LAUSR

The compartmentalization of enzymes into organelles is a promising strategy for limiting metabolic crosstalk and improving pathway efficiency; however, prokaryotes are unicellular organisms that lack membrane‐bound organelles. To mimic this natural compartmentalization, we present here the targeting of the reductive tricarboxylic acid (rTCA) pathway to the periplasm to enhance the production of malate. A multigene combination knockout strategy was used to construct a phosphoenolpyruvate (PEP) pool. Then, the genes encoding phosphoenolpyruvate carboxykinase and malate dehydrogenase were combinatorially overexpressed to construct a cytoplasmic rTCA pathway for malate biosynthesis; however, the efficiency of malate production was low. To further enhance malate production, the rTCA pathway was targeted to the periplasm, which led to a 100% increase in malate production to 18.8 mM. Next, dual metabolic engineering regulation was adopted to balance the cytoplasmic and periplasmic pathways, leading to an increase in malate production to 58.8 mM. The final engineered strain, GL2306, produced 193 mM malate with a yield of 0.53 mol/mol in 5 L of pH‐stat fed‐batch culture. The strategy described here paves the way for the development of metabolic engineering and synthetic biology in the microbial production of chemicals.