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Background  Whole cell-catalyzed biotransformation is a clear process option for the production of chiral alcohols via enantioselective reduction of precursor ketones. A wide variety of synthetically useful reductases are expressed heterologously in  Escherichia coli  to a high level of activity. Therefore, this microbe has become a prime system for carrying out whole-cell bioreductions at different scales. The limited capacity of central metabolic pathways in  E. coli  usually requires that reductase coenzyme in the form of NADPH or NADH be regenerated through a suitable oxidation reaction catalyzed by a second NADP +  or NAD +  dependent dehydrogenase that is co-expressed.  Candida tenuis  xylose reductase ( Ct XR) was previously shown to promote NADH dependent reduction of aromatic α-keto esters with high Prelog-type stereoselectivity. We describe here the development of a new whole-cell biocatalyst that is based on an  E. coli  strain co-expressing  Ct XR and formate dehydrogenase from  Candida boidinii  ( Cb FDH). The bacterial system was evaluated for the synthesis of ethyl  R -4-cyanomandelate under different process conditions and benchmarked against a previously described catalyst derived from  Saccharomyces cerevisiae  expressing  Ct XR.    Results  Gene co-expression from a pETDuet-1 vector yielded about 260 and 90 units of intracellular  Ct XR and  Cb FDH activity per gram of dry  E. coli  cell mass (g CDW ). The maximum conversion rate ( r  S ) for ethyl 4-cyanobenzoylformate by intact or polymyxin B sulphate-permeabilized cells was similar (2 mmol/g CDW h), suggesting that the activity of  Cb FDH was partly rate-limiting overall. Uncatalyzed ester hydrolysis in substrate as well as inactivation of  Ct XR and  Cb FDH in the presence of the α-keto ester constituted major restrictions to the yield of alcohol product. Using optimized reaction conditions (100 mM substrate; 40 g CDW /L), we obtained ethyl  R -4-cyanomandelate with an enantiomeric excess (e.e.) of 97.2% in a yield of 82%. By increasing the substrate concentration to 500 mM, the e.e. could be enhanced to ≅100%, however, at the cost of a 3-fold decreased yield. A recombinant strain of  S. cerevisiae  converted 100 mM substrate to 45 mM ethyl  R -4-cyanomandelate with an e.e. of ≥ 99.9%. Modifications to the recombinant  E. coli  (cell permeabilisation; addition of exogenous NAD + ) and addition of a water immiscible solvent (e.g. hexane or 1-butyl-3-methylimidazolium hexafluorophosphate) were not useful. To enhance the overall capacity for NADH regeneration in the system, we supplemented the original biocatalyst after permeabilisation with also permeabilised  E. coli  cells that expressed solely  Cb FDH (410 U/g CDW ). The positive effect on yield (18% → 62%; 100 mM substrate) caused by a change in the ratio of FDH to XR activity from 2 to 20 was invalidated by a corresponding loss in product enantiomeric purity from 86% to only 71%.    Conclusion  A whole-cell system based on  E. coli  co-expressing  Ct XR and  Cb FDH is a powerful and surprisingly robust biocatalyst for the synthesis of ethyl  R -4-cyanomandelate in high optical purity and yield. A clear requirement for further optimization of the specific productivity of the biocatalyst is to remove the kinetic bottleneck of NADH regeneration through enhancement (≥ 10-fold) of the intracellular level of FDH activity.

microbial cell factories

Whole-cell bioreduction of aromatic α-keto esters using Candida tenuis xylose reductase and Candida boidinii formate dehydrogenase co-expressed in Escherichia coli