To minimize glycerol production during 2,3-BD fermentation by the engineered Saccharomyces cerevisiae, the Lactococcus lactis water-forming NADH oxidase gene (noxE) was expressed at five different levels. The expression of NADH oxidase substantially decreased the intracellular NADH/NAD(+) ratio. The S. cerevisiae BD5_T2nox strain expressing noxE produced 2,3-BD with yield of 0.359 g 2,3-BD/g glucose and glycerol with 0.069 g glycerol/g glucose, which are 23.8% higher and 65.3% lower than those of the isogenic strain without noxE. These results demonstrate that the carbon flux could be redirected from glycerol to 2,3-BD through alteration of the NADH/NAD+ ratio by the expression of NADH oxidase. (C) 2015 Elsevier Ltd. All rights reserved.