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

摘要

With the aid of the recently developed Sensor reactor system, a series of three subsequent C-13 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 (luring 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.