Dual-dynamic control and metabolic rebalancing guided by toxic target discovery for efficient dencichine production

Abstract

Construction of microbial cell factories often requires extensive reconfiguration of metabolic networks, which frequently compromises cell growth. Here, we engineered E. coli to produce dencichine (β-ODAP), a plant-derived hemostatic agent. Knocking out serB blocks the competing L-serine pathway, causing L-serine auxotrophy and severely impairing cell growth. This growth defect was only partially restored by supplementation with L-serine or L-glycine, suggesting the presence of additional growth-limiting factors. Through adaptive laboratory evolution and reverse engineering, we further uncovered that overexpression of serA to enhance the supply of the precursor L-2,3-diaminopropionate led to accumulation of the toxic byproduct 2-hydroxyglutarate (2-HG) from α-ketoglutarate. To resolve these two growth constraints, we developed a growth-phase-dependent dual-dynamic regulation circuit. This circuit gradually activates serA expression and progressively represses serB expression as cells enter the stationary phase, thereby reducing 2-HG accumulation and alleviating L-serine auxotrophy. Combined with pathway balancing and cofactor optimization, the final engineered strain produced 13.46 g L-1 of β-ODAP with a yield of 0.32 g g-1 in 3-L bioreactors. This study reveals a toxicity mechanism in serine-pathway engineering and provides a dynamic regulation strategy applicable to the biosynthesis of serine-pathway-derived metabolites.