Metabolic control analysis of microbial fed-batch production of L-tryptophan

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Microbial production processes are subject to regulatory mechanisms on the cellular level. They determine the maximum specific production rate as well as the overall process duration. Therefore, modern approaches for process optimization use biological (metabolic design) and engineering techniques (process design). By using microbial L-phenylalanine production from glycerol with E. coli as an example, the application of Metabolic Control Analysis (MCA) successfully revealed the limiting enzymatic reactions at various phases of a standardized fed-batch process. For this purpose, rapid media transition and short-time experiments (20 min) were established in parallelized small-scale stirred-tank reactors. This enabled the perturbation of the metabolism of cells from the production process in different directions by feeding various carbon sources. By expression of suitable phosphate-antiporter systems, also phosphorylated substrates like phosphoenolpyruvate could be applied during the short-time experiments. The combination of the stochastic methods from MCA and a thermodynamic analysis of the reaction network allowed the determination of control parameters in E. coli metabolism from metabolome and fluxome data that were generated from the short-time experiments. For the first time, it was thus possible to show changes in the metabolic control during L-phenylalanine production in the course of the production process. This new method for MCA of cells from production processes was established in a successful cooperation between the project partners (Georg Sprenger, University of Stuttgart and Dirk Weuster-Botz, Technische Universität München) and it shall now be extended and applied to microbial production of the amino acid L-tryptophan from glycerol with E. coli. The analysis of microbial L-tryptophan production in a production process highly increases the complexity of the metabolic network to be analyzed. The main reasons for the choice of this topic are the additional crosslinks between aromatic amino acid biosynthesis and central metabolism caused by the required synthesis of L-serine and 5-phospho-D-ribose-1-diphosphate. The aim of this research project is to identify the controlling metabolic reactions in the producing organism at specific process times during a fed-batch process for L-tryptophan production from glycerol with E. coli. Strains and process strategies for L-tryptophan production will be established. Furthermore, transporter systems for the uptake of important metabolic intermediates shall be implemented. This will enable the identification of controlling steps in E. coli metabolism during the production process by applying short-time experiments, fluxome, metabolome as well as transcriptome analyses and MCA. These results will be used for further strain improvement and validation in the fed-batch production process.