Degree Type

Thesis

Date of Award

2011

Degree Name

Master of Science

Department

Chemical and Biological Engineering

First Advisor

Jacqueline V. Shanks

Abstract

Metabolic engineering has evolved to the point of fulfilling the dream of having industrial chemicals produced renewably. Carboxylic acids [e.g., short chain fatty acids (SCFAs) such as octanoic acid (C8)] are such chemical intermediates that can be produced by Escherichia coli engineered with thioesterases specific for short chain fatty acids. However, C8 is toxic to E. coli at concentration greater than 10 mM. To design a better host strain for the production of large amount of SCFAs, 13C metabolic flux analysis of E. coli was performed for both C8 stress and control condition (without C8). To investigate central carbon metabolism for the culture environmental change, a mixture of labeled and unlabeled glucose was used as the sole carbon source for bacterial growth and proteinogenic amino acid isotopomers were measured using two-dimensional (13C, 1H) HSQC NMR spectroscopy. Notable differences of several amino acids isotopomer abundance were observed between the control condition and C8 stress condition, suggesting that the precursor nodes of these amino acids in metabolic pathways were responding to the stress. More specifically, the difference suggested that the distribution of fluxes among the tricarboxylic acid (TCA) cycle, pentose phosphate pathway, pyruvate node, alpha-ketoglutarate node and oxaloacetate node changed. By comparing the metabolic flux maps of E. coli MG1655 grown at different conditions, pathways that have flux change under stress were identified. Inhibition effect for several pathways, resulting in a reduction in carbon flux, was found under stress: the TCA cycle flux by ~ 44%; the malic enzyme pathway by ~ 80%; the phosphoenolpyruvate carboxylase pathway ~ 60%; the CO2 production rate by 18%; and pyruvate dehydrogenase pathway by ~50%. Meanwhile, a few pathways were activated under C8 stress: the pyruvate dehydrogenase flux (`PoxB') became active; the malate dehydrogenase pathway (`mdh') in TCA cycle increased by ~55%; and the extracellular acetate production increased by ~ 80%. Based on these results, a hypothesis was proposed that low activity of NADH dehydrogenase may lead to low ratio of NAD+/NADH which in turn causes the low activity of two major pathways (TCA cycle, and pyruvate dehydrogenase). The inefficiency of regeneration of NAD+ from NADH may occur when the cell membrane is disrupted, which has been proven by membrane fluidity study (L. Royce, L. Jarboe, unpublished data) as well as transcriptome (L. Royce, L. Jarboe, unpublished data) and proteomic (M Rodriquez, R. Gonzalez, unpublished data) data. Besides, the other reason for low ratio of NAD+/NADH is that the electron transport chain may be disrupted under stress. It is also possible that the PdhR (pyruvate dehydrogenase complex regulator) is activated by low concentration of intracellular pyruvate, which in turn represses ndh and cyoABCDE that encode two major enzymes in the electron transport chain. This hypothesis is supported by the observation that supplementation of additional pyruvate in the media helps the cell partially recover from the stress.

DOI

https://doi.org/10.31274/etd-180810-2244

Copyright Owner

Yanfen Fu

Language

en

Date Available

2012-04-06

File Format

application/pdf

File Size

104 pages

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