<i>De Novo</i> Biosynthesis of Biochanin A in <i>Saccharomyces cerevisiae</i> via Integrated Metabolic and Organelle Engineering

Biochanin A, a valuable <i>O</i>-methylated isoflavone, requires efficient P450 activity, regioselective methylation, and substantial cofactor availability. Here, we established a systematic strategy for its high-level production in <i>Saccharomyces cerevisiae</i>. We first constructed a genistein platform strain (16.10 mg/L) by optimizing the 2-hydroxyisoflavanone synthase gene (<i>PlIFS</i>) copy number. Next, a screen identified <i>Pueraria lobata</i> PlOMT9 as the optimal 4'-<i>O</i>-methyltransferase, yielding 14.29 mg/L biochanin A. As methylation was constrained by ATP supply, we augmented the cellular energy budget by expressing <i>Vitreoscilla</i> hemoglobin, increasing biochanin A titer to 19.48 mg/L. We then expanded the endoplasmic reticulum membrane capacity and coexpressed protein-folding chaperones, which profoundly enhanced the functional expression of the membrane-associated enzymes, skyrocketing the biochanin A titer to 39.89 mg/L. Finally, we increased the titer to 49.86 mg/L by improving the heme supply. The final strain in this study will facilitate the production of <i>O</i>-methylated isoflavones through the biochanin A biosynthetic pathway.