Improvement of constraint-based flux estimation during L-phenylalanine production with Escherichia coli using targeted knock-out mutants

Michael Weiner, Julia Tröndle, Christoph Albermann, Georg A. Sprenger, Dirk Weuster-Botz

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

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 ΔmaeA pF81kan and E. coli FUS4.11 ΔmaeA ΔmaeB pF81kan). 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.

Original languageEnglish
Pages (from-to)1406-1416
Number of pages11
JournalBiotechnology and Bioengineering
Volume111
Issue number7
DOIs
StatePublished - Jul 2014

Keywords

  • Constraint-based flux estimation
  • Flux variability analysis
  • Glycerol
  • L-phenylalanine
  • Malic enzymes

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