Shuffling mutagenesis

Figure 2.5 Scheme for DNA shuffling illustrated for the case in which the parent genes originate from the WT by some form of mutagenesis [7e],... 

The Bacillus subtilis lipase A (BSLA) was the subject of two short directed evolution studies [19,47]. In one case systematic saturation mutagenesis at all of the ISlpositions of BSLA was performed [19]. Using meso-l,4-diacetoxy-2-cyclopentene as the substrate, reversed enantioselectivity of up to 83% ee was observed. In another study synthetic shuffling (Assembly of Designed Oligonucleotides) was tested using BSLA [47]. 

Initial approaches to directed evolution of enzymes rested upon the introduction of random mutations in random sites of the enzyme by the use of the error-prone PCR technique [92] or on the DNA-shuffling method [93]. Extensive research has also been reported in which every amino acid site in an enzyme was systematically subjected to saturation mutagenesis [94]. 

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. 

Figure 3.1 Overview of DNA library creation strategies. Random mutagenesis introduces mutations at positions throughout the gene sequence. Semi-rational design randomizes only the specific position(s) of interest. Gene shuffling brings existing sequence diversity from different parental DNA sequences together to form a chimeric library...

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). 

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. 

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. 

A library of parent DNA sequences encoding for the desired protein is chosen. Sequence diversity is created or increased through a mutagenesis step, either by introduction of random point mutations through error-prone PCR or by recombination of DNA fragments such as DNA shuffling or RACHITT. 

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

Fig. 3. Evolutionary progression of catalytic efficiency of pNB esterase (towards LCN-pNB) in 15% DMF through four generations of random mutagenesis, followed by two rounds of recombination by DNA shuffling of circled populations.

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