LAUSR

Chromosome‐based engineering is a superior approach for gene integration generating a stable and robust chassis. Therefore, an effective amplifier, T7 RNA polymerase (T7RNAP) from bacteriophage, has been incorporated into Escherichia coli W3110 by site‐specific integration. Herein, we performed the 5‐aminolevulinic acid (5‐ALA) production in four T7RNAP‐equipped W3110 strains using recombinant 5‐aminolevulinic synthase and further explored the metabolic difference in best strain. The fastest glucose consumption resulted in the highest biomass and the 5‐ALA production reached to 5.5 g/L; thus, the least by‐product of acetate was shown in RH strain in which T7RNAP was inserted at HK022 phage attack site. Overexpression of phosphoenolpyruvate (PEP) carboxylase would pull PEP to oxaloacetic acid in tricarboxylic acid cycle, leading to energy conservation and even no acetate production, thus, 6.53 g/L of 5‐ALA was achieved. Amino acid utilization in RH deciphered the major metabolic flux in α‐ketoglutaric acid dominating 5‐ALA production. Finally, the ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase were expressed for carbon dioxide recycling; a robust and efficient chassis toward low‐carbon assimilation and high‐level of 5‐ALA production up to 11.2 g/L in fed‐batch fermentation was established.