Application of radiolabeled tracers to biocatalytic flux analysis.

TitleApplication of radiolabeled tracers to biocatalytic flux analysis.
Publication TypeJournal Article
Year of Publication2001
AuthorsYanagimachi, KS, Stafford, DE, Dexter, AF, Sinskey, AJ, Drew, S, Stephanopoulos, G
JournalEur J Biochem
Date Published2001 Sep
KeywordsCarbon Radioisotopes, Catalysis, Chromatography, High Pressure Liquid, Fermentation, HIV Protease Inhibitors, Hydrolysis, Indenes, Indinavir, Kinetics, Molecular Probes, Oxygenases, Rhodococcus

Radiolabeled tracers can provide valuable information about the structure of and flux distributions in biocatalytic reaction networks. This method derives from prior studies of glucose metabolism in mammalian systems and is implemented by pulsing a culture with a radiolabeled metabolite that can be transported into the cells and subsequently measuring the radioactivity of all network metabolites following separation by liquid chromatography. Intracellular fluxes can be directly determined from the transient radioactivity count data by tracking the depletion of the radiolabeled metabolite and/or the accompanying accumulation of any products formed. This technique differs from previous methods in that it is applied within a systems approach to the problem of flux determination. It has been used for the investigation of the indene bioconversion network expressed in Rhodococcus sp. KY1. Flux estimates obtained by radioactive tracers were confirmed by macroscopic metabolite balancing and showed that indene oxidation in steady state chemostat cultures proceeds primarily through a monooxygenase activity forming (1S,2R)-indan oxide, with no dehydrogenation of trans-(1R,2R)-indandiol. These results confirmed the significance of indan oxide formation and identified the hydrolysis of indan oxide as a key step in maximizing the production of (2R)-indandiol, a chiral precursor of the HIV protease inhibitor, Crixivan.

Alternate JournalEur J Biochem
Citation Key122
PubMed ID11559364
Grant List2T32 GM08334-10 / GM / NIGMS NIH HHS / United States