Multilevel cytochrome P450 engineering integrating protein language model-guided evolution enables de novo biosynthesis of amentoflavone in Escherichia coli.

Amentoflavone-type biflavonoids exhibit potent neuroprotective activities but are limited by scarce natural abundance and inefficient chemical synthesis routes. Here, we establish a stable Escherichia coli platform for de novo amentoflavone biosynthesis through multilevel engineering of a cytochrome P450-dependent oxidative coupling module. Systematic optimization of redox partner pairing, N-terminal expression regulation, and intrinsic catalytic activity substantially enhances the performance of the key P450 enzyme GbCYP90J6. Protein language model-guided directed evolution identifies high-activity variants that, when combined with an optimized expression tag, markedly improve in vivo catalysis. Integration of precursor flux balancing, expression optimization, and enzyme evolution results in an overall 8.34-fold increase in de novo amentoflavone production, reaching 6.59 mg/L in shake-flask cultivation. Fed-batch fermentation in a 5-L bioreactor increases the amentoflavone titer to 22.01 mg/L, demonstrating pathway scalability while revealing cellular tolerance as a remaining bottleneck. Together, this study establishes a framework for engineering P450-dependent intermolecular C-C coupling and highlights the necessity of balancing catalytic efficiency with cellular robustness in microbial cell factories.