Directed evolution has proven to be a successful strategy for the modification of enzyme properties. To date, the preferred experimental procedure has been to apply mutations or crossovers randomly throughout the gene. With the emergence of powerful computational methods, it has become possible to develop focused combinatorial searches, guided by computer algorithms. Here, we describe several c...

We present a systematic computational framework, eCodonOpt, for designing parental DNA sequences for directed evolution experiments through codon usage optimization. Given a set of homologous parental proteins to be recombined at the DNA level, the optimal DNA sequences encoding these proteins are sought for a given diversity objective. We find that the free energy of annealing between the reco...

It has been 40 years since Spiegelman and co-workers demonstrated how RNA molecules can be evolved in the test tube. This result established Darwinian evolution as a chemical process and paved the way for the many directed evolution experiments that followed. Chemists can benefit from reflecting on Spiegelman's studies and the subsequent advances, which have taken the field to the brink of the ...

Generating diverse protein libraries that contain improved variants at a sufficiently high frequency is critical for improving the properties of proteins using directed evolution. Many studies have illustrated how random mutagenesis, cassette mutagenesis, DNA shuffling and similar approaches are effective diversity generating methods for directed evolution. Very few studies have explored random...

Directed evolution, the laboratory process by which biological entities with desired traits are created through iterative rounds of genetic diversification and library screening or selection, has become one of the most useful and widespread tools in basic and applied biology. From its roots in classical strain engineering and adaptive evolution, modern directed evolution came of age twenty year...

Enzymes offer cheap, environmentally responsible and highly efficient alternatives to chemical catalysts. The past two decades have seen a significant rise in the use of enzymes in industrial settings. Although many natural enzymes have been modified through protein engineering to better suit practical applications, these approaches are often insufficient. A key goal of enzyme engineers is to b...

Despite rapid advances in genome engineering technologies, inserting genes into precise locations in the human genome remains an outstanding problem. It has been suggested that site-specific recombinases can be adapted towards use as transgene delivery vectors. The specificity of recombinases can be altered either with directed evolution or via fusions to modular DNA-binding domains. Unfortunat...

Any single-enzyme directed evolution strategy has two fundamental requirements: the need to efficiently introduce variation into a gene of interest and the need to create an effective library from those variants. Generation of a maximally diverse gene library is particularly important when employing nontargeted mutagenesis strategies such as error-prone PCR (epPCR), which seek to explore very l...

The de novo design of protein-protein interfaces is a stringent test of our understanding of the principles underlying protein-protein interactions and would enable unique approaches to biological and medical challenges. Here we describe a motif-based method to computationally design protein-protein complexes with native-like interface composition and interaction density. Using this method we d...