| Summary: | Citronellol is a kind of unsaturated alcohol with rose-like smell and its (<i>S</i>)-enantiomer serves as an important intermediate for organic synthesis of (-)-<i>cis</i>-rose oxide. Chemical methods are commonly used for the synthesis of citronellol and its (<i>S</i>)-enantiomer, which suffers from severe reaction conditions and poor selectivity. Here, the first one-pot double reduction of (<i>E</i>/<i>Z</i>)-citral to (<i>S</i>)-citronellol was achieved in a multi-enzymatic cascade system: N-ethylmaleimide reductase from <i>Providencia stuartii</i> (NemR-PS) was selected to catalyze the selective reduction of (<i>E</i>/<i>Z</i>)-citral to (<i>S</i>)-citronellal, alcohol dehydrogenase from <i>Yokenella</i> sp. WZY002 (YsADH) performed the further reduction of (<i>S</i>)-citronellal to (<i>S</i>)-citronellol, meanwhile a variant of glucose dehydrogenase from <i>Bacillus megaterium</i> (BmGDH<sub>M6</sub>), together with glucose, drove efficient NADPH regeneration. The <i>Escherichia coli</i> strain co-expressing NemR-PS, YsADH, and BmGDH<sub>M6</sub> 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 (<i>S</i>)-citronellal and by-products. 0.4 mM NADP<sup>+</sup> was essential for maintaining high catalytic activity, while the feeding of the cells expressing BmGDH<sub>M6</sub> 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 (<i>S</i>)-citronellol within 36 h, demonstrating promise for industrial application.
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