Stimulation, Monitoring, and Analysis of Pathway Dynamics by Metabolic Profiling in the Aromatic Amino Acid Pathway

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.

Modular Engineering of l -Tyrosine Production in Escherichia coli

“…It is interesting that our production data and SRM analysis are consistent with a previous study in which pulse-feeding experiments and statistical analysis identified AroB, AroA, and AroL as promising metabolic engineering targets to alleviate flux control in L-phenylalanine-producing strains (32). In our study, we demonstrated, using the shikimate operons, that AroB expression remained relatively low unless its first 15 codons were optimized.…”

Metabolomics in Biotechnology (Microbial Metabolomics)

“…Successful applications cover a broad range of research topics and microbial systems, for example, in vivo NMR [23], optimization of amino acid formation [70,71], investigation of ethanol formation in yeast [72] and beta-lactam antibiotics [73][74][75].…”

Metabolomics—The State of Art

“…The most popular method that is used for microbial cells, is quenching with cold methanol, maintaining the sample temperature below -20 o C, followed by collection of the cells by a centrifugation step prior to extraction (19). This approach has been originally developed and applied for yeast, but has later also been used in studies of different bacteria (4,5,21,23,26,49,50). For animal and plant cells liquid nitrogen is commonly utilized for quenching (43).…”

“…Knowledge about the changes of the intra-cellular metabolite concentrations offers a direct access to the identification of enzymatic kinetics especially to the in vivo kinetics of the underlying metabolic network, (4,7,28,33,44,45,46) or may elucidate limiting or inhibiting biosynthetic steps, which can be used for iterative strain optimisation (26).…”