Fine-Tuned Transcription Factor Engineering and Precursor Rewiring Drive Enhanced Production of Berberine in Nicotiana benthamiana

Berberine is a pharmacologically active benzylisoquinoline alkaloid (BIA) with broad therapeutic potential. Although its heterologous biosynthesis has been achieved in microbial hosts, large-scale production remains hindered by the pathway complexity, metabolic burden, and inefficient expression of plant-derived cytochrome P450 enzymes. Here, we report the complete de novo biosynthesis of berberine in Nicotiana benthamiana through systematic pathway reconstruction and metabolic engineering. By increasing the availability of precursors, such as dopamine and 3,4-dihydroxyphenylacetaldehyde (3,4-DHPAA), and enhancing tyrosine biosynthesis via plastid-targeted strategies, we achieved a ( S )-reticuline yield of 17.76 μg·g −1 dry weight (DW). Introduction of downstream biosynthetic genes enabled the production of berberine at suboptimal titers. The accumulation of L- tyrosine-derived metabolites in competing pathways demonstrates substantial potential for flux optimization within the target pathway. However, additional transcription factors (TFs) lead to excessive transcriptional activation, resulting in leaf necrosis. This issue was resolved by fine-tuning TF expression using promoters of varying strengths. Subsequent enzyme engineering of the berberine bridge enzyme (BBE), specifically the Am BBE1 F398W-I431F variant, further increased the titer. Coexpression of this optimized BBE with the finely tuned TF Cj WRKY1 culminated in a final berberine yield of 13.91 μg·g −1 DW, which further increased to 15.03 μg·g −1 DW after postharvest heat treatment. To our knowledge, this represents the first complete reconstruction of the berberine biosynthetic pathway in a nonnatural plant chassis, establishing a robust and scalable platform for alkaloid production. Although current yields remain lower than those of microbial systems, our study provides a generalizable framework for the plant-based biosynthesis of complex natural products.