Automated continuous evolution of proteinsin vivo

Abstract

<jats:title>Abstract</jats:title><jats:p>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 <jats:italic>in vivo</jats:italic>, 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 multi-mutation 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.</jats:p>

Potential For Applying Continuous Directed Evolution To Plant Enzymes

“…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 [29].…”

“…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 highthroughput, automated systems, including some that can impose diverse and adjustable selection pressures to help cross fitness valleys [29].…”

“…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) [27,29,32]. Furthermore, particularly for OrthoRep, the target gene plasmid construct is tailor-made and cannot be easily repurposed for different genes of interest.…”

A high-throughput platform for feedback-controlled directed evolution

“…A key distinguishing feature of PRANCE from other automated evolution platforms [20][21][22] is that it enables real-time monitoring of not only bacterial density, but also fluorescence and luminescence within each lagoon. To do so, our method samples the lagoons in discrete intervals, measures these samples in a plate reader, and then either disposes of or preserves the samples for downstream analyses such as plaque assays, sequencing, or other activity measurements of the evolved biomolecule ( Fig.…”

“…19 As such, much effort has been spent developing new multiplexed evolution platforms. [20][21][22] To comprehensively address these challenges, we developed phage-androbotics-assisted near-continuous evolution (PRANCE), an automated platform for high-throughput directed evolution capable of activitydependent feedback control. Whereas traditional phage-assisted continuous evolution (PACE) houses a single evolving population of M13 bacteriophage in a sealed flask, 6 referred to as a "lagoon," PRANCE features individual evolving populations contained in the wells of a 96-well plate, enabling highly multiplexed evolution.…”