Directed evolution techniques

Combinatorial methods are often referred to as in vitro or directed evolution techniques. In nature, the random DNA mutations that lead to changes in protein sequences occur rarely and so evolution is usually a slow... 

Some researchers have begun to explore the possibihty of combining transition metal catalysts with a protein to generate novel synthetic chemzymes . The transition metal can potentially provide access to novel reaction chemistry with the protein providing the asymmetric environment required for stereoselective transformations. In a recent example from Reetz s group, directed evolution techniques were used to improve the enantioselectivity of a biotinylated metal catalyst linked to streptavidin (Scheme 2.19). The Asn49Val mutant of streptavidin was shown to catalyze the enantioselective hydrogenation of a-acetamidoacrylic acid ester 46 with moderate enantiomeric excess [21]. 

Recently, Turner et al. have shown tertiary amines can also be used as substrates by using a further variant of MAO-N (MAO-N-D5) which had also been developed by directed evolution techniques (Scheme 2.33). For example, racemic N-methyl-pyrrolidine 65 was subjected to deracemization, via the intermediate iminium ion... 

But there is still another point, not yet discussed but with considerable potential, which may also impact eventually on technical asymmetric catalysis. Even though biocatalysts are efficient, active, and selective, there still remains one big disadvantage At present, there is not yet an appropriate enzyme known or available for every given chemical reaction. It is estimated that about 25 000 enzymes exist in Nature, and 90% of these have still to be discovered [28, 29]. New biocatalysts are made available nowadays not only from screening known organism but also via screening metagenomic libraries and directed evolution techniques [30]. 

A combined approach of rational pathway assembly and directed evolution techniques opens the perspective for discovery and production of compounds that are either rare and hard to access in nature or even entirely non-natural, in simple laboratory organisms. 

Redesign of an Enzyme s Active Site KDPG Aldolase 331 11.5 Comparison of Directed Evolution Techniques 331... 

Just as nature uses evolution to naturally select enzyme variations that provide an advantage to the host, directed evolution of the amino acid substitution utilize techniques to screen or select for mutant enzymes that perform better than wild-type enzymes. Unlike site-directed mutagenesis, directed evolution techniques do not require a detailed understanding of the enzyme in order to identify useful variants. The techniques of applied molecular evolution however, do require a screening or selection step to identify the individual mutants of interest (Fig. 12.2). 

Fig. 8.10 X-ray crystal structure of an aspartate aminotransferase (AspAT) bound to its cofactor pyridoxal 5 -phosphate and aspartate. Directed evolution techniques produced changes in ligand specificity due to substitution of the disparate positions indicated. Coordinates from lART [25].

Interestingly, the arginine switch mechanism was first recognized when it was artificially induced in AAT. When AAT was mutated in six distinct positions, a substantial increase of activity with aromatic substrates was observed. The crystal structure of the engineered enzyme showed that the aromatic side chains could be accommodated at the active site as a result of R292 movement. A similar observation was made on an AAT mutant form whose substrate specificity was broadened using direct evolution techniques, in order to include branched chain and aromatic amino acids. 

Enzymes offer an environmentally benign alternative to chemical catalysts in many commercial and industrial applications. The expansion of their use depends on the development of new protein catalysts. This will imply the construction or identification of enzymes that have been genetically altered to improve their performance under defined, application-specific conditions. Directed evolution offers a fast and effective way of creating improved enzymes from relatively ineffective catalysts to commercially viable products by a variety of directed evolution techniques. 

Directed evolution techniques have been applied to Taq or other Stoffel Fragments to isolate mutants with improved function as previously reviewed. The first technique that will be discussed is a saeening technique called complementation. In addition, both phage display and compartmentalized self-replication (GSR) will be described. Loeb and co-workers have probed the mutability of Taq s active site by partially... 

Various examples were described, including whole cell-biocatalyzed reductions [4], especially employing baker s yeast [la,5]. However, there was always the problem that several active enzymes with different selectivities were present, although in particular cases excellent conversions and stereoselectivities were achieved. Later on, by directed evolution techniques [6], improvement of stereoselectivities and substrate specificities of enzymes was achieved, as well as several enzymatic properties (e.g., thermal stability) were modified. Moreover, large amounts of biocatalysts have been produced by fermentation of recombinant bacteria that express the gene, when their corresponding DNA has been doned. The drawback of the expensive cofactor recyding has also been overcome by the devdopment of effident techniques [7]. 

Further advancement of biocatalysis will require the use of directed evolution to bridge the functional gap between wild-type and desired biocatalyst properties. These studies underscore the power of directed evolution to create artificial enzymes derived from wild-type enzymes with the desired catalytic activity. Directed evolution techniques will continue to fulfill the promise of biocatalysis for industrial applications. 

A metabolic engineering approach [175] and directed evolution techniques [176] were evaluated to avoid side reactions, block degradative pathways, and enhance the desired reaction (conversion of indene to cxs-amino indanol 137 or ds-indanediol). Multiparameter flow cytometry was used to assess indene toxicity, and it was shown that concentrations up to 025 g/1 of indene (0.037g indene per gram dry cell wt.) in batch bioconversions did not influence reaction rate. Using this information, a single-phase... 

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