Hierarchical Flux‐Protection Engineering Stabilizes Oxidation‐Prone Metabolic Nodes for High‐Level Curcumin Biosynthesis

ABSTRACT Biosynthesis of complex natural products in engineered microbial hosts is frequently constrained by unstable oxidation‐prone intermediates that disrupt metabolic balance and lower carbon flux efficiency. In the synthesis pathways of polyphenolic compounds, redox‐sensitive nodes often function as bottlenecks due to intermediate accumulation and spontaneous oxidation under aerobic conditions. In this study, the oxygen‐dependent degradation of L‐3,4‐dihydroxyphenylalanine (L‐DOPA), a redox‐sensitive intermediate in curcumin biosynthesis, was characterized, and a hierarchical strategy was implemented to stabilize oxidation‐prone nodes while rebalancing intracellular carbon and electron fluxes. Adaptive evolution improved host robustness, respiratory chain modulation redistributed reducing equivalents under oxygen limitation, and directed evolution of 4‐hydroxyphenylacetate 3‐monooxygenase (HpaB) rebalanced intermediate distribution and enhanced productive hydroxylation. Combined with multistage pH–DO control, these strategies markedly improved carbon utilization efficiency and curcumin biosynthesis. In 5 L fed‐batch fermentation, curcumin production reached 1075.2 mg/L, the highest reported titer for single‐strain de novo curcumin biosynthesis. Extension of this framework to caffeic acid production increased the titer by approximately 69.1% and reduced A 400 ‐associated browning, supporting transferability to another oxidation‐prone phenolic product. This study demonstrates the effectiveness of flux protection in curcumin biosynthesis and supports the potential transferability of this framework to related HpaB‐dependent phenylpropanoid pathways.