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

Biermann M, Linnemann J, Knüpfer U, Vollstädt S, Bardl B, Seidel G, Horn U - Microb. Cell Fact. (2013)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Leibniz-Institute for Natural Product Research and Infection Biology (HKI), Beutenbergstrasse 11a, 07745 Jena, Germany. michael.biermann@hki-jena.de.

ABSTRACT

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.

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E. coli RV308 growth dynamics during different cultivation modes used in this study. The graphs illustrate experimental data for optical density (OD600nm), DOT and glucose concentration. (A) Glucose limited fed-batch cultivation on mineral salt medium with continuous feed of glucose at constant feed rate of 10 g L-1 and indicated time point of induction (dashed line). (B) and (C) same as (A) but with a downshift of DOT by a decrease of stirrer speed (arrow) and either without (B) or with (C) the addition of extra trace elements to the fermentation medium. Stirrer downshift in (B) and (C) was performed 20 min before the start of glucose feeding to ensure glucose excess during cultivations. Samples were collected from the beginning of continuous glucose feed.
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Figure 1: E. coli RV308 growth dynamics during different cultivation modes used in this study. The graphs illustrate experimental data for optical density (OD600nm), DOT and glucose concentration. (A) Glucose limited fed-batch cultivation on mineral salt medium with continuous feed of glucose at constant feed rate of 10 g L-1 and indicated time point of induction (dashed line). (B) and (C) same as (A) but with a downshift of DOT by a decrease of stirrer speed (arrow) and either without (B) or with (C) the addition of extra trace elements to the fermentation medium. Stirrer downshift in (B) and (C) was performed 20 min before the start of glucose feeding to ensure glucose excess during cultivations. Samples were collected from the beginning of continuous glucose feed.

Mentions: In consequence the aim of this work focussed on the so far disregarded influence of supplementary molybdenum, nickel and selenium on the formation of the rare amino acids norleucine and norvaline during an oxygen downshift in recombinant E. coli. We therefore chose an E. coli RV308 production strain, expressing the camelid antibody domain B10 as a model for possible target molecules underlying current clinical interest. In order to compare the obtained results to balanced cultivation conditions a glucose limited fed-batch reference set-up with dissolved oxygen tension (DOT) above 20% was performed (see Figure 1A). To ensure large-scale stress conditions, oxygen limitation and glucose excess during cultivation was provoked by a stepwise stirrer down-shift and continuous linear feeding in the scale-down fermentation set-ups (see Figure 1B,C). To allow proper analysis of the E. coli cultivation samples, the oxygen downshift was started at optical densities of about OD600nm = 35, since lower cell densities showed crucially varying results in amino acid analysis (unpublished data). The growth of E. coli during reference fermentation indicated typical substrate consumption and increasing biomass production up to a high cell density of OD600nm = 72 at the end of induction phase. In the experiments wherecells were exposed to an permanent oxygen downshift, cell growth during induction phase arrested at lower levels around OD600nm = 30 as expected. The oxygen limited cultivation with additional Mo, Ni and Se showed slightly lower cell densities between OD600nm = 26 and 32 during the induction phase compared to the cultivation without extra trace elements with an OD600nm between 30 and 36. This effect is very likely a consequence of an increased carbon loss through a higher carbon dioxide production related to active FHL or an increased lactate accumulation which was observed in similar experiments with permanent oxygen downshift [27]. Concerning the lowered biomass yield as a critical concern in industrial protein production, the addition of Mo, Ni and Se could still be an option to test, since the authors also reported no negative effects during experiments with oscillating oxygen and glucose concentrations.


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

Biermann M, Linnemann J, Knüpfer U, Vollstädt S, Bardl B, Seidel G, Horn U - Microb. Cell Fact. (2013)

E. coli RV308 growth dynamics during different cultivation modes used in this study. The graphs illustrate experimental data for optical density (OD600nm), DOT and glucose concentration. (A) Glucose limited fed-batch cultivation on mineral salt medium with continuous feed of glucose at constant feed rate of 10 g L-1 and indicated time point of induction (dashed line). (B) and (C) same as (A) but with a downshift of DOT by a decrease of stirrer speed (arrow) and either without (B) or with (C) the addition of extra trace elements to the fermentation medium. Stirrer downshift in (B) and (C) was performed 20 min before the start of glucose feeding to ensure glucose excess during cultivations. Samples were collected from the beginning of continuous glucose feed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842802&req=5

Figure 1: E. coli RV308 growth dynamics during different cultivation modes used in this study. The graphs illustrate experimental data for optical density (OD600nm), DOT and glucose concentration. (A) Glucose limited fed-batch cultivation on mineral salt medium with continuous feed of glucose at constant feed rate of 10 g L-1 and indicated time point of induction (dashed line). (B) and (C) same as (A) but with a downshift of DOT by a decrease of stirrer speed (arrow) and either without (B) or with (C) the addition of extra trace elements to the fermentation medium. Stirrer downshift in (B) and (C) was performed 20 min before the start of glucose feeding to ensure glucose excess during cultivations. Samples were collected from the beginning of continuous glucose feed.
Mentions: In consequence the aim of this work focussed on the so far disregarded influence of supplementary molybdenum, nickel and selenium on the formation of the rare amino acids norleucine and norvaline during an oxygen downshift in recombinant E. coli. We therefore chose an E. coli RV308 production strain, expressing the camelid antibody domain B10 as a model for possible target molecules underlying current clinical interest. In order to compare the obtained results to balanced cultivation conditions a glucose limited fed-batch reference set-up with dissolved oxygen tension (DOT) above 20% was performed (see Figure 1A). To ensure large-scale stress conditions, oxygen limitation and glucose excess during cultivation was provoked by a stepwise stirrer down-shift and continuous linear feeding in the scale-down fermentation set-ups (see Figure 1B,C). To allow proper analysis of the E. coli cultivation samples, the oxygen downshift was started at optical densities of about OD600nm = 35, since lower cell densities showed crucially varying results in amino acid analysis (unpublished data). The growth of E. coli during reference fermentation indicated typical substrate consumption and increasing biomass production up to a high cell density of OD600nm = 72 at the end of induction phase. In the experiments wherecells were exposed to an permanent oxygen downshift, cell growth during induction phase arrested at lower levels around OD600nm = 30 as expected. The oxygen limited cultivation with additional Mo, Ni and Se showed slightly lower cell densities between OD600nm = 26 and 32 during the induction phase compared to the cultivation without extra trace elements with an OD600nm between 30 and 36. This effect is very likely a consequence of an increased carbon loss through a higher carbon dioxide production related to active FHL or an increased lactate accumulation which was observed in similar experiments with permanent oxygen downshift [27]. Concerning the lowered biomass yield as a critical concern in industrial protein production, the addition of Mo, Ni and Se could still be an option to test, since the authors also reported no negative effects during experiments with oscillating oxygen and glucose concentrations.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Leibniz-Institute for Natural Product Research and Infection Biology (HKI), Beutenbergstrasse 11a, 07745 Jena, Germany. michael.biermann@hki-jena.de.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus