Advances in ultrahigh-throughput screening for directed enzyme evolution
Ulrich Markel, Khalil D. Essani, Volkan Besirlioglu, Johannes Schiffels, Wolfgang R. Streit and Ulrich Schwaneberg*, Chem. Soc. Rev., 2020, 49, 233-262. DOI:10.1039/C8CS00981C
Here, we review state-of-the-art and up-and-coming ultrahigh-throughput methods for the screening of large libraries in directed enzyme evolution.
Enzymes often need to be re-engineered or optimized in order to exploit their full potential. (Semi-) rational design requires detailed knowledge about structure function relationships. In turn, directed evolution methodologies can improve an enzyme’s properties without structural knowledge by iterative rounds of diversity generation and screening. Current diversity generation methods allow us to generate huge libraries but conventional screening on agar plates or in microtiter plates fails to interrogate the full generated diversity in reasonable time. Ultrahigh-throughput screening methods drastically increase the number of enzyme variants we can screen and speed up biocatalyst design ultimately widening our knowledge about sequence function relationships. In the present review, we summarize recent advances in ultrahigh-throughput screening for the directed evolution of enzymes. We discuss the importance of compartmentalization to link genotype and phenotype and illustrate how cells and biomimetic compartments can serve this function. Finally, we discuss how new functional metagenomics approaches can profit from ultrahigh-throughput screening to identify natural biocatalysts for novel chemical transformations.