Directed evolution drives the next generation of biocatalysts

Abstract

Enzymes are increasingly being used as biocatalysts in the generation of products that have until now been derived using traditional chemical processes. Such products range from pharmaceutical and agrochemical building blocks to fine and bulk chemicals and, more recently, components of biofuels. For a biocatalyst to be effective in an industrial process, it must be subjected to improvement and optimization, and in this respect the directed evolution of enzymes has emerged as a powerful enabling technology. Directed evolution involves repeated rounds of (i) random gene library generation, (ii) expression of genes in a suitable host and (iii) screening of libraries of variant enzymes for the property of interest. Both in vitro screening-based methods and in vivo selection-based methods have been applied to the evolution of enzyme function and properties. Significant developments have occurred recently, particularly with respect to library design, screening methodology, applications in synthetic transformations and strategies for the generation of new enzyme function.

Installing hydrolytic activity into a completely de novo protein framework

“…43,44 We envisage improving the efficiency and selectivity of our designs in the future through directed evolution of residues close to the active site, 12 and/or via the precise installation of a surrogate for the oxyanion hole to stabilize the tetrahedral intermediates. 19 In conclusion, this is the first report of catalytically active Cys-His-Glu triads being installed into a completely de novo protein framework.…”

“…Often only moderate efficiencies are achieved, although these can be further improved by directed evolution. [12][13][14][15][16] Computational approaches have also been used to design esterases, again with moderate activities being achieved through the placement of a histidine (His) residue into the active site of thioredoxin, 17 the C-terminal domain of calmodulin 18 and cysteine-His (Cys-His) dyads installed into existing / protein folds. 19 By contrast, much less has been demonstrated for the design of completely de novo catalysts; that is, where both the protein scaffold and the catalytic activity are built from scratch.…”

Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention

“…[1][2][3] For instance, intermolecular interactions can lead to significant aggregation and subsequent precipitation, a phenomenon which is difficult to predict. Directed evolution [4][5][6][7][8][9] offers an alternative method for improving the robustness of proteins in biotechnology, 10 with error-prone polymerase chain reaction (epPCR) and/or DNA shuffling being the most popular gene mutagenesis techniques in this endeavor. In many cases this approach is superior to rational design, although at the expense of having to screen large mutant libraries, which is still considered to be the bottleneck of directed evolution in general.…”

Directed evolution combined with synthetic biology strategies expedite semi-rational engineering of genes and genomes

“…2 Therefore, natural enzymes always need to be engineered to possess the desirable catalytic properties that are required for practical applications. 3 In this regard, many directed evolution techniques such as error-prone PCR, site-directed saturation mutagenesis, iterative saturation mutagenesis, and DNA shuffling have been developed and widely used to optimize many catalytic parameters including thermostability, activity, substrate specificity, and enantioselectivity in artificial environments. 4,5 In addition, the construction of robust cell factories for whole-cell biocatalysis or de novo synthesis of the target products with an optimized background is also attractive and indispensable.…”