Engineering the Activity of Old Yellow Enzyme NemR-PS for Efficient Reduction of (E/Z)-Citral to (S)-Citronellol

The cascade catalysis of old yellow enzyme, alcohol dehydrogenase and glucose dehydrogenase has become a promising approach for one pot, two-step reduction of (E/Z)-citral to (S)-citronellol, serving as a chiral alcohol with rose fragrance. During the multi-enzymatic cascade catalysis, old yellow enzyme is responsible for the reduction of the conjugated C=C and the introduction of the chiral center, requiring high activity and (S)-enantioselectiviy. Herein, to improve the activity of the old yellow enzyme from Providencia stuartii (NemR-PS) with strict (S)-enantioselectivity, the semi-rational design on its substrate binding pocket was performed through a combination of homology modeling, molecular docking analysis, alanine scanning and iterative saturation mutagenesis. The NemR-PS variant D275G/F351A with improved activity was obtained and then purified for characterization, obeying the substrate inhibition kinetics. Compared with the wild type, the parameters K-i and K-cat/K-m were increased from 39.79 mM and 2.09 s(-1)mM(-1) to 128.50 mM and 5.01 s(-1)mM(-1), respectively. Moreover, the variant D275G/F351A maintained strict (S)-enantioselectivity, avoiding the trade-off effect between activity and enantioselectivity. Either the enzyme NemR-PS or the variant D275G/F351A was co-expressed with alcohol dehydrogenase from Yokenella sp. WZY002 (YsADH) and glucose dehydrogenase from Bacillus megaterium (BmGDH(M6)). In contrast to the whole-cell biocatalyst co-expressing NemR-PS, that co-expressing the variant D275G/F351A shortened the reaction time from 36 h to 12 h in the reduction of 400 mM (E/Z)-citral. In the manner of substrate constant feeding, the accumulated product concentration reached up to 500 mM and completely eliminate the residual intermediate and by-product, suggesting the effectiveness of protein engineering and substrate engineering to improve catalytic efficiency.

Automated summary

In this study, the old yellow enzyme was improved through protein engineering, resulting in increased activity and strict (S)-enantioselectivity. The modified enzyme showed higher catalytic efficiency in the reduction of (E/Z)-citral compared to the wild type. Furthermore, co-expression of the modified enzyme with alcohol dehydrogenase and glucose dehydrogenase shortened the reaction time and increased the product concentration.