Artificial cell-free system for the sustainable production of acetoin from bioethanol

The present work introduces and validates an artificial cell free system for the synthesis of acetoin from ethanol, representing a greener alternative to conventional chemical synthesis. The one pot multi-enzymatic system, which employs pyruvate decarboxylase from Zymobacter palmae (ZpPDC), alcohol dehydrogenase from Saccharomyces cerevisiae (ScADH), and NADH oxidase from Streptococcus pyogenes (SpNOX), achieves nearly 100 % substrate conversion and reaction yield within 6 h under optimal conditions (pH 7.5, enzyme activities: ZpPDC 100 U·mL^-1, ScADH 50 U·mL^-1, SpNOX 127 U·mL^-1, and 1 mM NAD⁺). Using air for oxygen supply mitigates enzyme inactivation while effectively accelerating the regeneration of NAD⁺. The use of bioethanol as a substrate demonstrates the robustness and sustainability of the bioprocess, enabling the production of natural acetoin from renewable resources. This environmentally friendly approach offers significant advantages for industrial applications, aligning with green chemistry principles.