Directed saturation mutagenesis

Cephalosporins are a class of antibiotic produced via the intermediate 7-aminocephalos-poranic acid (7-ACA), or 7-aminodesacetoxycephalosporanic acid (7-ADCA). Directed evolution has been used to improve the activity of cephalosporin acylases to produce these intermediates from adipyl-7-ACA or cephalosporin C [68]. Using site-directed saturation mutagenesis and a selection system whereby the E. coli host is dependent on leucine liberated from derivatives of the cephalosporin side-chains, a mutant was found that increased the catalytic efficiency toward adipyl-7-ADCA by 36-fold. 

Improvement of enzyme performance by mutation can be classified into two categories 1) rational mutation such as point mutation and site-directed saturation mutagenesis (change of one amino acid to all 20 naturally occurring amino acids at a specific site within a protein) and 2) random mutation such as directed evolution (change of amino acids at random to create a large library of mutants proteins followed by the selection of the proteins possessing the desired property the process is repeated as in nature). When the structure of the enzyme is not known or cannot be predicted, only random mutation can be performed. 

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]. 

Wong, T.S., Tee, K.L., Hauer, B. and Schwaneberg, U. (2004) Sequence saturation mutagenesis (SeSaM) a novel method for directed evolution. Nucleic Acids Research, 32, e26. 

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. 

J. M. Short, Saturation Mutagenesis in Directed Evolution, US patent 6,171,820, 2001. 

The improvement in enantioselectivity by the directed evolution of Pseudomonas aeruginosa lipase is shown in Figure 8(b).8 The combination of different mutagenesis methods (error-prone PCR and site-specific saturation mutagenesis) improved the enantioselectivity from E=l.l in wild type to E=25.8. 

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