4.7 Article

The Metabolic Flux Probe (MFP)-Secreted Protein as a Non-Disruptive Information Carrier for 13C-Based Metabolic Flux Analysis

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MDPI
DOI: 10.3390/ijms22179438

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C-13-labeling; metabolic flux analysis; metabolic flux probe; isotope mapping

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Novel cultivation technologies require adaptation of analytical concepts, such as metabolic flux analysis (MFA), which traditionally relies on stable-isotope labeling of biomass-bound protein. This study introduces a non-disruptive approach using secreted protein as an information carrier for C-13-based metabolic flux analysis. Results show faster incorporation of isotope information from labeled glucose into recombinant reporter protein compared to biomass-bound protein, suggesting potential for analyzing fast metabolic dynamics under isotopically nonstationary conditions.
Novel cultivation technologies demand the adaptation of existing analytical concepts. Metabolic flux analysis (MFA) requires stable-isotope labeling of biomass-bound protein as the primary information source. Obtaining the required protein in cultivation set-ups where biomass is inaccessible due to low cell densities and cell immobilization is difficult to date. We developed a non-disruptive analytical concept for C-13-based metabolic flux analysis based on secreted protein as an information carrier for isotope mapping in the protein-bound amino acids. This metabolic flux probe (MFP) concept was investigated in different cultivation set-ups with a recombinant, protein-secreting yeast strain. The obtained results grant insight into intracellular protein turnover dynamics. Experiments under metabolic but isotopically nonstationary conditions in continuous glucose-limited chemostats at high dilution rates demonstrated faster incorporation of isotope information from labeled glucose into the recombinant reporter protein than in biomass-bound protein. Our results suggest that the reporter protein was polymerized from intracellular amino acid pools with higher turnover rates than biomass-bound protein. The latter aspect might be vital for C-13-flux analyses under isotopically nonstationary conditions for analyzing fast metabolic dynamics.

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