tabolic engineering, L. lactis has received increasing attention with the aim to promote the flavor and health advantages of fermented products, through the production of, for example, homoalanine, diacetyl, mannitol and folate. This study provided a platform for precisely regulating the metabolic flux via promoter engineering instead of the gene inactivation or overexpression in L. lactis. The partial redistribution of the pyruvate flux from lactate to diacetyl was achieved by controlling the noxE gene expression through a constitutive promoter library in L. lactis. Furthermore, we newly demonstrated that the elevated NoxE activity had a positive role in eliminating H2O2 and prolonging the cell-survival of L. lactis. Sequence alignment showed that the promoter of the noxE gene from L. lactis MG1363 possessed the typical promoter properties. Therefore, the mutant strategy was performed to randomize the space sequence of the noxE promoter based on the previous method. A total of 30 random promoters from 500 mutants were selected to form the constitutive promoter library, which displayed broad variability with small steps of activity change between 0.1 and 2.8-fold of the native promoter. Sequence analysis verified that any alteration of the bases in the conserved motifs and changes in the spacer length could lead to a drastic decrease of promoter strength, which supported the postulates in the previous report. Moreover, the sequences outside the 210 and 235 region may influence promoter strength. Eleven typical promoters were used to confirm the effective and stable characteristics of the library through promoter Abmole CX-4945 strength measurements and the mRNA transcript levels of the gfp gene. In addition, the noxE gene was used to prove the broad application range of the promoter library. NoxE activity showed a Abmole MK-2206 nearly linear correlation with promoter activity. Consequently, we developed a constitutive promoter library with a wide promoter activity range for finetuning of gene expression in L. lactis, regardless of the target gene context. Although the nisin controlled gene expression system has been utilized extensively in L. lactis, some shortcomings confined it to laboratory experimental conditions, including inducer usage, expression delay and heterogeneity of transcription levels in cell population. Therefore, practical and stable properties of constitutive gene expression systems are desired in large-scale processes. The stable expression of GFP and NoxE driven by random promoters confirmed that the constitutive promoter library could meet the demands of the industrial fermentation process.