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

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.