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The acetic acid bacterium  Gluconobacter oxydans  is known for its unique incomplete oxidation and therefore widely applied in the industrial production of many compounds, e.g., 2-keto-L-gulonic acid (2-KLG), the direct precursor of vitamin C. However, few molecular tools are available for metabolically engineering  G. oxydans , which greatly limit the strain development. Promoters are one of vital components to control and regulate gene expression at the transcriptional level for boosting production. In this study, the low activity of SDH was found to hamper the high yield of 2-KLG, and enhancing the expression of SDH was achieved by screening the suitable promoters based on RNA sequencing data. We obtained 97 promoters from  G. oxydans ’s genome, including two strong shuttle promoters and six strongest promoters. Among these promoters, P 3022  and P 0943  revealed strong activities in both  Escherichia coli  and  G. oxydans , and the activity of the strongest promoter (P 2703 ) was about threefold that of the other reported strong promoters of  G. oxydans . These promoters were used to overexpress SDH in  G. oxydans  WSH-003. The titer of 2-KLG reached 3.7 g/L when SDH was under the control of strong promoters P 2057  and P 2703 . This study obtained a series of gradient promoters, including two strong shuttle promoters, and expanded the toolbox of available promoters for the application in metabolic engineering of  G. oxydans  for high-value products.

Identification of Gradient Promoters of Gluconobacter oxydans and Their Applications in the Biosynthesis of 2-Keto-L-Gulonic Acid