Combinatorial optimization engineering for the high-level production of shikimic acid in Escherichia coli

Shikimic acid is a crucial precursor for the production of indole derivatives and chiral drugs, and the biomanufacturing offers reduced cost and simplified process compared to traditional plant extraction. Current research on industrial-scale production of shikimic acid achieves notable progress, yet the limitations associated with high production cost, complex operation process, and excessive byproduct formation still persist. To tackle these difficulties, this study aimed at establishing a high-efficient shikimic acid-producer through systematic modification and fermentation optimization in Escherichia coli. First, the route for the synthesis of shikimic acid was divided into three modules (i.e., sugar uptake, bioproduct synthesis, and metabolite catabolism), of which the disruption of glucose uptake system, reinforcement of bioproduct synthesis, and block of its catabolism rewired metabolic fluxes and provided the possibility for efficient synthesis. Next, series of systematic engineering involving in deleting pyruvate bypass, reinforcing the pentose phosphate (PP) pathway, and adjusting promoter strength promoted precursor supply and avoided metabolic overflow. The best strain EC022 generated 5.27 g/L of shikimic acid, highlighting the significance of precursor availability. Then, fermentation conditions including substrate concentration, inoculation size, cultivation temperature, induction time, and C/N ratio were comprehensively optimized, which boosted the bioproduction to 8.70 g/L in the optimized conditions. Finally, a final titer of 61.87 g/L shikimic acid was achieved in a 7 L bioreactor. This study developed an efficient shikimic acid-producer with industrial potential through metabolic engineering and bioprocess optimization.