Optimization and Scale-up of a Bioreduction Process for Preparation of Ethyl ( S )-4-Chloro-3-hydroxybutanoate

Ethyl 4-chloro-3-oxobutanoate (CUBE) was asymmetrically reduced with Escherichia coli cells expressing a reductase (ScCR) from Streptomyces coelicolor to afford enantiopure ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE], which is an important precursor for preparing the drug atorvastatin. The substrate load was fixed at 100 g/L, and the concentration of coenzyme NAD(+) was limited to 0.1 mM based on cost considerations. Under these conditions, the other reaction parameters were optimized as 25 degrees C and pH 6.5, with a biocatalyst dose of 10 kU/L in the presence of isopropanol (1.5 equiv of CUBE), which acted as a cosubstrate for regenerating NADH. The reaction was performed in a toluene aqueous biphasic system (1:1, v/ v), with agitation at the maximal linear rate of 0.88 m/s. Finally, the bioreaction was performed on a pilot scale using a SO L thermostated stirred-tank-reactor, affording (S)-CHBE in 85.4% yield and 99.9% ee, and a total turnover number (TTN) of 6060 for the cofactor NAD(+). The specific production was calculated to be 36.8 g(product)/g(dcw), which is the highest value reported to date among the whole-cell-mediated processes for producing (S)-CHBE.