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• SDH_1927 has the highest specific activity (56.72 ± 1.2 U/mg) among the eight L-sorbose dehydrogenases. • The 2-KGA production can be significantly increase by PMS under the condition of avoiding light. • The product 2-KGA could inhibit SDH activity by competing for the same hydrogen bonding sites with the substrate of L-sorbose. • The cell-recycling catalysis system is proposed to convert L-sorbose to 2-KGA, with the yield of 80.12 g/L and the productivity of 6.68 g/L/h. There are five L-sorbose dehydrogenases (SDH_2764, SDH_2744, SDH_0718, SDH_1927, SDH_0337) in Ketogulonicigenium vulgare SPU B805 and three L-sorbose dehydrogenases (SDH_02251, SDH_02271, SDH_P100164) in K. robustum SPU_B003, which play essential roles in the classical two-step fermentation for 2-keto-L-gulonic acid (2-KGA, the precursor of vitamin C) synthesis. In this work, the above eight SDH enzymes were cloned and purified, and their optimal pH values and reaction temperatures, kinetic properties (K m and K cat), thermal stabilities, substrate spectra, and effects of metal ions were determined. Eight SDH-harboring recombinant E.coli strains were verified to be capable to catalyze L-sorbose to 2-KGA. Additionally, the molecular docking results showed that the product 2-KGA could inhibit SDH activity by competing for the same hydrogen bonding sites with substrate L-sorbose. Finally, a novel and efficient whole-cell recycling catalysis system to overcome the product inhibition was proposed, the yield of 2-KGA reached up to 80.12 g/L with the productivity of 6.68 g/L/h. The results provide a promising prospect for selection and modification sorbose dehydrogenase for construction a one-step biotransformation system of 2-KGA. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • A one-step fermentation system was established in P. putida KT2440. • Enhanced electron transport efficiency increased the 2-KGA production. • The provision of a stable catalytic environment can enhance 2-KGA production. • Multi-strategy leads to a 15.48-fold enhancement in one-step fermentation. 2-KGA, a precursor for the synthesis of Vitamin C, is currently produced in China utilizing the "two-step fermentation" technique. Nevertheless, this method exhibits many inherent constraints. This study presents a comprehensive metabolic engineering strategy to establish and optimize a one-step 2-KGA fermentation process from D-sorbitol in Pseudomonas putida KT2440. In general, the endogenous promoters were screened to identify promoter P1 for subsequent heterologous gene expression in KT2440. Following the screening and confirmation of suitable heterologous gene elements such as sldh , sdh , cytc 551, pqq AB, and irr E, genetic recombination was performed in KT2440. In comparison to the initial achievement of expressing only sldh and sdh in KT2440, a yield of merely 0.42 g/L was obtained. However, by implementing four metabolic engineering strategies, the recombinant strain KT20 exhibited a significant enhancement in its ability to produce 2-KGA with a remarkable yield of up to 6.5 g/L – representing an impressive 15.48-fold improvement. [ABSTRACT FROM AUTHOR]