Abscisic acid is a sesquiterpene phytohormone with extensive applications in agriculture and human health. Currently, it is produced through fermentation of , a plant pathogenic filamentous fungus. The process requires morphology controls, which complicates production and strain optimization. In this study, the abscisic acid production strain SM309 was optimized by enhancing the precursor supply using a "push-pull-restrain" strategy focusing on acetyl-CoA, which increased abscisic acid production from 266.34 to 328.51 mg/L. Subsequently, prediction and analysis were used to obtain the docking conformations and binding affinity of ABC transporters for abscisic acid. Overexpression of ABC transporter Gcn20 further enhanced abscisic acid production by 10.88%, reaching 354.21 mg/L. Additionally, low temperature and dodecane addition were employed as auxiliary strategies to promote abscisic acid synthesis, resulting in a titer of 605.92 mg/L. Finally, the engineered strain achieved an abscisic acid titer of 2056.64 mg/L in a 5 L bioreactor, representing the highest titer reported for a yeast de novo synthesis system to date.
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