Balancing Precursor Supply and Enhancing Enzyme Solubility for Efficient Production of (S)-Reticuline in Escherichia coli.

Benzylisoquinoline alkaloids (BIAs) are pharmacologically valuable natural products, yet their microbial production in Escherichia coli is hindered by two major bottlenecks: (1) imbalanced metabolic flux between the two upstream precursors, 3,4-dihydroxyphenylacetaldehyde (3,4-DHPAA) and dopamine, and (2) poor soluble expression of plant-derived enzymes, particularly norcoclaurine synthase (NCS) and the methyltransferase Ps4'OMT2. To address these challenges, we systematically engineered E. coli for efficient synthesis of (S)-reticuline, the universal precursor to diverse BIAs. First, to resolve the flux imbalance, we introduced a heterologous ω-transaminase (ω-TA) that dynamically interconverts 3,4-DHPAA and dopamine, thereby coordinating precursor supply without tedious tuning of branch pathways. Second, to overcome enzyme insolubility, we combined solubility-enhancing strategiesincluding SUMO fusion, ProteinMPNN-guided design, and N-terminal truncationwhich markedly improved the soluble expression of CjNCS and Ps4'OMT2. Furthermore, strengthening the S-adenosylmethionine (SAM) regeneration cycle through integration of luxS, mtn, and metF genes enhanced the efficiency of the three sequential methylation reactions. The final engineered strain achieved a (S)-reticuline titer of 347 mg/L in shake-flask fermentation. Overall, this work establishes an efficient E. coli chassis for sustainable BIAs production by systematically tackling flux imbalance and enzyme solubility.