Abstract
Using a concerted approach of biochemical standard preparation,
analytical access via LC-MS/MS, glucose pulse, metabolic profiling, and
statistical data analysis, the metabolism dynamics in the aromatic amino
acid pathway has been stimulated, monitored, and analyzed in different
tyrosine-auxotrophic L-phenylalanine-producing Escherichia coli strains.
During the observation window from -4 s (before) up to 27 s after the
glucose pulse, the dynamics of the first five enzymatic reactions in the
aromatic amino acid pathway was observed by measuring intracellular
concentrations of 3-deoxy-D-arabino-heptulosonate 7-phosphate DAH(P),
3-dehydroquinate (3-DHQ), 3-dehydroshikimate (3-DHS), shikimate
3-phosphate (S3P), and shikimate (SHI), together with the pathway
precursors phosphoenolpyruvate (PEP) and P5P, the lumped pentose
phosphate pool as an alternative to the nondetectable erythrose
4-phosphate (E4P). Provided that a sufficient fortification of the
carbon flux into the pathway of interest is ensured, respective
metabolism dynamics can be observed. On the basis of the intracellular
pool measurements, the standardized pool velocities were calculated, and
a simple, data-driven criterion-called ``pool efflux capacity''
(PEC)-is derived. Despite its simplifying system description, the
criterion managed to identify the well-known AroB limitation in the E.
coli strain A (genotype Delta(pheA tyrA aroF)/pJF119EH aroF(fbr)
pheA(fbr) amp) and it also succeeded to identify AroL and AroA (in
strain B, genotype Delta(pheA tyrA aroF)/pJF119EH aroF(fbr) pheA(fbr)
aroB amp) as promising metabolic engineering targets to alleviate
respective flux control in subsequent L-Phe producing strains.
Furthermore, using of a simple correlation analysis, the reconstruction
of the metabolite sequence of the observed pathway was enabled. The
results underline the necessity to extend the focus of glucose pulse
experiments by studying not only the central metabolism but also
anabolic pathways.
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