Corynebacterium ammoniagenes is an important industrial organism that is widely used to produce nucleotides and the potential for industrial production of coenzyme A by C. ammoniagenes ATCC 6871 has been shown. However, the yield of coenzyme A needs to be improved, and the available constitutive promoters are rather limited in this strain. In this study, 20 putative DNA promoters derived from genes with high transcription levels and 6 promoters from molecular chaperone genes were identified. To evaluate the activity of each promoter, red fluorescence protein (RFP) was used as a reporter. We successfully isolated a range of promoters with different activity levels, and among these a fragment derived from the upstream sequence of the 50S ribosomal protein L21 (Prpl21) exhibited the strongest activity among the 26 identified promoters. Furthermore, type III pantothenate kinase from Pseudomonas putida (PpcoaA) was overexpressed in C. ammoniagenes under the control of Prpl21, CoA yield increased approximately 4.4 times. This study provides a paradigm for rational isolation of promoters with different activities and their application in metabolic engineering. These promoters will enrich the available promoter toolkit for C. ammoniagenes and should be valuable in current platforms for metabolic engineering and synthetic biology for the optimization of pathways to extend the product spectrum or improve the productivity in C. ammoniagenes ATCC 6871 for industrial applications.

中文翻译：

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产氨棒杆菌 ATCC 6871 启动子的分离及其在 CoA 合成中的应用


产氨棒杆菌是一种重要的工业生物体，广泛用于生产核苷酸，并且产氨棒杆菌 ATCC 6871 已显示出工业生产辅酶 A 的潜力。然而，辅酶A的产量有待提高，并且该菌株可用的组成型启动子相当有限。在这项研究中，鉴定了 20 个来自高转录水平基因的推定 DNA 启动子和 6 个来自分子伴侣基因的启动子。为了评估每个启动子的活性，使用红色荧光蛋白（RFP）作为报告基因。我们成功分离出一系列具有不同活性水平的启动子，其中来自50S核糖体蛋白L21（Prpl21）上游序列的片段在所鉴定的26个启动子中表现出最强的活性。此外，来自恶臭假单胞菌（PpcoaA）的III型泛酸激酶在Prpl21的控制下在产氨梭菌中过表达，CoA产量增加了约4.4倍。该研究为合理分离具有不同活性的启动子及其在代谢工程中的应用提供了范例。这些启动子将丰富产氨梭菌可用的启动子工具包，并且在当前的代谢工程和合成生物学平台中应该具有价值，可用于优化途径以扩展产氨梭菌 ATCC 6871 的产品谱或提高产氨梭菌 ATCC 6871 的生产率，以用于工业应用。