The H2Oforming NADH oxidase specifically utilizes NADH and provides an extra route for the regeneration of NAD + when O2 is available. In this study, eleven typical promoters from the promoter library were chosen to precisely control the noxE expression and the intracellular NADH/NAD + ratios were pinpointly regulated. The direct oxidation of NADH necessary for pyruvate reduction by the increased NoxE activity resulted in a diminished pyruvate flux towards lactate via LDH, and the pyruvate flux was redistributed to the ALS pathway. Subsequently, Pempidine a-acetolactate was decarboxylated into diacetyl in the presence of O2. The Metabolic Control Analysis prediction and experimental observation showed that the glycolytic flux to the a-acetolactate branch was less than 0.1% in wild-type L. lactis. However, the increasing NoxE activity driven by the eleven promoters led to the increase of 5.98% to 23.88% flux towards a-acetolactate and retained 67.29% to 33.96% flux to lactate. Acetate production exhibited a slight increase, probably due to the specific PDH activity which catalyzed the conversion of pyruvate to acetyl-CoA with the regeneration of NADH under aerobic conditions. Moreover, neither formate nor ethanol was detected, indicating that no flux was distributed to the pyruvate formate lyase pathway and the alcohol dehydrogenase pathway, which was in agreement with the previous report. In the milk fermentation process, the carbon flux was apportioned to lactate and diacetyl in 4:1 proportion in L. lactis DA/pB6nox, in which the diacetyl yield was significantly improved as compared to the wild-type strain. Accordingly, through the precise control of the noxE gene expression levels by the constitutive promoter library, the tight constraint on the end-product fluxes in the wild type was alleviated by the gradual lowering of NADH/NAD + ratios, yielding a series of recombinant strains with small differences in the proportion of lactate and diacetyl production among the end metabolites, which provide potential strains to optimize metabolite distribution. Generally, the tolerance of lactic acid bacteria to O2 requires the presence of either catalase, NADH oxidases, superoxidase dismutase or thiol-active enzyme system. There are more than seven NADH oxidase and dehydrogenase genes in the L. lactis genome, including noxA, noxB, noxC, noxD, noxE, yphA and aphF. NoxA and NoxB are two membraneintegrated NADH-dehydrogenases and have been demonstrated to be components of the electron transfer chain. NoxC and NoxD are described as Nortriptyline H2O2-forming NADH oxidases, however there is no experimental evidence to support this. NoxE is a well characterized H2O-forming NADH oxidase.