Integrated constraints based analysis of an engineered violacein pathway in Escherichia coli

摘要

Strategies towards optimal violacein biosynthesis, a potential drug molecule, need systems level coordination of enzymatic activities of individual genes in a multigene operon vioABCDE. Constraints-based flux balance analysis of an extendediAF1260 model (iAF1260vio) with a reconstructed violacein module predicted growth and violacein yields inEscherichia coliaccurately. Shadow price (SP) analysis identified tryptophan metabolism and NADPH as limiting. Increased tryptophan levels in Δpgi& ΔpheA were validated usingin silicogene deletion analysis. Phenotypic phase plane (PhPP) analysis highlighted sensitivity between tryptophan and NADPH for violacein synthesis at molar growth yields. A synthetic VioABCDE operon (SYNO) sequence was designed to maximize Codon Adaptive Index (CAI: 0.9) and tune translation initiation rates (TIR: 2–50 fold higher) inE. coli. All pSYNE. colitransformants produced higher violacein, with a maximum six-fold increase in yields. The rational designE. coli:ΔpheASYN: gave the highest violacein titers (33.8?mg/l). Such integrated approaches targeting multiple molecular hierarchies in the cell can be extended further to increase violacein yields.