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

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 A400-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.