METABOLIC ENGINEERING OF ESCHERICHIA COLI FOR CHORISMATE OVERPRODUCTION

There is a growing interest in microbial metabolic engineering as a more environmentally friendly approach to the production of bioactive compounds and their biosynthetic precursors. Chorismate is an important compound in the shikimate pathway and serves as a key branch point in the biosynthesis of aromatic amino acids and other specialized metabolites. Among the latter, echinosporin has shown promise as an anticancer agent, and therefore, the modulation of chorismate metabolism presents a promising approach in biotechnology. The current research aims to develop a genetically engineered Escherichia coli that can effectively manipulate the metabolic flux in the shikimate pathway to increase chorismate levels. In this study, we used E. coli K-12 MG1655 and E. coli BL21(DE3) Gold strains to develop a dual-module approach that combines metabolic pathway attenuation and heterologous gene expression. The deletion of the E. coli trpDE locus in E. coli K-12 was successfully carried out using the λ Red recombineering approach, a homologous recombination–based genome engineering technique, and the integration of the resistance cassette was verified using colony PCR and molecular analysis. However, the presence of the pheA and tyrA branches was retained, leading to a partially attenuated background rather than an optimized chorismate background. In order to modulate the aromatic pathway flux, the engineered strain was grown under phenylalanine and tyrosine starvation conditions. Biochemical analysis revealed that, due to the presence of the PheA and TyrA enzymes, chorismate was most likely rapidly converted to prephenate under starvation conditions. In addition, the use of strong acidification conditions may have led to the spontaneous conversion of prephenate to phenylpyruvate before organic extraction. Therefore, the definitive confirmation of chorismate accumulation was not possible without biochemical analysis. In parallel, the BL21(DE3) Gold strain was successfully transformed with a pET28a-His8 plasmid carrying the EchC gene, thereby establishing the second component of the precursor conversion module. Although enzymatic assays were not carried out within the study period, the genetic basis for downstream biosynthetic integration was successfully established. In summary, this study outlines the methodological basis for the development of a precursor-directed metabolic engineering approach targeting chorismate-derived biosynthetic pathways. Although the attenuation and biochemical validation of this approach remain to be accomplished, this study identifies key metabolic constraints and provides a basis for the development of microbial strains for the bioproduction of echinosporin and related compounds.