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

Abstract

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 (BmGDHM6), together with glucose, drove efficient NADPH regeneration. The Escherichia coli strain co-expressing NemR-PS, YsADH, and BmGDHM6 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 BmGDHM6 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.

Engineering of Yeast Old Yellow Enzyme OYE3 Enables Its Capability Discriminating of (E)-Citral and (Z)-Citral

“…On the one hand, the supplement of exogenous NADP + significantly increased the catalytic efficiency, and the NADP + concentration (>0.4 mM) was required to maintain high catalytic activity (Figure S5a). On the other hand, the addition of FMN did not increase the conversion (Figure S5b), suggesting that the binding of FMN was tight enough for maintaining the activity of the variant S296/W116G [36]. In addition, the amounts of GDH and glucose were important for the efficiency of NADPH regeneration.…”

Applications of Ene-Reductases in the Synthesis of Flavors and Fragrances

“…WZY002), and BmGDH M6 (from Bacillus megaterium) to catalyze the biotransformation of (E/Z)-citral to (S)-citronellol. 57 Due to limited cell capacity, the expression level of BmGDH M6 is reduced in coexpressed bacteria, resulting in insufficient activity to meet the requirements of two reductases. After 24 h of reaction, feeding additional wet cells expressing BmGDH M6 achieved a complete substrate conversion of up to 400 mM.…”

Engineering aBacillus subtilisesterase for selective hydrolysis ofd,l-menthyl acetate in an organic solvent-free system

“…The cell envelope offered the protection of intracellular enzymes but also the permeability of hydrophobic substrates to keep the reaction going. [35][36][37] Compared with isolated enzymes, the use of whole-cell catalysts could simplify the procedure of biocatalyst preparation and facilitate the biocatalyst reuse, offering a cost-effective advantage. More importantly, further catalytic experiments revealed that the e.e.…”