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

Abstract

BackgroundWhole 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 (CtXR) 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 CtXR and formate dehydrogenase from Candida boidinii (CbFDH). 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 CtXR.ResultsGene co-expression from a pETDuet-1 vector yielded about 260 and 90 units of intracellular CtXR and CbFDH activity per gram of dry E. coli cell mass (gCDW). The maximum conversion rate (rS) for ethyl 4-cyanobenzoylformate by intact or polymyxin B sulphate-permeabilized cells was similar (2 mmol/gCDWh), suggesting that the activity of CbFDH was partly rate-limiting overall. Uncatalyzed ester hydrolysis in substrate as well as inactivation of CtXR and CbFDH 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 gCDW/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 CbFDH (410 U/gCDW). 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%.ConclusionA whole-cell system based on E. coli co-expressing CtXR and CbFDH is a powerful and surprisingly robust biocatalyst for the synthesis of ethyl R-4-cyanomandelate in high op...

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

“…Therefore, the FDH activity in E. coli XR_FDH was just one-fifth that of the reference strain, E. coli FDH. It was previously shown by us (Kratzer et al, 2008b) and others (Weckbecker and Hummel, 2004) that functional expression of the CbFDH gene in E. coli is markedly attenuated by co-expression of another (reductase) gene. XR activity in S. cerevisiae XR2 m was 91 U/g CDW and half of the appendant ADH activity.…”

“…E. coli_FDH is the JM109 strain containing plasmid pBTac1 that encodes CbFDH (Krahulec et al, 2008). E. coli XR_FDH is a BL21 (DE3) strain containing the plasmid vector pETDuet-1 used for co-expression of the genes encoding CtXR and CbFDH (Kratzer et al, 2008b). S. cerevisiae XR2m is the parent CEN.PK strain harboring the yeast 2m expression plasmid p426GPD that contains the gene for CtXR under control of the constitutive glyceraldehyde-3-phosphate dehydrogenase (TDH3) promoter (Kratzer et al, 2008a).…”

“…Experiments with E. coli cells were carried out as described previously (Kratzer et al, 2008b) with the exception that the pH in all reactions was 6.5. In reactions where a water-immiscible organic co-solvent was used, ochloroacetophenone was dissolved in the co-solvent first and added in a 1:1 volumetric ratio to the aqueous phase containing the cells.…”

“…Results of a reaction engineering study are reported in which two CtXR whole-cell preparations, previously developed in E. coli (Kratzer et al, 2008b) and S. cerevisiae (Kratzer et al, 2008a) host systems, were compared for o-chloroacetophenone reduction. A key task of reaction optimization was to (partly) overcome the pronounced ''toxicity'' of the hydrophobic substrate, a problem often encountered in the field of whole-cell biotransformations.…”

Direct bioelectrocatalysis at the interfaces by genetically engineered dehydrogenase

“…43 Furthermore, FDHs have been used for sensing applications to detect formate quantitatively as an important fermentation product of aerobic and anaerobic bacteria. 44,45 The capability to transfer electron pairs between specific substrates and NAD + molecules offers another interesting potential for FDHs as power bioelectronic devices. 46 The industrial importance of NAD + -dependent FDH has led researchers to develop different kinds of immobilisation strategies through a variety of support materials to efficiently enable the desired use of the enzyme.…”