De Novo Production of Plant 4′-Deoxyflavones Baicalein and Oroxylin A from Ethanol in Crabtree-Negative Yeast

Baicalein and oroxylin A are well-known medicinal 4′-deoxyflavones found mainly in the roots of traditional medicinal plant Scutellaria baicalensis Georgi. However, extraction from plants is time-consuming, environmentally unfriendly, and insufficient. Although microbial synthesis of flavonoids has been extensively reported, synthesis of downstream modified 4′-deoxyflavones has not, and their yields are extremely low. Here, we reassembled the S. baicalensis 4′-deoxyflavone biosynthetic pathway in a Crabtree-negative yeast, Pichia pastoris, with activity analysis and combinatorial expression of eight biosynthetic genes, allowing production of 4′-deoxyflavones like baicalein, oroxylin A, wogonin, norwogonin, 6-methoxywogonin, and the novel 6-methoxynorwogonin. De novo baicalein synthesis was then achieved by complete pathway assembly. Toxic intermediates highly impaired the cell production capacity; hence, we alleviated cinnamic acid growth inhibition by culturing the cells at near-neutral pH and using alcoholic carbon sources. To achieve pathway balance and improve baicalein and oroxylin A synthesis, we further divided the pathway into five modules. A series of ethanol-induced and constitutive transcriptional amplification devices were constructed to adapt to the modules. This fine-tuning pathway control considerably reduced byproduct and intermediate accumulation and achieved high-level de novo baicalein (401.9 mg/L with a total increase of 1182-fold, the highest titer reported) and oroxylin A (339.5 mg/L, for the first time) production from ethanol. This study provides new strategies for the microbial synthesis of 4′-deoxyflavones and other flavonoids.