DIRECTED EVOLUTION OF AN ESTERASE FOR THE STEREOSELECTIVE RESOLUTION OF A KEY INTERMEDIATE IN THE SYNTHESIS OF EPOTHILONES

The directed evolution of an esterase from Pseudomonas fluorescens usi ng the mutator strain Epicurian coli XL1-Red was investigated. Mutants were assayed for their ability to hydrolyze a sterically hindered 3-h ydroxy ester, which can serve as a building block in the synthesis of epothilones. Screening was performed by plating esterase producing col onies derived from mutation cycles onto minimal media agar plates cont aining indicator substances (neutral red and crystal violet). Esterase -catalyzed hydrolysis of the 3-hydroxy ester (ethyl or glycerol ester) was detected by the formation of a red color due to a pH decrease cau sed by the released acid. Esterases isolated from positive clones were used in preparative biotransformations of the ethyl ester. One varian t containing two mutations (A209D and L181V) stereoselectively hydroly zed the ethyl ester resulting in 25% ee for the remaining ester. (C) 1 998 John Wiley & Sons, Inc.