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

Abstract

The bioconversion of indene to cis-(1S,2R) indandiol, a potential key intermediate in the synthesis of Merck's HIV protease inhibitor, CRIXIVAN trade mark, can be achieved using a Rhodococcus strain. This study using Rhodococcus I24 reports on the application of multiparameter flow cytometry for the measurement of cell physiological properties based on cytoplasmic membrane (CM) integrity and membrane depolarization as indicators of toxic effects of the substrate, indene. Quantification of intact polarized CM, intact depolarized CM and permeabilized CM of a large population of bacterial cells has been conducted using specific intracellular and membrane-binding fluorescent stains. Measurements of oxygen uptake rate (OUR) and optical density (OD) as indicators of metabolic activity and biomass growth, respectively, were also made. Indene concentrations of up to 0.25 g/L (0.037 g indene/g dry cell weight) did not significantly (<5% compared to control) affect cell light-scattering properties, intact CM, membrane polarization, respiratory activity, or biomass growth. Between this value and 1.5 g/L (0.221 g indene/g dry cell weight), the changes in intact CM, respiratory activity and biomass growth were relatively insignificant (<5% compared to control), although dissipation of the membrane potential of a significant proportion of the cell population occurred at 0.50 g/L (0.074 g indene/g dry cell weight). At 2.5 g/L (0.368 g indene/g dry cell weight) there was a significant increase in the dead cell population, accompanied by changes in the extracellular cationic concentrations and substantial decrease in respiratory activity. The primary effect of indene toxicity was the disruption of the proton motive force across the cytoplasmic membrane which drives the formation of ATP. The disruption of the proton motive force may have been due to the measured changes in proton permeability across the membrane. In addition, indene may have directly inhibited the membrane-bound enzymes related to respiratory activity. The overall consequence of this was reduced respiratory activity and biomass growth. The cell physiological properties measured via flow cytometry are important for understanding the effects of toxicity at the cellular level which neither measurements of biomass growth or indandiol formation rates can provide since both are cell averaged measurements. The technique described here can also be used as a generic tool for measuring cell membrane properties in response to toxicity of other indene-resistant strains that may be possible to use as recombinant hosts to perform the biotransformation of indene. This study has demonstrated that flow cytometry is a powerful tool for the measurement of cell physiological properties to assess solvent toxicity on whole cell biocatalysts.