Serial 13C-Based Flux Analysis of an L-Phenylalanine-Producing E. coli Strain Using the Sensor Reactor

Abstract

With the aid of the recently developed Sensor reactor system, a series of three subsequent (13)C labeling experiments was performed mirroring the l-phenylalanine (l-Phe) production phase of a recombinant E. coli strain that was cultivated under industry-like conditions in a 300 L bioreactor. On the basis of the data from NMR labeling analysis, three subsequent flux patterns were successfully derived monitoring the l-Phe formation during an observation window from 14 to 23.3 h process time. Linear programming was performed to identify optimal flux patterns for l-Phe formation. Additionally, flux sensitivity analysis was used to identify the most promising metabolic engineering target. As a result, high rates of phosphoenolpyruvate (PEP) to pyruvate (PYR) conversion were identified as the most important reason for deterioration of the l-Phe/glucose yield from 20 to finally 11 mol %. Considering the characteristics of the enzyme kinetics involved, the working hypothesis was formulated that phosphoenolpyruvate synthase activity was increasingly hampered by rising oxaloacetate and 2-oxoglutarate concentrations, while at the same time pyruvate kinase activity arose due to activation by fructose 1,6-diphosphate. Hence, pps overexpression should be performed to optimize the existing production strain.

Stationary versus non-stationary 13C-MFA: A comparison using a consistent dataset

“…The predominating industrial process conditions are batch and fed-batch. It has been shown in several studies that 13 C-MFA can be applied under these conditions (Costenoble et al, 2007;Drysch et al, 2004;Fischer et al, 2004;Iwatani et al, 2007;Wahl et al, 2004;Wittmann and Heinzle, 2002). 3.…”

“…Analytical equipment. In principle, labeling information can be provided by measuring isotopic patterns of proteinogenic amino acids or metabolic intermediates using NMR Marx et al, 1996;Sauer et al, 1997;Wahl et al, 2004) or MS devices (Antoniewicz et al, 2007a;Fischer et al, 2004;Huege et al, 2007;van Winden et al, 2005;. When applying the isotopically non-stationary approach measurements from low concentrated intermediates (including labeling fractions and pool sizes, cf.…”

Metabolic flux analysis in biotechnology processes

“…Phenylalanine, for example is used for the production of the sweetener, Aspartame. Wahl et al (2004) conducted a series of three subsequent labeling experiments mirroring the phenylalanine production phase of E. coli grown under industry-like conditions in a 300 l reactor. The large flux from PEP to pyruvate was suggested to be responsible for the decrease in phenylalanine yield, since PEP is a pathway precursor for phenylalanine, and that PEP synthase was an ideal target for overexpression.…”

Metabolic Engineering in the -omics Era: Elucidating and Modulating Regulatory Networks

“…The product of this gene, carbon storage regulator protein, binds several species of mRNA molecules responsible for carbon metabolism, specifically repressing pckA and pps (encoding phosphoenolpyruvate carboxykinase and phosphoenolpyruvate synthase), thus inhibiting the formation of phosphoenolpyruvate, an essential precursor of aromatic amino acid biosynthesis. Not surprisingly, an independent study reported increased titers for phenylalanine in E. coli strains in which the pps gene was overexpressed due to increased phosphoenolpyruvate availability (227). Similarly, disrupting three repressors of galactose uptake (Gal6p, Gal80p, and Mig1p) in S. cerevisiae increased galactose consumption (161).…”