Dataset Information

Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor

ABSTRACT: Metabolic heterogeneity modulates productivity, antibiotic resistance and cancer aggressiveness. Since metabolic fluxes represent the functional output of metabolism, with glycolytic flux correlating with highly-productive phenotypes and cancer, such flux map will be indicative of the cellular metabolic state. Therefore, the quantification of metabolic fluxes is vital to identify the existence of metabolic subpopulations and to understand the process of their emergence at the single-cell level. However, so far inference of metabolic fluxes in individual cells is not possible as no method is available. Here, we developed a biosensor for glycolytic flux measurements in single yeast cells drawing on the robust correlation between fructose-1,6-bisphosphate (FBP) and flux levels in yeast, and using the B. subtilis FBP-binding transcription factor CggR. We followed a systematic engineering approach starting from promoter design, computational protein design and protein engineering, accompanied by strict characterization of the biosensor using different biochemical methods, proteomics, metabolomics and physiological analyses. As proof of principle, we applied the biosensor in vivo in the search for metabolic subpopulations in yeast cultures and, using fluorescence microscopy, we demonstrated that quiescent yeast cells have low glycolytic fluxes in comparison to coexisting dividing cells. We anticipate that our biosensor will contribute with unprecedented resolution for the study of metabolic subpopulations, to understand how and why metabolic subpopulations emerge and, very importantly, give clues on how to counteract the undesirable effects of such.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Cell Culture

SUBMITTER: Alexander Schmidt

LAB HEAD: Alexander Schmidt

PROVIDER: PXD012964 | Pride | 2019-12-17

REPOSITORIES: Pride

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Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor.

Monteiro Francisca F Hubmann Georg G Takhaveev Vakil V Vedelaar Silke R SR Norder Justin J Hekelaar Johan J Saldida Joana J Litsios Athanasios A Wijma Hein J HJ Schmidt Alexander A Heinemann Matthias M

Molecular systems biology 20191201 12

Metabolic heterogeneity between individual cells of a population harbors significant challenges for fundamental and applied research. Identifying metabolic heterogeneity and investigating its emergence require tools to zoom into metabolism of individual cells. While methods exist to measure metabolite levels in single cells, we lack capability to measure metabolic flux, i.e., the ultimate functional output of metabolic activity, on the single-cell level. Here, combining promoter engineering, com ...[more]

PMID: 31885198

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