Synthetic Carbon-Efficient Pathway and Programmable Microenvironment for Sustainable Caffeic Acid Production

Abstract Microbial production of phenolic acids offers a sustainable alternative to plant extraction, reducing carbon loss and environmental impact. Here, we present a multi-level engineering strategy in Escherichia coli integrating carbon-efficient pathways, enzyme optimization, and subcellular compartmentalization. A synthetic TktA–Rpe–tyrosinase (TRT) pathway increased the theoretical carbon yield from 0.5 to 0.6 mol CaA per mol of glucose. Structure-guided engineering of tyrosinase enhanced catalytic efficiency toward p-coumaric acid by 5.07-fold. Programmable alkaline condensates created intracellular microenvironments optimized for alkaliphilic enzymes. This approach enabled the highest reported space–time yield of 211 mg L–1 h–1 for CaA. This work demonstrates a sustainable, high-efficiency microbial production strategy that maximizes carbon utilization, enhances enzyme performance, and precisely controls intracellular reactions.