Objective To explore the RecET-Cre/ loxP system for chromosomal replacement of promoter and its application on enhancement l -leucine production in Corynebacterium glutamicum ( C. glutamicum ) ATCC14067. Results The RecET-Cre/ loxP system was used to achieve the chromosomal replacement of promoter in C. glutamicum ATCC14067 to adjust the metabolic flux involving the l -leucine synthetic pathway. First, leuA r _13032 from C. glutamicum ATCC13032 which carried two mutations was overexpressed to release enzyme feedback inhibition. Then, comparing different mutations in ilvBNC gene clusters, the results indicated that ilvBNC_ CP was most effective to enhance the metabolic flux of pyruvate towards l -leucine synthesis. The promoters of pck, odx and pyk 2 were overexpressed under the strong promoter P eftu or P sod to improve the supply of pyruvate. Besides, the promoter P ilvBNC was employed to dynamically control the transcription level of icd due to its attenuation mechanism by responding to the concentration of l -leucine. The final engineered strain produced 14.05 g l -leucine/L in flask cultivation. Conclusion The RecET-Cre /loxP system is effective for gene manipulation in C. glutamicum ATCC14067. Besides, the results demonstrate the potential of C. glutamicum ATCC14067 for l -leucine production and provide new targets and strategies for strain development.

中文翻译：

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RecET-Cre/loxP系统在谷氨酸棒杆菌ATCC14067生产L-亮氨酸中的应用

目的探讨RecET-Cre/loxP启动子染色体置换系统及其在增强谷氨酸棒杆菌ATCC14067中L-亮氨酸生产中的应用。结果利用RecET-Cre/loxP系统实现谷氨酸棒杆菌ATCC14067启动子染色体置换，调节L-亮氨酸合成途径的代谢通量。首先，携带两个突变的谷氨酸棒杆菌ATCC13032的leuA r_13032被过表达以释放酶反馈抑制。然后，比较ilvBNC基因簇中的不同突变，结果表明ilvBNC_CP对于增强丙酮酸向L-亮氨酸合成的代谢通量最有效。pck、odx 和pyk 2 的启动子在强启动子P eftu 或P sod 下过表达以改善丙酮酸的供应。此外，由于其衰减机制，启动子PilvBNC通过响应l-亮氨酸的浓度来动态控制icd的转录水平。最终的工程菌株在烧瓶培养中产生 14.05gl-亮氨酸/L。结论 RecET-Cre /loxP 系统对谷氨酸棒杆菌 ATCC14067 的基因操作有效。此外，结果证明了谷氨酸棒杆菌 ATCC14067 在 l-亮氨酸生产方面的潜力，并为菌株开发提供了新的目标和策略。谷氨酸 ATCC14067。此外，结果证明了谷氨酸棒杆菌 ATCC14067 在 l-亮氨酸生产方面的潜力，并为菌株开发提供了新的目标和策略。谷氨酸 ATCC14067。此外，结果证明了谷氨酸棒杆菌 ATCC14067 在 l-亮氨酸生产方面的潜力，并为菌株开发提供了新的目标和策略。