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Phosphoenolpyruvate Transporter Enables Targeted Perturbation During Metabolic Analysis of L‐Phenylalanine Production With Escherichia coli
Author(s) -
Tröndle Julia,
Albermann Christoph,
Weiner Michael,
Sprenger Georg A.,
WeusterBotz Dirk
Publication year - 2018
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201700611
Subject(s) - escherichia coli , phosphoenolpyruvate carboxykinase , transporter , phenylalanine , metabolism , biochemistry , intracellular , chemistry , glycerol , metabolic engineering , biology , biophysics , enzyme , amino acid , gene
Usually perturbation of the metabolism of cells by addition of substrates is applied for metabolic analysis of production organisms, but perturbation studies are restricted to the endogenous substrates of the cells under study. The goal of this study is to overcome this limitation by making phosphoenolpyruvate (PEP) available for perturbation studies with Escherichia coli producing L‐phenylalanine. A production strain overexpressing a PEP‐transporter variant (UhpT‐D388C) is applied in a standardized fed‐batch production‐process on a 42 L‐scale. Four parallel short‐term perturbation experiments of 20 min are performed with glucose and glycerol as fed‐batch carbon sources after rapid media transition of cells from the production‐process. PEP is added after 9 min and is immediately consumed by the cells with up to 1.5 mmol g CDW −1 h −1 . L‐phenylalanine production rates increased by up to 200% after addition of PEP. This clearly indicates an intracellular PEP‐limitation in the L‐phenylalanine production strain under study. Thus, it is shown that overexpressing specific transporters for analytical reasons makes exogenous substrates available as perturbation substrates for metabolic analyses of cells sampled from production‐processes and thereby allows a very targeted perturbation of whole‐cell metabolism.