Bioethanol-to-acetoin conversion through an enzymatic cascade

Biocatalysis offers a sustainable route for producing high-value chemicals under mild and environmentally friendly conditions. In this study, a multienzymatic cascade was developed for acetoin production from bioethanol. Alcohol oxidases from Pichia pastoris (PpAOX) and Phanerochaete chrysosporium (PcAOX), expressed in Escherichia coli, were evaluated for ethanol oxidation into acetaldehyde using enzymatic extracts, fresh and lyophilized whole cells, and His-tag immobilized enzymes. To address the accumulation of hydrogen peroxide generated during oxidation, two co-enzymes were studied: catalase or unspecific peroxygenase (UPO). In the second step, benzaldehyde lyase from Pseudomonas fluorescens was applied as lyophilized whole cells to synthesize acetoin. Lyophilized cells achieved up to 99% conversion at 100 mM and 90% at 200 mM ethanol after 24 h. Sequential and simultaneous cascade strategies were compared, both reaching complete substrate consumption. These results demonstrate an efficient and sustainable platform for acetoin production from renewable feedstock, supporting the synthesis of value-added chemicals.