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Rapid Fluorescence Assay for Polyphosphate in Yeast Extracts Using JC-D7.

Alexander Deitert1, Jana Fees1, Anna Mertens1

  • 1Institute of Applied Microbiology-iAMB, Aachen Biology and Biotechnology-ABBt, RWTH Aachen University, Aachen, Germany.

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Summary

A new fluorescent dye, JC-D7, offers a simple method for semi-quantifying polyphosphate (polyP) in yeast extracts. This assay aids in screening microbes for enhanced biotechnological polyP production and sustainable phosphorus use.

Keywords:
JC‐D7Saccharomyces cerevisiaepolymerspolyphosphateyeast

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Area of Science:

  • Biochemistry
  • Microbiology
  • Analytical Chemistry

Background:

  • Polyphosphate (polyP) is a ubiquitous molecule with diverse cellular roles and industrial applications.
  • Accurate polyP quantification remains analytically challenging, with 31P NMR as the current gold standard.
  • Existing methods limit high-throughput screening for polyP production.

Purpose of the Study:

  • To develop a simple, sensitive, and cost-effective method for semi-quantitative polyP analysis.
  • To evaluate the fluorescent dye JC-D7 for polyP detection in microbial extracts.
  • To enable high-throughput screening of polyP production in yeast.

Main Methods:

  • A novel staining method using the fluorescent dye JC-D7 was developed for polyP evaluation.
  • The fluorescence response of JC-D7 was analyzed concerning polyP concentration and chain length.
  • Interference from inorganic phosphate, trace elements, and mineral salts was assessed.
  • The assay was validated using yeast extracts from strains with altered (poly)phosphate homeostasis.

Main Results:

  • JC-D7 fluorescence correlated with polyP concentration (0.5–500 µg/mL) and chain length, enabling semi-quantification.
  • The dye showed no significant interference from inorganic phosphate up to 50 mM.
  • Certain trace elements and toxic mineral salts diminished JC-D7 fluorescence, impacting polyP-metal complex analysis.
  • The assay proved robust and sensitive for detecting polyP in yeast extracts.

Conclusions:

  • JC-D7 staining provides a simple and sensitive method for semi-quantitative polyP detection in yeast and potentially other microbes.
  • The assay's simplicity facilitates high-throughput screening for enhanced biotechnological polyP production.
  • This method contributes to sustainable phosphorus utilization by optimizing polyP bioproduction.