Cover Picture

Abstract

The cover picture shows a schematic representation of the positive genetic selection of an error-prone T7 RNA polymerase (T7 RNAP) mutant (shown in green). A system of two compatible plasmids (blue) carrying a T7 RNAP mutant library (bottom left) or an inactive mutant of the gene coding for tetracycline resistance under the exclusive control of a T7 promoter (top) rewarded inaccurate transcription with the survival of bacteria (symbolized by the smileys in the flask). Following a single round of selection, the survivors expressed a T7 RNAP variant with a nucleotide substitution error rate at least 20-fold greater than that of the wild-type enzyme. The heterogeneity of RNA products synthesized by this enzyme was demonstrated in vitro, andÐmost impressivelyÐin vivo by employing the transcription of Green Fluorescent Protein (GFP): In contrast to the uniform distribution of fluorescence that was observed after transcription by the wild-type T7 RNAP (left inset diagram), transcription by the mutant enzyme induced large deviations from the standard fluorescence behavior (right inset diagram), which were attributed to contributions of GFP mutants with altered excitation wavelengths. More about this successful application of the principle of directed evolution can be found in the paper by S. Brakmann and S. Grzeszik on p. 212 ff.

Interactions between Physics and Biodisciplines within the Framework of Molecular Sciences

“…Stability may be sometimes enhanced by introducing an enzyme into a hydrogel matrix [7]. From this perspective effort to make progress on the road to synthetic enzymes appears promising [8]. Not only increasing enzyme stability is very topical, but increasing selectivity and catalytic efficiency is as well and can be achieved by that which is called "directed evolution" or "evolution engineering" [9].…”

Discovery of Superior Enzymes by Directed Molecular Evolution

“…Most often, this is achieved by genetic complementation of hosts that are deficient in a certain pathway or activity. In other cases, the positive feedback coupling between a property of interest and cell survival may be achieved with alteration of genetic contexts, for example, employing enzyme‐specific control elements like transcriptional promoters 42. Selective enrichment of only those clones that express the particular enzyme function can be very efficient.…”

“…The narrow range of substrates that are accepted by natural enzymes often retards or prevents their use in new synthetic and commercial applications. By far most results therefore reflect the efficient tuning of catalytic efficiency toward nonnatural substrates 34, 42, 43, 65, 68–88. Similarly, the enantioselectivity of specific bioconversions has been significantly improved by using evolutionary approaches 89–91.…”