Application of radiolabeled tracers to biocatalytic flux analysis

Abstract

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, Crixivanw.Keywords: metabolic flux; radiolabeled tracers; indene; Rhodococcus; metabolic engineering.Rational improvement of cellular properties through metabolic engineering requires detailed knowledge of cell physiology, in particular as expressed by fluxes and flux distributions through key intracellular pathways. Metabolic flux analysis provides a framework for determining pathway fluxes from the stoichiometry of the pathway reactions in combination with extracellular metabolite measurements [1][2][3]. Rates of metabolite consumption and production, conveniently measured in chemostat experiments, usually provide the necessary information to calculate the fluxes. Batch and fed-batch systems have also been used occasionally. This framework has been applied to many biological systems, such as lysine production in Corynebacterium glutamicum [4,5], and central carbon metabolism in Escherichia coli [6 -9], yeast [10,11], hybridoma cells [12][13][14], and organ tissue [15], among others.Metabolic flux analysis can be further enhanced with the inclusion of additional measurement techniques providing more detailed flux information than that embedded in typical extracellular metabolite data. Such information is particularly important for the study of underdetermined systems, which occur when the number of unknown fluxes exceeds the number of available flux measurements and metabolite balances [2]. This is usually the case with highly interconnected networks or networks comprising metabolic cycles [16]. The basis of such measurement methods is the use of isotope labels. For example, 13 C-labeling can yield flux information from ...

Optimizing bioconversion pathways through systems analysis and metabolic engineering

“…Indene metabolite concentrations were measured by using a reverse-phase HPLC assay with a Zorbax RX-C8 column (4.6 mm ϫ 25 cm, Hewlett-Packard) and a separate chiral assay using a normal phase HPLC Chiralpak AD column (Chiral Technologies, Exton, PA) as described by Chartrain et al (4). 14 C radioactive counts were measured as described elsewhere (8). The ee of the cis-[ C-tracer studies was calculated by using the total counts of each enantiomer separated by using an HPLC assay with a Chiralpak OJ column running an isocratic elution of 90% hexane and 10% isopropyl alcohol as described elsewhere (9).…”

“…1 for a representative steady state (0.065 h Ϫ1 dilution rate and 100 ppm indene air feed concentration; ref. 8). Additional information was generated by 14 C-tracer experiments in the form of (i) measures of the flux split ratio to trans-and cis-(2R)-indandiol formation from indan oxide hydrolysis, (ii) redundant measurement of the indene uptake flux determined from the chemostat gas-phase measurements, and (iii) direct calculation of the trans-(1R,2R)-indandiol dehydrogenase flux (8).…”

Metabolic flux analysis in plants using dynamic labeling technique: Application to tryptophan biosynthesis in cultured rice cells

“…The timedependent decrease in the labeling level of the precursor (Okazaki et al, 2004;Yanagimachi et al, 2001) as well as the increase in that of a down-stream metabolite in the pathway (Matsuda et al, 2005a) is monitored as a dynamic process. The value of metabolic flux is determined by fitting a model describing the labeling dynamics of the pathway to the observed labeling data.…”

Engineering GST M2-2 for High Activity with Indene 1,2-Oxide and Indication of an H-Site Residue Sustaining Catalytic Promiscuity

“…IDO is a synthon in the assembly of the drug Crixivan, and the mutant enzyme may find use in fine-chemical synthesis. 13 However, the only substrate available for our studies was the racemic mixture, and the enantioselectivity in the reaction has not yet been elucidated. Considering the possibility of glutathione addition to either of carbons 1 or 2 in the oxirane structure, four isomeric products are possible as shown with other epoxides.…”