Periplasmic Engineering Enhances Terminal Hydroxylation for Efficient Caffeic Acid Biosynthesis in Escherichia coli

Caffeic acid (CA) is a valuable phenylpropanoid with applications in food, pharmaceutical, and chemical industries. Microbial production of CA is often limited by the terminal hydroxylation catalyzed by 4-hydroxyphenylacetate 3-monooxygenase (HpaBC). Here, we constructed a de novo CA biosynthetic pathway in Escherichia coli and enhanced production through systematic metabolic and spatial engineering. Optimization of l-tyrosine supply and HpaBC expression increased CA production to 61.0 mg/L. However, further enhancement of glucose uptake and precursor supply was insufficient, indicating that hydroxylation remained a major limitation. To address this, HpaBC was relocated to the periplasm via the Tat pathway, increasing CA production 4.9-fold to 299.1 mg/L with reduced byproduct formation. Further lpp+14 mediated periplasmic remodeling and fermentation optimization increased shake-flask production to 463.7 mg/L. Finally, fed-batch fermentation achieved 5.1 g/L CA in a 3 L bioreactor. This study highlights periplasmic engineering as an effective strategy for improving oxidation-dependent phenylpropanoid biosynthesis.