Mutagenesis and DNA shuffling

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. 

Figure 11.3 Evolution of enzymes with random mutagenesis and DNA shuffling (adapted from Arnold, 1996).

The directed evolution of proteins by random mutagenesis and DNA shuffling has proven extremely powerful to modify and optimize functional and physicochemical properties of existing proteins [6, 79 - 85] (for an up-dated list on targets see http //www.che.caltech.edu / groups / fha/Enzyme/directed.html). 

Normal sequential error-prone PCR mutagenesis and DNA shuffling can not efficiently recombine or dissect two or more mutations if they are very close to each other [18]. In contrast, the Walk-Through approach allows recombination to occur at every position of templates, and therefore provides the possibihty of recombining or dissecting two or more mutations, although they may be very close to each other. 

Pseudomonas aeruginosa lipase-catalyzed hydrolysis of racemic ester 23 proceeds with very low enantioselectivity E = 1.1). Sequential use of error-prone PCR, saturation mutagenesis at chosen spots and DNA shuffling resulted in the formation of a mutant whose enantioselectivity was over 50. 

In contrast with error-prone PCR and DNA shuffling, saturation mutagenesis enables randomization of a particular residue (or several residues) in the protein of interest with any of the 20 natural amino acids [4]. In this process, however, additional information is required to determine interesting positions for randomization. 

Parikh, M.R. and Matyoumara, I., Site-saturation mutagenesis is more efficient than DNA shuffling for the directed evolution of / -fucosidase. J. Mol Biol, 2005, 352, 621-628. 

To date, an impressive number of gene mutagenesis methods are available for application in directed evolution 17-20,36,37). However, it is currently not clear how they compare in terms of efficiency and ease of performance. It is also not obvious when and how to apply a given method in a directed evolution project 36-38). The fact that a few of the methods constitute proprietary intellectual property, such as DNA shuffling, poses a different kind of problem for potential users in industry. Some of the most important gene mutagenesis methods are described briefly here (for complete coverage, the reader is referred to recent reviews 17-20,36,37). 

The sequence of chapters mirrors the steps in a standard directed-evolution experiment. In the beginning, various methods for the creation of molecular diversity are considered. S. Brakmann and B.F. Lindemann (Chapter 2) present protocols for the generation of mutant libraries by random mutagenesis. Two chapters deal with the particularly powerful approach of in-vitro recombination. H. Suenaga, M. Goto, and K. Furukawa (Chapter 3) describe the application of DNA shuffling, and M. Ninkovic (Chapter 4) presents DNA recombination by the S tEP method. 

---