Tsai, Philip S. (1995) Understanding the physiological consequences of Vitreoscilla hemoglobin (VHb) expression in Escherichia coli. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-10292007-135708
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Physiological effects of Vitreoscilla hemoglobin (VHb) expression in recombinant Escherichia coli were investigated to expand the understanding of the means by which VHb enhances oxygen-limited cell growth and recombinant protein production. The proton pumping efficiency (protons extruded per oxygen reduced) of VHb-synthesizing E. coli [...] grown under microaerobic conditions (dissolved oxygen concentration less than 2% air saturation) was 1.5 times the respective value of a control strain not expressing VHb [...]. Deconvolution of VHb, cytochrome o and d bands from absorption spectra revealed a 5-fold increase in cytochrome o content and a 1.5-fold increase in cytochrome d content of the [...] strain relative to the VHb parent. Substrate-utilization kinetic measurements of E. coli mutants lacking one of the two terminal oxidases disclosed enhancement by VHb of the specific activity of cytochrome o but not that of cytochrome d. These results are consistent with a proposed model of VHb action in E. coli hypothesizing that VHb increases the effective dissolved oxygen concentration, resulting in an increase of cytochrome o activity and energy synthesis efficiency.
On-line NAD(P)H fluorescence measurement of [...] and [...] cultures subjected to several high/low oxygen transients showed that microaerobic E. coli expressing VHb maintained a more oxidized state by affording a 2.4-fold smaller NAD(P)H utilization rate under decreasing oxygen tensions and a steady NAD(P)H level of 1.8-fold less under non-aeration than those of the isogenic controls. A rudimentary simulation of oxygen oscillations typical of large-scale bioreactors showed dampened fluorescence response by VHb-containing E. coli to sudden changes of oxygen tension. The oxygen uptake rate, estimated from culture redox potential of cells under submicromolar oxygen tension, was 25% higher for [...] cells relative to the [...] controls.
The possible involvement of the E. coli global oxygen-sensing regulators, Fnr and the Arc system, in modulating the activity of the vhb promoter, [...], were explored using E. coli fnr and arc mutants. Expression of VHb and the activity of [...] were activated by Fnr but relatively unaffected by the Arc system. The presence of VHb increased the activity of [beta]-galactosidase from a cytochrome d promoter-lacZ fusion by 1.5-fold relative to the wild-type control.
VHb expression was modulated over a broad range to study the response of E. coli physiology to VHb dosage. Final cell density increased stepwise with increasing VHb concentration until saturation of cell density at a 2.7-fold increase over the cell density of the [...] culture was observed. The presence of VHb reduced CO2 evolution, by-product excretions, and enhanced growth rate, growth yield and respiration rate. Metabolic flux distribution analysis revealed increased and decreased carbon fluxes to the pentose phosphate pathway and tricarboxylic acid cycle, respectively, of [...] E. coli under oxygen-limited conditions. This analysis also predicted higher overall [...] and ATP fluxes for the [...] cells.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Major Option:||Chemical Engineering|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||10 April 1995|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||13 Nov 2007|
|Last Modified:||26 Dec 2012 03:07|
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