A Systems Approach for Metabolic Engineering of Tryptophan in Escherichia Coli

Biosynthesis of L-tryptophan is strictly controlled at several steps in Escherichia coli. The economic importance of this amino acid and its role as a precursor in many downstream pathways for secondary metabolites, makes it imperative to metabolically engineer a host strain for its over production. Prospective design analysis using in silico metabolic network models is essential to the success of identifying target genes for optimization of desired metabolite production. More over, combination of prospective design, high throughput data generation and evolutionary engineering hold great promise. This talk focuses on the development and analysis of a tryptophan over producing strain of E. coli using a gene deletion strategy to channel flux through the aromatic amino acid production branch to tryptophan. LC-MS analysis showed about ten-fold increase in tryptophan biosynthesis in a multiple knockout ΔTrpRΔpgi E. coli strain. LC-MS has been further used to characterize the intracellular profiles of amino acid production to study non-intuitive coupling between biosynthesis of amino acids. Further, in order to make the strain function efficiently in a hemi-cellulosic environment, directed evolution under suitable selection pressures activated a cryptic operon for native biosynthesis of a putative β xylosidase in the E. coli strain.