We noticed the aggrecan transcript decreased during the differentiation in comparison to the undifferentiated NC-like cells. Aggrecan is possibly regulated by a negative feedback loop, in which this core protein makes cells to attenuate its transcript; but it may not necessarily lower the protein production or comprise GAGs accumulation. Immunohistochemical and biochemical and assays confirmed the significant deposition of aggrecan and GAGs in the matrix. Importantly, a high ratio of GAGs to hydroxyproline was detected which is much closer to that of native NP tissue rather than hyaline cartilage. The herein excessive pyruvate was channeled to acetoin or diacetyl via ALS, whereas the flux from pyruvate to lactate was almost abolished. Considering that lactate is an important metabolite that prevents fermented products from spoilage and contributes to the texture of dairy products, the strong overexpression or complete deletion of target genes limits the application of genetically modified lactic acid bacteria as starter cultures for industrial production of dairy products. Thus, strategies for the fine-tuning of gene expression are required to control the aimed metabolic fluxes. Promoter engineering is interpreted as the creation of a functional promoter library for precisely controlling gene expression to perform metabolic optimization or control analysis. It has promising perspectives with respect to the research of functional genomics, cell network analysis and synthetic biology. For example, a series of mutant L. lactis strains have been constructed based on synthetic promoters to demonstrate that LDH exerted virtually no control on the glycolytic flux at the wild-type enzyme level and the lactate production. In addition, a synthetic promoter library has been used to evaluate the influence of different production levels of glucose-6-phosphate dehydrogenase on xylose fermentation and ethanol yield in Saccharomyces cerevisiae. These previous studies have illustrated the valuable applications of the promoter engineering in the precise control and the quantitative assessment of gene expression level. L. lactis is a facultatively anaerobic bacterium and O2 has negative effects on both cell growth and survival, because in vivo O2 is converted into reactive oxygen species which cause protein, lipid and nucleic acid damage. To cope with oxygen toxicity, L. lactis could grow via respiratory metabolism when heme is available. Some lactic acid bacteria have antioxidant enzymes to detoxify O2-derived compounds. Aside from the toxic effects of O2, aeration could induce the metabolic shift from homolactic to mixed-acid fermentation, making pyruvate metabolism more flexible. Therefore, lowering cytoplasmic O2 is economically significant in improving cell growth and survival under aerobic conditions. Here we constructed a constitutive promoter library by randomizing the space sequence between the two conserved motifs of the promoter of the H2O-forming NADH oxidase gene in L. lactis. Under the control of individual random promoters, the fine tuning of lactate and diacetyl production was achieved by precisely regulating the intracellular NADH/NAD + ratios in L. lactis. Furthermore, the beneficial effects of the increased NoxE activity on cell survival were also investigated in this study.