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, ScADH 50 U·mL, SpNOX 127 U·mL, and 1 mM NAD).