Design and Optimization for Efficient Production of (S)-Canadine in Escherichia coli

The biosynthesis of benzylisoquinoline alkaloids (BIAs) in microorganisms has attracted considerable attention, but achieving high yields of downstream BIAs remains challenging. (S)-canadine, a downstream BIA exhibiting potential therapeutic applications, holds particular importance as a common precursor for berberine and noscapine. Here, the de novo synthesis of (S)-canadine was achieved in E. coli at 165.74 mg/L. First, we focused on a key rate-limiting reaction and found that tyramine inhibits rat tyrosine hydroxylase. After overexpressing 4-hydroxyphenylacetate 3-monooxygenase mutant (HpaBC-D11) and purine-nucleoside phosphorylase (DeoD), we reduced the feedback inhibition of tyramine and increased the supply of dopamine, which optimized (S)-reticuline titer by 92-fold to 100.13 mg/L. Finally, we utilized the semirational design and protein scaffold to overcome the unfunctional expression of berberine bridge enzyme (BBE) and canadine synthase (CAS), resulting in the efficient synthesis of (S)-canadine. This study provides a promising platform for large-scale biomanufacturing of BIAs and affords guidelines for the synthesis of complicated natural compounds.