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

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.

Automated summary

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.