ANALYSIS OF METABOLIC PATHWAYS BY THE GROWTH OF CELLS IN THE PRESENCEOF ORGANIC-SOLVENTS

A new approach to the analysis of metabolic pathways involving poorly water-soluble intermediates is proposed, It relies upon the ability of the hydrophobic intermediates formed by a sequence of intracellular r eactions to cross the membrane(s) and partition between aqueous and or ganic phases, when cells are incubated in the presence of a nonpolar a nd nontoxic organic solvent, As a result of this thermodynamically dri ven efflux of the formed intermediates from the cell, they accumulate in the organic medium in sufficient quantities for CC-MS analysis and identification, This enables direct determination of the sequence of c hemical reactions involved with no requirement for the isolation of ea ch individual metabolite from a cell-free extract. The feasibility of the proposed methodology has been demonstrated by the elucidation of t he biosynthesis of (R)-gamma-decalactone from (R)-ricinoleic acid cata lyzed by the yeast Sporidiobolus ruinenii grown in the presence of dec ane. The corresponding 4-hydroxy-acid intermediates, formed in the cou rse of beta-oxidation of (R)-ricinoleic acid, were simultaneously obse rved in a single experiment on the same chromatogram, Potential applic ations of this proposed methodology are briefly discussed.