Serial 13C-based flux analysis of an L-phenylalanine-producing E. coli strain using the sensor reactor.
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.
Wahl
Aljoscha
A
Institute of Biotechnology, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.
El Massaoudi
Mohamed
M
Schipper
Dick
D
Wiechert
Wolfgang
W
Takors
Ralf
R
D019149:Bioreactors / Q000382:microbiology*; D002247:Carbon Isotopes / Q000378:metabolism; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D003198:Computer Simulation; D004734:Energy Metabolism / Q000502:physiology; D004926:Escherichia coli / Q000145:classification / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000378:metabolism; D010649:Phenylalanine / Q000096:biosynthesis*; D011382:Programming, Linear; D013045:Species Specificity
D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D010649:Phenylalanine