Understanding cellular metabolism using stable isotopic labeling, metabolic flux analysis and kinetic modeling

Neelanjan Sengupta, Purdue University

Abstract

There is focus on increasing scent production in flowers by metabolic engineering due to its broad impact in the perfume and horticulture industry. Metabolic engineering of flowers to enhance the scent production can be done in an efficient and targeted manner, if there is mechanistic information about the kinetics and regulation of the pathways involved in scent production. Thus a kinetic model of the benzenoid (phenylpropanoid) network was developed using Michaelis-Menten type equations to simulate whole network responses to different concentrations of supplied phenylalanine (Phe) in petunia flowers. The in vivo kinetic parameters were obtained by network decomposition and non-linear least squares optimization of data from petunia flowers supplied with 75 and 150 mM 2H5-Phe. The generated kinetic model was validated using flowers from transgenic petunia plants. Further, the model was used for metabolic control analysis, where flux control coefficients were calculated for fluxes around key branch points, and revealed that phenylacetaldehyde synthase activity is the primary controlling factor for the phenylacetaldehyde branch of the benzenoid network, while control of flux through the β-oxidative and non-β-oxidative pathways is highly distributed (pathways involved in production of key scent compound methyl benzoate). Further, experimental methods for compartment specific labeling information were developed and applied to Snapdragon (a flower) for generation of compartment specific labeling information required for MFA. Chlamydomonas reinhardtii is a well researched alga that is capable of synthesizing lipids, and thus is a promising source of the liquid fuel biodiesel. It has been reported that C. reinhardtii has enhanced lipid production in mixotrophic growth on acetate, particularly under nitrogen starvation. One of the key goals for successful biofuel production from algae is to maximize lipid content while not impairing biomass production. Thus, as a base case, it is important to characterize the way C. reinhardtii uses acetate in terms of its intracellular reaction rates (metabolic fluxes) and its effect on lipid and biomass production. Feeding 13C labeled acetate and performing metabolic flux analysis, we were able to evaluate the fluxes involved in central metabolism and conversion to biomass. Flux analysis indicated that two citrate synthases were involved in acetyl-coA metabolism; one localized in the mitochondria and the other outside the mitochondria. Acetyl-coA synthesized in the plastid was directly incorporated in synthesis of fatty acids. Despite having a complete TCA cycle in the mitochondria, it was also found that a majority of the malate flux is shuttled to the cytosol and plastid where it is converted to oxaloacetate providing reducing equivalents to these compartments. We performed metabolic flux analysis of CHO cell cultures in stationary phase by isotopomer modeling, fed with 13C labeled glucose. Flux analysis of the glycolysis and oxidative pentose phosphate pathway (PPP) indicated that almost all of the uptaken glucose is diverted towards PPP with a high level of NADPH production. We hypothesize that the probable function of this high NADPH production is to counteract the oxidative stress during the late stationary phase. A second study on CHO cells was done where fluxes were evaluated under two process conditions that yield different lactic acid profiles (high and low). An untargeted proteomic analysis was also performed to understand the causes of the underlying shifts in the fluxes and lactic acid production profiles. Partial least squares analysis was used, for correlating the changes in metabolic proteins to key fluxes and split ratios. For the high lactic acid production process, changes in the protein profiles related to mitochondrial function and ATP production were correlated to the TCA cycle flux and lactic acid production. The lactate dehydrogenase expression pattern was not correlated or had a minor effect on lactic acid production flux, in both processes. This indicated the control of the lactic acid production, was largely allosteric and dependent on the substrate availability. Further, for both the processes, proteins that play a role in oxidative stress were found to be correlated to the flux profiles. This indicated that the proteins that affect redox potential and energy state of the cells play a role in changing flux profiles over the cell culture. (Abstract shortened by UMI.)

Degree

Ph.D.

Advisors

Morgan, Purdue University.

Subject Area

Cellular biology|Chemical engineering|Biophysics

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