Abstract
L-tryptophan production from glycerol with Escherichia coli was analysed by perturbation studies and metabolic control analysis. The insertion of a non-natural shikimate transporter into the genome of an Escherichia coli L-tryptophan production strain enabled targeted perturbation within the product pathway with shikimate during parallelised short-term perturbation experiments with cells withdrawn from a 15 L fed-batch production process. Expression of the shikimate/H+-symporter gene (shiA) from Corynebacterium glutamicum did not alter process performance within the estimation error. Metabolic analyses and subsequent extensive data evaluation were performed based on the data of the parallel analysis reactors and the production process. Extracellular rates and intracellular metabolite concentrations displayed evident deflections in cell metabolism and particularly in chorismate biosynthesis due to the perturbations with shikimate. Intracellular flux distributions were estimated using a thermodynamics-based flux analysis method, which integrates thermodynamic constraints and intracellular metabolite concentrations to restrain the solution space. Feasible flux distributions, Gibbs reaction energies and concentration ranges were computed simultaneously for the genome-wide metabolic model, with minimum bias in relation to the direction of metabolic reactions. Metabolic control analysis was applied to estimate elasticities and flux control coefficients, predicting controlling sites for L-tryptophan biosynthesis. The addition of shikimate led to enhanced deviations in chorismate biosynthesis, revealing a so far not observed control of 3-dehydroquinate synthase on L-tryptophan formation. The relative expression of the identified target genes was analysed with RT-qPCR. Transcriptome analysis revealed disparities in gene expression and the localisation of target genes to further improve the microbial L-tryptophan producer by metabolic engineering.
Highlights
Metabolic engineering has been widely used to reroute fluxes towards the desired products to overcome production limitations of biotechnological processes due to constraints and regulations in cell metabolism
The successful integration and expression of the shiA-transporter gene was verified by real-time quantitative PCR (RT-qPCR) (Supplementary Fig. 1) and the shikimate uptake by L-tryptophan producing cells without shikimate-transporter (E. coli NT1259 pF112aroFBLKan was analysed in comparison to cells with the integrated shikimate-transporter (E. coli NT1259 shiACg pF112aroFBLKan) in parallel cultivations in shake-flasks
The integration of the shiA-gene from Corynebacterium glutamicum enabled the uptake of shikimate with a measured rate of 0.26 mmol gCDW−1 h−1 in shake-flask, whereas the concentration of shikimate remained constant during the cultivation of the cells without transporter (Supplementary Fig. 2)
Summary
Metabolic engineering has been widely used to reroute fluxes towards the desired products to overcome production limitations of biotechnological processes due to constraints and regulations in cell metabolism. Rational design is impeded by the complexity of biosynthesis pathways, and in vitro analyses of single enzymes cannot display processes in vivo realistically. Perturbation experiments have opened up the possibility of characterising metabolic pathways. As a reaction to the disturbance, the metabolism will be shifted, and a new metabolic steady state can be established within minutes inside the cells [1, 5]. Rapid Media Transition is one option for conducting metabolic analyses [4]. Interesting time points in continuous or semicontinuous fed-batch processes serve as reference states from which cell broth is sampled, centrifuged and rapidly transferred with fresh media into smaller stirred-tank bioreactors used for short-term perturbation studies. Disturbance of the reference state can be avoided by separation
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