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Serial 13 C‐Based Flux Analysis of an l ‐Phenylalanine‐Producing E. coli Strain Using the Sensor Reactor
Author(s) -
Wahl Aljoscha,
Massaoudi Mohamed El,
Schipper Dick,
Wiechert Wolfgang,
Takors Ralf
Publication year - 2004
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp0342755
Subject(s) - phosphoenolpyruvate carboxykinase , flux (metallurgy) , metabolic flux analysis , pyruvate kinase , chemistry , biochemistry , strain (injury) , metabolic engineering , bioreactor , flux balance analysis , citrate synthase , atp synthase , enzyme , kinetics , phenylalanine , pyruvate decarboxylase , glycolysis , biology , metabolism , organic chemistry , physics , amino acid , quantum mechanics , anatomy , alcohol dehydrogenase
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.