Integration of enzyme, strain and reaction engineering to overcome limitations of baker's yeast in the asymmetric reduction of α‐keto esters

Abstract

We report on the development of a whole-cell biocatalytic system based on the popular host Saccharomyces cerevisiae that shows programmable performance and good atom economy in the reduction of alpha-keto ester substrates. The NADPH-dependent yeast reductase background was suppressed through the combined effects of overexpression of a biosynthetic NADH-active reductase (xylose reductase from Candida tenuis) to the highest possible level and the use of anaerobic reaction conditions in the presence of an ethanol co-substrate where mainly NADH is recycled. The presented multi-level engineering approach leads to significant improvements in product optical purity along with increases in the efficiency of alpha-keto ester reduction and co-substrate yield (molar ratio of formed alpha-hydroxy ester to consumed ethanol). The corresponding alpha-hydroxy esters were obtained in useful yields (>50%) with purities of > or =99.4% enantiomeric excess. The obtained co-substrate yield reached values of greater than 1.0 with acetate as the only by-product formed.

The Aldo-Keto Reductase Superfamily and its Role in Drug Metabolism and Detoxification

“…Aldehdye reductase activity is also readily measurable in insects (Drosophila) and yeast, and the enzymes from each species showed molecular weight between 30 and 40 kDa (Davidson et al, 1978). Most studies on AKRs have been performed on mammalian proteins with the exception of xylose reductase from yeast (AKR2B), which has potential biotechnological applications such as xylose fermentation to ethanol and organic synthesis (Kratzer et al, 2008;Nidetzky et al, 1996). The proteins encoded by Akr genes catalyze a variety of metabolic oxidation-reduction reactions ranging from the reduction of glucose, glucocorticoids and small carbonyl metabolites to glutathione conjugates and phospholipid aldehydes.…”

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

“…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). All strains were cultivated using the conditions described in the given references.…”

“…Ethanol was added as required to obtain a homogeneous liquid phase, cells were then removed by centrifugation and the supernatant was used for further analysis. Bioreductions catalyzed by S. cerevisiae XR2m were carried out exactly as described previously (Kratzer et al, 2008a). A preparative synthesis of S-1-(o-chlorophenyl)-ethanol was performed using 40 g CDW /L of E. coli XR_FDH for the reduction of 100 mM o-chloroacetophenone in a total volume of 10 mL.…”

“…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.…”

Integration of newly isolated biocatalyst and resin-based in situ product removal technique for the asymmetric synthesis of (R)-methyl mandelate

“…Whole cells are favored for these reactions owing to the internal cofactor regeneration and the easy product isolation [16]. Recently, various microorganisms have been used to catalyze the enantioselective reduction of benzoylformates or their substituted derivatives to the corresponding (R)-isomers with high optical purity and moderate to high yield, which including Saccharomyces cerevisiae [17][18][19], Geotrichum candidum [20], Bacillus pumilus [21] and recombinant Escherichia coli [22,23]. However, weaknesses frequently met were the limited substrate concentration and the long reaction time.…”