Methods for the directed evolution of proteins

Abstract

Directed evolution has proved to be an effective strategy for improving or altering the activity of biomolecules for industrial, research and therapeutic applications. The evolution of proteins in the laboratory requires methods for generating genetic diversity and for identifying protein variants with desired properties. This Review describes some of the tools used to diversify genes, as well as informative examples of screening and selection methods that identify or isolate evolved proteins. We highlight recent cases in which directed evolution generated enzymatic activities and substrate specificities not known to exist in nature.

High-throughput enzyme screening platform for the IPP-bypass mevalonate pathway for isopentenol production

“…A correlation emerged between 388 growth rates (and/or titers) and PMD activity when an observed turnover number was calculated 389 at 100 mM MVAP using the analytical expression for noncompetitive substrate inhibition, kobs 390 (Supplementary Figure S7). This turnover number (kobs) depends upon each mutant's kcat, KM, and 391 KI, perhaps yielding a more accurate assessment of the turnover conditions in vivo (Kang et In this regard, growth-based selection is powerful because it does not require extensive tests of an 400 individual design, and designs with the desirable activity are enriched if the activity is essential 401 for growth of the organism (Packer and Liu, 2015). In this study, we designed a growth-based 402 screening platform to improve PMD decarboxylation activity toward MVAP for isopentenol 403 production.…”

Are in vivo selections on the path to extinction?

“…There are many advantages to using microbial selections, such as their low cost and high throughput compared with the equivalent screening assays (Packer and Liu, 2015). However, their most relevant property is the provision of the genotype–phenotype linkage required for evolution (Colin et al ., 2015b; van Vliet et al ., 2015).…”

Molecular Modeling in Enzyme Design, Toward In Silico Guided Directed Evolution

“…Directed evolution (DE), has proven to be a powerful tool for adapting enzymes to wider applications [50][51][52][53].…”

“…Regrettably, our knowledge of enzyme activity is still incomplete which makes our attempts to modifying them often limited. Detailed understanding of the enzymatic structure/function relation is, however, not necessary in directed evolution, an alternative engineering technique based on massive mutations and selective evolution.Directed evolution (DE), has proven to be a powerful tool for adapting enzymes to wider applications [50][51][52][53].Briefly, in DE diversity is first created through mutagenesis or recombination, followed by screening for improvements in desired properties. One of the main advantages of DE is most certainly that it does not require a thorough understanding of structure/function relationships, unlike rational or de novo design.…”