Abstract:AbstractWe 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 … Show more
“…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].…”
Section: Continuous Directed Evolutionmentioning
confidence: 99%
“…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].…”
Section: Orthorepmentioning
confidence: 99%
“…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.…”
Section: Limitations Of Continuous Directed Evolution Systemsmentioning
SUMMARYPlant evolution has produced enzymes that may not be optimal for maximizing yield and quality in today’s agricultural environments and plant biotechnology applications. By improving enzyme performance, it should be possible to alleviate constraints on yield and quality currently imposed by kinetic properties or enzyme instability. Enzymes can be optimized faster than naturally possible by applying directed evolution, which entails mutating a target gene in vitro and screening or selecting the mutated gene products for the desired characteristics. Continuous directed evolution is a more efficient and scalable version that accomplishes the mutagenesis and selection steps simultaneously in vivo via error-prone replication of the target gene and coupling of the host cell’s growth rate to the target gene’s function. However, published continuous systems require custom plasmid assembly, and convenient multipurpose platforms are not available. We discuss two systems suitable for continuous directed evolution of enzymes, OrthoRep in Saccharomyces cerevisiae and EvolvR in Escherichia coli, and our pilot efforts to adapt each system for high-throughput plant enzyme engineering. To test our modified systems, we used the thiamin synthesis enzyme THI4, previously identified as a prime candidate for improvement. Our adapted OrthoRep system shows promise for efficient plant enzyme engineering.
“…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].…”
Section: Continuous Directed Evolutionmentioning
confidence: 99%
“…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].…”
Section: Orthorepmentioning
confidence: 99%
“…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.…”
Section: Limitations Of Continuous Directed Evolution Systemsmentioning
SUMMARYPlant evolution has produced enzymes that may not be optimal for maximizing yield and quality in today’s agricultural environments and plant biotechnology applications. By improving enzyme performance, it should be possible to alleviate constraints on yield and quality currently imposed by kinetic properties or enzyme instability. Enzymes can be optimized faster than naturally possible by applying directed evolution, which entails mutating a target gene in vitro and screening or selecting the mutated gene products for the desired characteristics. Continuous directed evolution is a more efficient and scalable version that accomplishes the mutagenesis and selection steps simultaneously in vivo via error-prone replication of the target gene and coupling of the host cell’s growth rate to the target gene’s function. However, published continuous systems require custom plasmid assembly, and convenient multipurpose platforms are not available. We discuss two systems suitable for continuous directed evolution of enzymes, OrthoRep in Saccharomyces cerevisiae and EvolvR in Escherichia coli, and our pilot efforts to adapt each system for high-throughput plant enzyme engineering. To test our modified systems, we used the thiamin synthesis enzyme THI4, previously identified as a prime candidate for improvement. Our adapted OrthoRep system shows promise for efficient plant enzyme engineering.
“…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.…”
Section: Development Of An Automated Evolution Platformmentioning
confidence: 99%
“…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.…”
Continuous directed evolution rapidly implements cycles of mutagenesis, selection, and replication to accelerate protein engineering. However, individual experiments are typically cumbersome, reagent-intensive, and require manual readjustment, limiting the number of evolutionary trajectories that can be explored. We report the design and validation of Phage-and-Robotics-Assisted Near-Continuous Evolution (PRANCE), an automation platform for the continuous directed evolution of biomolecules that enables real-time activitydependent reporter and absorbance monitoring of up to 96 parallel evolution experiments. We use this platform to characterize the evolutionary stochasticity of T7 RNA polymerase evolution, conserve precious reagents with miniaturized evolution volumes during evolution of aminoacyl-tRNA synthetases, and perform a massively parallel evolution of diverse candidate quadruplet tRNAs. Finally, we implement a feedback control system that autonomously modifies the selection strength in response to real-time fitness measurements. By addressing many of the limitations of previous methods within a single platform, PRANCE simultaneously enables multiplexed, miniaturized, and feedback-controlled continuous directed evolution. from the National Cancer Institute (F32 CA247274-01).
Directed
evolution aims to expedite the natural evolution process
of biological molecules and systems in a test tube through iterative
rounds of gene diversifications and library screening/selection. It
has become one of the most powerful and widespread tools for engineering
improved or novel functions in proteins, metabolic pathways, and even
whole genomes. This review describes the commonly used gene diversification
strategies, screening/selection methods, and recently developed continuous
evolution strategies for directed evolution. Moreover, we highlight
some representative applications of directed evolution in engineering
nucleic acids, proteins, pathways, genetic circuits, viruses, and
whole cells. Finally, we discuss the challenges and future perspectives
in directed evolution.
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