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Improvement of constraint‐based flux estimation during L‐phenylalanine production with Escherichia coli using targeted knock‐out mutants
Author(s) -
Weiner Michael,
Tröndle Julia,
Albermann Christoph,
Sprenger Georg A.,
WeusterBotz Dirk
Publication year - 2014
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.25195
Subject(s) - phenylalanine , escherichia coli , flux (metallurgy) , mutant , biochemistry , metabolic flux analysis , intracellular , metabolic engineering , carbon flux , metabolism , malic enzyme , chemistry , biology , enzyme , amino acid , gene , dehydrogenase , organic chemistry , ecology , ecosystem
Fed‐batch production of the aromatic amino acid L‐phenylalanine was studied with recombinant Escherichia coli strains on a 15 L‐scale using glycerol as carbon source. Flux Variability Analysis (FVA) was applied for intracellular flux estimation to obtain an insight into intracellular flux distribution during L‐phenylalanine production. Variability analysis revealed great flux uncertainties in the central carbon metabolism, especially concerning malate consumption. Due to these results two recombinant strains were genetically engineered differing in the ability of malate degradation and anaplerotic reactions ( E. coli FUS4.11 Δ maeA pF81 kan and E. coli FUS4.11 Δ maeA Δ maeB pF81 kan ). Applying these malic enzyme knock‐out mutants in the standardized L‐phenylalanine production process resulted in almost identical process performances (e.g., L‐phenylalanine concentration, production rate and byproduct formation). This clearly highlighted great redundancies in central metabolism in E. coli . Uncertainties of intracellular flux estimations by constraint‐based analyses during fed‐batch production of L‐phenylalanine were drastically reduced by application of the malic enzyme knock‐out mutants. Biotechnol. Bioeng. 2014;111: 1406–1416. © 2014 Wiley Periodicals, Inc.