| Summary: | <i>Trans</i>-2-decenoic acid has a wide range of applications, including those in medicine, food, and health care. Therefore, the industrial production of <i>trans</i>-2-decenoic acid is particularly important. However, few studies have focused on medium-chain unsaturated fatty acids. Therefore, we aimed to optimize the fermentation process of decanoic acid biocatalysis to synthesize <i>trans</i>-2-decenoic acid using an engineered <i>Escherichia coli</i> constructed in the laboratory. Early-stage culture and the effect of the seed liquid culture time, culture temperature, inoculum amount, induction temperature, dissolution effects of the substrate solvent, metal ions, and substrate loading on the titer of <i>trans</i>-2-decenoic acid were evaluated. Based on a single-factor experimental optimization, a Box–Behnken design (BBD) was used for response surface testing using the substrate feeding concentration, inducer concentration, and MnCl<sub>2</sub> concentration as response variables and <i>trans</i>-2-decenoic acid production as the response value. The optimal fermentation process was as follows: Seed culture time of 20 h, culture temperature of 37 °C, inoculation amount of 1%, induction temperature of 30 °C, substrate flow of 0.15 g/L, inducer concentration of 5.60 g/L, and MnCl<sub>2</sub> concentration of 0.10 mM. Under these conditions, the average production of <i>trans</i>-2-decenoic acid was 1.982 ± 0.110 g/L, which was 1.042 g/L higher than that obtained in the basic LB medium. Compared with that of the previous period, the titer of the <i>trans</i>-2-decenoic acid studied increased by 1.501 ± 0.110 g/L, providing a basis for further research on the fermentation process of the biocatalytic decanoic acid synthesis of <i>trans</i>-2-decenoic acid.
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