Cassette mutagenesis

Finally, the region of accessible protein sequence space was extended by developing a modified version of Stemmer s combinatorial multiple-cassette mutagenesis (CMCM)... 

Figure 2.9 Schematic summary of the directed evolution of enantioselective lipase variants originating from the WT PAL used as catalysts in the hydrolytic kinetic resolution of ester rac-1. CMCM = Combinatorial multiple-cassette mutagenesis [8c,22],...

Using X-ray structure data, amino acids which were believed to affect the desired characteristics were identified. Using either site-directed mutagenesis or "cassette" mutagenesis (8) different amino acid substitutions were made in the subtilisin structural gene (2,9). 

If the slopes of the absorption/time curves differ considerably, a positive hit is indicated (i.e., an enantioselective lipase-variant has been identified) (16). Figure 5 shows two typical experimental plots, illustrating the presence of a non-selective lipase (top) and a hit (bottom) (16). As a consequence of the crudeness of the test, quantitative evaluation is not possible. Therefore, the hits need to be investigated separately in laboratory-scale reactions and evaluated quantitatively by conventional chiral GC. About 800 plots of this kind can easily be recorded per day. A total of 40 000 lipase-variants were generated by epPCR, saturation mutagenesis, cassette mutagenesis, and DNA shuffling and screened in the model reaction. 

Fig. 15. Extended combinatorial multiple-cassette mutagenesis in the evolution of an (S)-selective lipase-variant from P. aeruginosa as a catalyst in the kinetic resolution of 1 (A position 20 A position 161 A position 234 O position 53 position 180 position 272) (49).

Fig. 16. Binding pocket of the lipase from P. aeruginosa for the acid part of 2-methyldecanoic acid p-nitrophenyl ester showing the geometric position of amino acids 160-163 which were randomized in cassette mutagenesis (49).

Identification of hot regions empirically or by rational design and subsequent application of cassette mutagenesis. 

Boyer PL, Ferris AL, Hughes SH. Cassette mutagenesis of the reverse transcriptase of human immunodeficiency virus type 1. J Virol 1992 66 1031-1039. 

Subsequent directed evolution work on Pseudomonas aeruginosa demonstrates that protein sequence space with respect to enantioselectivity is best explored by a three-step procedure (Reetz, 2001) (i) generation of mutants by error-prone PCR at a high-mutation rate (ii) identification of hot regions and spots in the enzyme by error-prone PCR and substantiation by simplified combinatorial multiple-cassette mutagenesis (iii) extension of the process of combinatorial multiple-cassette mutagenesis to cover a defined region of protein sequence space. 

K. -E. Jaeger, Directed evolution of an enantioselective enzyme through combinatorial multiple-cassette mutagenesis, Angew. Chem. Inti Ed. 2001b, 40, 3589-3591. 

II) has also proven to be viable and is more convenient when using cassette mutagenesis to perform many manipulations at once. For example, a short linker modeled after the last module of the rapamycin PKS has been used to delete many of the (I-keto processing domains of DEBS [55]. Introduction of p-keto-processing activities, or gain-of-function mutagenesis, represents a more difficult... 

Because the peptide sequences are very short in such systems, few (if any) errors are introduced by the polymerase and even error-prone PCR techniques are not ideal for introducing sequence changes. Therefore, unless a cassette mutagenesis strategy is followed, the benefit of ribosome display for an evolutionary approach is not exploited with displayed peptides. What is exploited, however, is the possibility to use larger libraries than those possible with in vivo selection systems. 

Reidhaar-Olson JF, Sauer RT, Combinatorial cassette mutagenesis as a probe of the informational content of protein sequences, Science, 241 53-57, 1988. 

The starting point for further exploration of protein sequence space was the conjecture that Stemmer s method of Combinatorial Multiple Cassette Mutagenesis (CMCM) [74] can be applied in appropriately modified form. It is a special type of DNA-shuffling which can be used to generate mutant gene libraries in which cassettes composed of random or defined sequences and the wild-type are incorporated randomly. CMCM had been developed for use in the area of functional antibodies [74],... 

Fig. 11.16. Schematic representation of modified Combinatorial Multiple Cassette Mutagenesis of lipase genes [43],...

In the case of the most (S)-selective variant 1H8 observed so far, which was evolved by Combinatorial Multiple Cassette Mutagenesis [43] (see above), the analysis based on molecular modeling and molecular dynamics calculations has not been completed. However, based on the results of the sequencing experiments and the X-ray structure... 

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