The last strategy required the gene encoding the enzyme and an efficient expression system. Fortunately, our lab had a gene encoding a PFE from Pseudomonas fluorescens, which was expressed well in E. coli using a rhamnose-inducible promoter. Since the structure of PFE is not known, positions for site-directed mutagenesis are difficult to predict. Alternatively, a random mutagenesis of the gene combined with an assay system also allows detection of the desired variants.
Recent methods are described for the directed evolution of enzymes by random mutagenesis using error-prone PCR or DNA-shuffling.5,6 For both of these methods, ligation of the PCR products is critical. In addition, the mutation bias must be optimized. We chose to use the mutator strain, Epicurian Coli XL1-Red (lacking DNA repair mechanisms), which should result in random mutations generated within a clone of interest.7
To identify the desired variants from the numerous clones produced by random mutagenesis, we supplemented agar plates containing E. coli colonies with rhamnose, substrate 1, and indicators. A color change, caused by the release of substrate 1s corresponding acid, indicated hydrolysis. To further select for the mutants, the corresponding glycerol ester 2 was introduced. When this ester is hydrolyzed, the carbon source glycerol is released, thereby speeding up bacterial growth and producing active esterases on minimal media. With this strategy, we identified an esterase variant capable of stereoselectively hydrolysing the sterically-hindered 3-hydroxy ester 1.8 This compound can serve as a building block in the synthesis of Epothilones, a new class of macrolides showing taxol-like biological activity.9