Reductive enzymatic dynamic kinetic resolution affording 115 g/L (S)-2-phenylpropanol

Abstract

Background: Published biocatalytic routes for accessing enantiopure 2-phenylpropanol using oxidoreductases afforded maximal product titers of only 80 mM. Enzyme deactivation was identified as the major limitation and was attributed to adduct formation of the aldehyde substrate with amino acid residues of the reductase.

Results: A single point mutant of Candida tenuis xylose reductase (CtXR D51A) with very high catalytic efficiency (43·10³ s^-1 M^-1) for (S)-2-phenylpropanal was found. The enzyme showed high enantioselectivity for the (S)-enantiomer but was deactivated by 0.5 mM substrate within 2 h. A whole-cell biocatalyst expressing the engineered reductase and a yeast formate dehydrogenase for NADH-recycling provided substantial stabilization of the reductase. The relatively slow in situ racemization of 2-phenylpropanal and the still limited biocatalyst stability required a subtle adjustment of the substrate-to-catalyst ratio. A value of 3.4 g_substrate/g_{cell-dry-weight} was selected as a suitable compromise between product ee and the conversion ratio. A catalyst loading of 40 g_{cell-dry-weight} was used to convert 1 M racemic 2-phenylpropanal into 843 mM (115 g/L) (S)-phenylpropanol with 93.1% ee.

Conclusion: The current industrial production of profenols mainly relies on hydrolases. The bioreduction route established here represents an alternative method for the production of profenols that is competitive with hydrolase-catalyzed kinetic resolutions.

Keywords: Aldo–keto reductase engineering; Biocatalyst stability; Enantiopure 2-aryl-1-propanol; Reductive dynamic kinetic resolution.

Fig. 1

Reductive enzymatic dynamic kinetic resolution of racemic 2-phenylpropanal

Fig. 2

Conversions and product enantiopurities for the reduction of 100 mM racemic 2-phenylpropanal using a lyophilized and rehydrated whole-cell catalyst. The effects of catalyst loading on product concentration (mM, bars) and product ee-value (%, crosses) were studied. The NAD⁺ concentration was 6 mM and the reaction time was 24 h. (The details are summarized in the Supplementary data, Table S1)

Fig. 3

Active site of wild-type CtXR with NAD⁺ (PDB 1MI3, [33]) and modelled substrates. A Xylose (blue carbons, red oxygens), B (S)- and (R)-2-phenylpropanal (S-enantiomer yellow carbons, R-enantiomer brown carbons, red oxygens), C (S)-2-phenylpropanal (yellow carbons, red oxygen), D (R)-2-phenylpropanal (brown carbons, red oxygen). Possible hydrogen bonds between substrates and the enzyme are shown as dashed lines, with distances in Å

Fig. 4

Effect of the substrate-to-biocatalyst ratio (whole-cell biocatalyst) on product enantiopurity. Blue diamonds show the conversion of 100 mM rac-2-phenylpropanal with 4 and 10 g_CDW biocatalyst, green diamonds with 1 M substrate and 40 g_CDW, red diamonds with 1 M substrate and 20 g_CDW, black crosses with 2 M substrate and 40 g_CDW. (Data with error bars from reactions with 6 mM NAD⁺ are depicted. See also the section Optimization of 2-phenylpropanal bioreduction, Table 3)