Enzymatic reduction of the α, β-unsaturated carbon bond in citral

Bacteria, yeasts and filamentous fungi were screened for enantio-specific reduction of the α, β-unsaturated carbon bond in citral to produce citronellal. While a traditional aqueous screening system revealed only Zymomonas mobilis as positive, citronellal was produced in an aqueous/organic two liquid phase system by 11 of the 46 tested strains, which demonstrates the relevance of applying two-phase systems to screening strategies. Z. mobilis and Citrobacter freundii formed 1 mM citronellal in 3 h in the presence of a NADPH regenerating system and 20% (v/v) toluene. In comparison to these bacteria, the eukaryotic strains showed at least five-fold lower citral reductase activities. The bacterial strains produced preferentially the (S)-enantiomer of citronellal with e.e. values of >99% for Z. mobilis and 75% for Citrobacter freundii. In contrast the yeasts produced preferentially (R)-citronellal, i.e. Candida rugosa with an enantiomeric excess value of more than 98%. Many strains formed alcoholic by-products, viz. geraniol, nerol and citronellol. For Z. mobilis the production of these alcohols was suppressed in the presence of various organic solvents, e.g. toluene, and further decreased after EDTA addition.