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[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]