Enzyme identification and development of a whole‐cell biotransformation for asymmetric reduction of o ‐chloroacetophenone

Chiral 1‐( o ‐chlorophenyl)‐ethanols are key intermediates in the synthesis of chemotherapeutic substances. Enantioselective reduction of o ‐chloroacetophenone is a preferred method of production but well investigated chemo‐ and biocatalysts for this transformation are currently lacking. Based on the discovery that Candida tenuis xylose reductase converts o ‐chloroacetophenone with useful specificity ( k cat / K m  = 340 M −1  s −1 ) and perfect S ‐stereoselectivity, we developed whole‐cell catalysts from Escherichia coli and Saccharomyces cerevisiae co‐expressing recombinant reductase and a suitable system for recycling of NADH. E. coli surpassed S. cerevisiae sixfold concerning catalytic productivity (3 mmol/g dry cells/h) and total turnover number (1.5 mmol substrate/g dry cells). o ‐Chloroacetophenone was unexpectedly “toxic,” and catalyst half‐life times of only 20 min ( E. coli ) and 30 min ( S. cerevisiae ) in the presence of 100 mM substrate restricted the time of batch processing to maximally ∼5 h. Systematic reaction optimization was used to enhance the product yield (≤60%) of E. coli catalyzed conversion of 100 mM o ‐chloroacetophenone which was clearly limited by catalyst instability. Supplementation of external NAD + (0.5 mM) to cells permeabilized with polymyxin B sulfate (0.14 mM) resulted in complete conversion providing 98 mM S ‐1‐( o ‐chlorophenyl)‐ethanol. The strategies considered for optimization of reduction rate should be generally useful, however, especially under process conditions that promote fast loss of catalyst activity. Biotechnol. Bioeng. 2011; 108:797–803. © 2010 Wiley Periodicals, Inc.