Trace element associated reduction of norleucine and norvaline accumulation during oxygen limitation in a recombinant Escherichia coli fermentation

BACKGROUND: Norleucine and norvaline belong to a group of non-canonical amino acids which are synthesized as byproducts in the branched chain amino acid metabolism of Escherichia coli. The earlier observed misincorporation of these rare amino acids into recombinant proteins has attracted increasing attention due to the rising use of protein based biopharmaceuticals in clinical application. Experimental data revealed pyruvate overflow inducing conditions, which typically occur in oxygen limited zones of large-scale fermentations as a major reason leading to norvaline and norleucine synthesis during E. coli cultivation. Previous approaches to suppress misincorporation of norleucine and norvaline considered growth media supplementation with the relevant canonical isostructural compounds, but no research was performed on the impact of the overflow metabolism related trace elements molybdenum, nickel and selenium. These elements form essential parts of the formate hydrogen lyase (FHL) metalloprotein complex, which is a key enzyme of anaerobic pyruvate metabolism in E. coli and could therefore represent a crucial connection to the pyruvate accumulation associated biosynthesis of rare amino acids. RESULTS: In this study, the trace element associated response of recombinant antibody producing E. coli to oxygen limitation at high glucose concentration with a special focus on non-canonical amino acids was analysed. During fed-batch cultivation with provoked oxygen limitation and glucose excess norleucine and norvaline were only accumulated in the absence of molybdenum, nickel and selenium. In contrast, the trace element supplemented stress fermentation showed significantly reduced concentrations of these rare amino acids and the major signature fermentation product formate, supporting the correlation between a functional formate hydrogen lyase complex and low unspecific amino acid synthesis under oxygen limitation at high glucose concentration. CONCLUSIONS: The formation of norleucine and norvaline by recombinant E. coli during cultivation with provoked oxygen limitation and glucose excess can be reduced to levels at the detection limit by adding the trace elements molybdenum, selenium and nickel to the fermentation medium. Even under the metabolic burden during induction phase the physiologically available concentrations of non-canonical amino acids remained low. Since our results allow facile process changes that can be easily implemented to avoid the undesirable accumulation of norleucine and norvaline, we consider this study highly interesting for improved process development in E. coli based recombinant drug production and the future development of possible mechanisms to reduce misincorporation events into protein based biopharmaceuticals.