Directed evolution goals

Directed evolution methods, as well as rational structure-based mutagenesis approaches, have been successful in broadening the substrate tolerance of aldolases. A common goal is to... 

Prerequisites to this approach are methods of incorporating non-natural moieties at predetermined positions in the biopolymer. This volume is meant to serve as a source in this respect describing the state of the art of some major lines of attack with this goal in mind. As a modern alternative, the creation of novel catalysts by directed evolution of nucleic acid aptamers is included. In this case, too, it is of prime importance to learn about the structural details which cooperate to bring about the catalytic function underlying the selection process. 

Thus, because the test can be carried out on 96-well microtiter plates, high throughput is possible. Of course, the inherent disadvantage noted above for some of the colorimetric tests also applies here, namely, the fact that the optimization of a potential catalyst is focused on a specific substrate 11 modified by incorporation of a probe, in this case the fluorogenic moiety 14. However, one can expect the test to be useful in directed evolution projects in which proof of principle is the goal. Moreover, this kind of approach can be used in very practical applications, namely as a pre-test for the activity of enzymes. 

However, efficient directed evolution is not a matter of generating huge libraries which then require considerable efforts in screening for the desired property. The goal is to create a maximum in structural diversity while minimizing the size of the libraries (17,23,24,37). 

Therefore tailoring the enzyme to the processing conditions would be preferred recent advances in random mutagenesis and directed evolution, notable features conceptually illustrated in Figure 11.3, offer the possibility of achieving this goal in the absence of detailed structural information on the protein. 

All these approaches have been used to alter protein function, to increase the activity or solubility of proteins, or to adapt enzymes for industrial applications. The goal of artificial man-made proteins with tailor-made activities is, however, still far away and none of the currently existing approaches provides the ultimate solution to the directed evolution of proteins. Nevertheless, numerous examples of successfully altered and improved proteins clearly show the power of directed evolution for protein design. 

Directed evolution has previously been used to generate enzyme-variants displaying improved properties such as higher activity and stability [11 - 17,19,81,91,92], The work summarized in this chapter shows that the difficult goal of controlling enantios-electivity of enzymes can also be reached by directed evolution [8 - 10]. In doing so, two major challenges had to be dealt with ... 

A major goal of directed evolution of DNA polymerases has been to elucidate the structural elements that confer high fidelity during DNA replication. If DNA polymerases were to rely solely on the stability of nucleotides that aligned with template for discrimination of correct template-directed polymerization, the error frequency would be in the order of one mispaired nucleotide per 100 incorporated [23], The measured error rate for incorporation and extension of a mismatched nucleotide attributable to DNA polymerases lacking an error correcting exonucleolytic activity range... 

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. 

When improved catalytic activity is the goal of the directed evolution process every variant is often screened following spatial distribution (e.g., single bacterial colonies in microtiter wells), and this generally involves the measurement of an optical change in each well. This allows the screening of 102-106 variants, and is a reliable but labor-intensive method. 

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