Abstract: Efficient production of 3-hydroxypropionic acid (3-HP) using acetate as a low-cost carbon source is crucial for cost-effective biomanufacturing. In this study, engineered Escherichia coli ZWR23 (M*DA) served as the parent strain. We combined strain modification with fermentation process optimization to improve 3-HP synthesis. Carbon flux diversion was suppressed by overexpressing fabF, a key gene in fatty acid biosynthesis. Meanwhile, critical process parameters were systematically optimized, including dissolved oxygen (DO) level, substrate feeding strategy, pH control method, and salt system composition. The optimal conditions were determined as follows: 20% DO, fed-batch feeding at 8.0 g/L, pH maintained at 7.0 with 3.0 M sulfuric acid, and an ammonium acetate-sodium bicarbonate salt system. Under these optimized conditions, the recombinant strain ZWR23 (M*DA, FabF) achieved a 3-HP titer of 20.55 g/L and a yield of 0.56 g/g. Compared with the parent strain, these values increased by 67.21% and 180%, respectively. Additionally, the highest 3-HP titer (22.02 g/L) was obtained in an all-potassium salt system, representing a 79.17% increase relative to the parent strain. This work provides a theoretical basis and technical reference for efficient 3-HP biosynthesis from acetate, without the need for exogenous inhibitors.