E. coli strain NT1259 /pF112aroFBLkan was able to produce 14.3 g L-1 L-tryptophan within 68 h in a fed-batch process from glycerol on a 15 L scale. To gain detailed insight into metabolism of this E. coli strain in the fed-batch process, a sample of L-tryptophan producing cells was withdrawn after 47 h, was separated rapidly and then resuspended in four parallel stirred-tank bioreactors with fresh media. Four different carbon sources (glucose, glycerol, succinate, pyruvate) were supplied individually with varying feeding rates within 19 min and the metabolic reactions of the cells in the four parallel reactors were analyzed by quantification of extracellular and intracellular substrate, product and metabolite concentrations. Data analysis allowed the estimation of intracellular carbon fluxes and of thermodynamic limitations concerning intracellular concentrations and reaction energies. Carbon fluxes and intracellular metabolite concentrations enabled the estimation of elasticities and flux control coefficients by applying metabolic control analysis making use of a metabolic model considering 48 enzymatic reactions and 56 metabolites. As the flux control coefficients describe connections between enzyme activities and metabolic fluxes, they reveal genetic targets for strain improvement. Metabolic control analysis of the recombinant E. coli cells withdrawn from the fed-batch production process clearly indicated that (i) the supply of two precursors for L-tryptophan biosynthesis, L-serine and phosphoribosyl-pyrophosphate, as well as (ii) the formation of aromatic byproducts and (iii) the enzymatic steps of igps and trps2 within the L-tryptophan biosynthesis pathway have major impact on fed-batch production of L-tryptophan from glycerol and should be the targets for further strain improvements.
Keywords: Escherichia coli; Glycerol; L-Tryptophan; Metabolic analysis; Metabolic control analysis; Perturbation experiment.
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