with indole analogs.…”