Directed Evolution Fundamentals

Isotope labeling forms the basis of a different MS-based ee-assay (92,93). It involves a fundamentally new way to measure enantiopurity. The method has been parallelized for high throughput and used a number of times in the directed evolution of enantioselective enzymes (46,64,94-96). In this MS-based approach. 

The chapter by Reetz includes many examples and explanations of methods illustrating how directed evolution has been applied to prepare new enzyme catalysts. Directed evolution of enantioselective enzymes has emerged as a fundamentally new approach to asymmetric catalysis. It involves the combination... 

Directed evolution bypasses the bottleneck of rational design and mimics natural evolution in a test tube to evolve proteins without knowledge of their structures. What fundamentally differentiates directed evolution from natural evolution is its power to significantly accelerate the process of evolution. As shown in Fig. 1, directed evolution uses various methods to generate a collection of random protein variants, called a library, at the DNA level. Followed by screening/selection of the library, protein variants with improvement in desired phenotypes are obtained. Usually, the occurrence of these functionally improved protein variants is a rare event thus, this two-step procedure has to be iterated several rounds until the goal is achieved or no further improvement is possible. 

Directed evolution has been successfully used to alter existing enzyme properties and even to create novel enzyme functions. In addition to creating enzymes for specific industrial applications, directed evolution has also been increasingly used to address fundamental questions in biology, such as the evolutionary mechanisms of novel protein functions, protein structure-function relationship, and protein folding mecha-... 

A fundamentally new approach toward the development of better activities and new drugs that has been developed within the past few years is the concept of directed evolu tion of enzymes and pathways (74-76). The principle of directed evolution is to mimic the natural evolution process, but also to accelerate it in vitro, directing it toward an applied goal, such as an enhanced etuyinatic activity, broader or altered substrate specificity, or in the case of application of this principle to pathways, even completely new compounds. 

While CAM-6 is somewhat limited in its ability to perform large-scale simulations of physical systems (it is a much less capable system than its follow-on, the CAM-8, for example see discussion below), its fundamental historical importance cannot be overstated. CAM-6 allowed researchers to directly experience, for the first time and in real time, the evolution of CA systems theretofore undertsood only as purely conceptual models. Margolus and Toffoli recall that when Pomeau, one of... 

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