Cascading Old Yellow Enzyme, Alcohol Dehydrogenase and Glucose Dehydrogenase for Selective Reduction of (E/Z)-Citral to (S)-Citronellol

Citronellol is a kind of unsaturated alcohol with rose-like smell and its (S)-enantiomer serves as an important intermediate for organic synthesis of (-)-cis-rose oxide. Chemical methods are commonly used for the synthesis of citronellol and its (S)-enantiomer, which suffers from severe reaction conditions and poor selectivity. Here, the first one-pot double reduction of (E/Z)-citral to (S)-citronellol was achieved in a multi-enzymatic cascade system: N-ethylmaleimide reductase from Providencia stuartii (NemR-PS) was selected to catalyze the selective reduction of (E/Z)-citral to (S)-citronellal, alcohol dehydrogenase from Yokenella sp. WZY002 (YsADH) performed the further reduction of (S)-citronellal to (S)-citronellol, meanwhile a variant of glucose dehydrogenase from Bacillus megaterium (BmGDH(M6)), together with glucose, drove efficient NADPH regeneration. The Escherichia coli strain co-expressing NemR-PS, YsADH, and BmGDH(M6) was successfully constructed and used as the whole-cell catalyst. Various factors were investigated for achieving high conversion and reducing the accumulation of the intermediate (S)-citronellal and by-products. 0.4 mM NADP(+) was essential for maintaining high catalytic activity, while the feeding of the cells expressing BmGDH(M6) effectively eliminated the intermediate and by-products and shortened the reaction time. Under optimized conditions, the bio-transformation of 400 mM citral caused nearly complete conversion (>99.5%) to enantio-pure (S)-citronellol within 36 h, demonstrating promise for industrial application.

Automated summary

A novel approach for the one-pot double reduction of (E/Z)-citral to (S)-citronellol was achieved in a multi-enzymatic cascade system, using N-ethylmaleimide reductase, alcohol dehydrogenase, and a variant of glucose dehydrogenase. The engineered E. coli strain co-expressing the enzymes displayed high conversion rates and enantioselectivity, demonstrating potential for industrial application.