Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

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

Version 1 : Received: 15 July 2021 / Approved: 16 July 2021 / Online: 16 July 2021 (14:49:34 CEST)

How to cite: Dusny, C.; Schmid, A. The Metabolic Flux Probe (MFP) - Secreted Protein as a Non-Disruptive Information Carrier for 13C-Based Metabolic Flux Analysis. Preprints 2021, 2021070381 (doi: 10.20944/preprints202107.0381.v1). Dusny, C.; Schmid, A. The Metabolic Flux Probe (MFP) - Secreted Protein as a Non-Disruptive Information Carrier for 13C-Based Metabolic Flux Analysis. Preprints 2021, 2021070381 (doi: 10.20944/preprints202107.0381.v1).

Abstract

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 scarce or inaccessible due to low cell densities and cell immobilization is difficult to date. We developed a non-disruptive analytical concept for 13C-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 13C-flux analyses under isotopically nonstationary conditions for analyzing fast metabolic dynamics.

Subject Areas

13C-labeling; Metabolic flux analysis; Metabolic flux probe; Isotope mapping

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