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

Abstract

BackgroundNorleucine 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.ResultsIn 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.ConclusionsThe 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.

Comparative Study on Trace Element Excretions between Nonanuric and Anuric Patients Undergoing Continuous Ambulatory Peritoneal Dialysis

“…Zhang et al reported a relationship between Mo and Se utilization in prokaryotes [64]. Furthermore, both Se and Mo play a similar role in a recombinant Escherichia coli fermentation [65]. Based on the studies mentioned above, our study further indicated a close association between Se and Mo excretion, and the association might even exist in their metabolism and physiological function in human beings.…”

Heterogeneous oxygen availability affects the titer and topology but not the fidelity of plasmid DNA produced by Escherichia coli

“…While several previous studies have focused on the impact of oxygen availability on E. coli , only a few have studied the relevance of this parameter on product quality. For example, it has been demonstrated that oxygen limitation contributes to the synthesis of the non-canonical amino acid norvaline by E. coli [ 21 , 22 ]. This may affect the quality of the recombinant protein produced.…”

Strain engineering to prevent norleucine incorporation during recombinant protein production in Escherichia coli

“…The extent of norleucine incorporation in the recombinant protein during an E. coli fermentation depends on the nature of the recombinant protein being produced and the fermentation process (e.g., medium composition, process parameters such as pH, temperature, etc.) . When methionine was not fed during the fermentation, the recombinant proteins produced in the AF1, AF2, and AF3 fermentation processes accumulated 0.7–10% of norleucine in place of methionine residues (Table ).…”

“…The extent of norleucine incorporation in the recombinant protein during an E. coli fermentation depends on the nature of the recombinant protein being produced and the fermentation process (e.g., medium composition, process parameters such as pH, temperature, etc.). 19 When methionine was not fed during the fermentation, the recombinant proteins produced in the AF1, AF2, and AF3 fermentation processes accumulated 0.7-10% of norleucine in place of methionine residues ( Table 4). Contrary to the previous observations, 3,20 these results indicate that the norleucine incorporation is not random, as some positions had higher levels of norleucine incorporation than others in recombinant proteins produced in all three processes ( Table 4).…”