title: Analysis of metabolic pathways by the growth of cells in the presence of organic solvents.
abstract: 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 reactions to cross the membrane(s) and partition between aqueous and organic phases, when cells are incubated in the presence of a nonpolar and nontoxic organic solvent. As a result of this thermodynamically driven efflux of the formed intermediates from the cell, they accumulate in the organic medium in sufficient quantities for GC-MS analysis and identification. This enables direct determination of the sequence of chemical reactions involved with no requirement for the isolation of each individual metabolite from a cell-free extract. The feasibility of the proposed methodology has been demonstrated by the elucidation of the biosynthesis of (R)-gamma-decalactone from (R)-ricinoleic acid catalyzed by the yeast Sporidiobolus ruinenii grown in the presence of decane. The corresponding 4-hydroxy-acid intermediates, formed in the course of beta-oxidation of (R)-ricinoleic acid, were simultaneously observed in a single experiment on the same chromatogram. Potential applications of this proposed methodology are briefly discussed.
authors.affiliation: Institut National de la Recherche Agronomique, Laboratoire de Recherches sur les Arômes, Dijon, France.
authors.forename: H E
authors.initials: HE
authors.lastname: Spinnler
authors.forename: C
authors.initials: C
authors.lastname: Ginies
authors.forename: J A
authors.initials: JA
authors.lastname: Khan
authors.forename: E N
authors.initials: EN
authors.lastname: Vulfson