Application of radiolabeled tracers to biocatalytic flux analysis

Radiolabeled tracers can provide valuable information about the structure o f and flux distributions in biocatalytic reaction networks. This method der ives from prior studies of glucose metabolism in mammalian systems and is i mplemented 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. Intr acellular fluxes can be directly determined from the transient radioactivit y count data by tracking the depletion of the radiolabeled metabolite and/o r the accompanying accumulation of any products formed. This technique diff ers from previous methods in that it is applied within a systems approach t o 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 me tabolite balancing and showed that indene oxidation in steady state chemost at 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 o f (2R)-indandiol, a chiral precursor of the HIV protease inhibitor, Crixiva n(R).