LAUSR

This study reports on the development of effective strain engineering strategies for the high-level bio-based production of coproporphyrin (CP), a porphyrin pigment compound with various applications, using Escherichia coli as a production host. Our approach involves heterologous implementation of the Shemin/C4 pathway in an E. coli host strain with an enlarged intracellular pool of succinyl-CoA. To regulate the expression of the key pathway genes, including hemA/B/D/E/Y, we employed a plasmid system comprising two operons regulated by strong trc and gracmax promoters, respectively. Using the engineered E. coli strains for bioreactor cultivation under aerobic conditions with glycerol as the carbon source, we produced up to 353 mg/L CP with minimal byproduct formation. The overproduced CP was secreted extracellularly, posing minimal physiological toxicity and impact on the producing cells. To date, targeted bio-based production of CP by E. coli has yet to be reported. In addition to the demonstration of high-level bio-based production of CP, our study underscores the importance of identifying key enzymatic reactions limiting the overall metabolite production for developing differential expression strategies for pathway modulation and even optimization. This investigation paves the way for the development of effective metabolic engineering strategies based on targeted manipulation of key enzymes to customize engineered strains for effective large-scale bio-based production.