Application of multi-parameter flow cytometry using fluorescent probes to study substrate toxicity in the indene...

Area of Science:

• Biotechnology
• Microbial Biotransformation
• Cellular Toxicology

Background:

• Rhodococcus strain I24 bioconverts indene to cis-(1S,2R) indandiol, a key intermediate for Merck's HIV protease inhibitor.
• Assessing indene toxicity on microbial biocatalysts is crucial for optimizing biotransformation processes.

Purpose of the Study:

• To apply multiparameter flow cytometry for measuring cell physiological properties of Rhodococcus I24.
• To evaluate the toxic effects of indene on cytoplasmic membrane integrity and function.
• To correlate cellular responses with indene concentration during bioconversion.

Main Methods:

• Utilized multiparameter flow cytometry with fluorescent stains to quantify bacterial cell membrane integrity (polarized, depolarized, permeabilized).
• Measured oxygen uptake rate (OUR) for metabolic activity and optical density (OD) for biomass growth.
• Exposed Rhodococcus I24 to varying indene concentrations (up to 2.5 g/L).

Main Results:

• Indene concentrations up to 1.5 g/L showed minimal impact on cell scattering, membrane integrity, respiration, and growth.
• Indene at 0.50 g/L caused significant membrane potential dissipation, indicating proton motive force disruption.
• At 2.5 g/L, indene significantly increased dead cells, altered cation concentrations, and reduced respiration, primarily by disrupting the proton motive force.

Conclusions:

• Flow cytometry provides a powerful tool for assessing solvent toxicity at the cellular level, offering insights beyond averaged measurements of biomass or product formation.
• The study demonstrates flow cytometry's utility in measuring cell membrane properties to evaluate toxicity in whole-cell biocatalysts.
• This technique can be adapted for screening indene-resistant strains for improved biotransformation efficiency.