Automated Continuous Evolution of Proteins in Vivo

ACS Synth Biol. 2020 Jun 19;9(6):1270-1276. doi: 10.1021/acssynbio.0c00135. Epub 2020 May 12.


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, multiparameter 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.

Keywords: OrthoRep; continuous culture; continuous evolution; eVOLVER; orthogonal replication.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Directed Molecular Evolution / methods*
  • Mutagenesis
  • Plasmodium falciparum / enzymology
  • Protozoan Proteins / antagonists & inhibitors
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism
  • Saccharomyces cerevisiae / metabolism*
  • Tetrahydrofolate Dehydrogenase / chemistry
  • Tetrahydrofolate Dehydrogenase / genetics
  • Tetrahydrofolate Dehydrogenase / metabolism
  • Thermotoga maritima / metabolism


  • Bacterial Proteins
  • Protozoan Proteins
  • Tetrahydrofolate Dehydrogenase