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Vitreoscilla hemoglobin. In vitro mutagenesis and transcript profiling to improve its biotechnological usefulness

Andersson, Charlotte LU (2002)
Abstract
Vitreoscilla is an obligate aerobic bacterium found in oxygen-poor environments such as stagnant ponds and decaying vegetable matter. Vitreoscilla induces the production of a homodimeric hemoglobin (VHb) when subjected to oxygen-limiting conditions. It is thought that VHb facilitates oxygen delivery to the respiratory apparatus of its host by increasing the local oxygen concentration under oxygen-limiting conditions. Furthermore, VHb may increase the level and activity of terminal oxidases, preferably o, thus enhancing respiration and thereby ATP formation, or alternatively act as an alternative terminal oxidase itself. VHb has been shown to have numerous positive effects on diverse cellular processes which are affected by the availability... (More)
Vitreoscilla is an obligate aerobic bacterium found in oxygen-poor environments such as stagnant ponds and decaying vegetable matter. Vitreoscilla induces the production of a homodimeric hemoglobin (VHb) when subjected to oxygen-limiting conditions. It is thought that VHb facilitates oxygen delivery to the respiratory apparatus of its host by increasing the local oxygen concentration under oxygen-limiting conditions. Furthermore, VHb may increase the level and activity of terminal oxidases, preferably o, thus enhancing respiration and thereby ATP formation, or alternatively act as an alternative terminal oxidase itself. VHb has been shown to have numerous positive effects on diverse cellular processes which are affected by the availability of oxygen or subjected to oxygen-poor conditions. Heterologous VHb expression has led to higher cell density and faster growth, higher yield of recombinant proteins or higher production of secondary metabolites, altered by-product formation, and enhanced bioremediation of toxic wastes.



This thesis focuses on development of novel, improved forms of VHb, a set of point mutated VHb forms and a VHb-VHb fusion protein, and on the characterization of their growth properties in Escherichia coli. We have thus demonstrated that error-prone PCR can be used to generate VHb mutants able to further promote growth rates in microaerobic E. coli cultures. In addition, these mutants produce higher levels of active ribosomes in their host cells. The VHb-VHb fusion protein also enhances E. coli cell growth under fully aerobic conditions. In order to delineate the metabolic consequences of VHb expression, transcript profiling using high-density DNA arrays has been carried out. These data have revealed that previously obtained metabolic results using e.g. NMR methodologies, are supported by global gene expression measurements. (Less)
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author
opponent
  • Professor Nielsen, Jens, Center for Process Biotechnology, Biocentrum-DTU, Technical University of Denmark, Lyngby, Denmark
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Biochemical technology, Biokemisk teknik, gene expression analysis, transcript profiling, microarray, DNA chips, microaerobic, hypoxia, oxygen-limited, bacterial hemoglobin, Escherichia coli, Vitreoscilla hemoglobin (VHb)
pages
220 pages
publisher
Pure and Applied Biochemistry, Lund University
defense location
Hall D at Center for Chemistry and Chemical Engineering
defense date
2003-01-22 10:30
ISBN
91-628-5483-6
language
English
LU publication?
yes
id
ec7123f0-f45a-40c9-a6bd-9e1290fb507e (old id 465345)
date added to LUP
2007-10-13 13:50:46
date last changed
2016-09-19 08:45:08
@misc{ec7123f0-f45a-40c9-a6bd-9e1290fb507e,
  abstract     = {Vitreoscilla is an obligate aerobic bacterium found in oxygen-poor environments such as stagnant ponds and decaying vegetable matter. Vitreoscilla induces the production of a homodimeric hemoglobin (VHb) when subjected to oxygen-limiting conditions. It is thought that VHb facilitates oxygen delivery to the respiratory apparatus of its host by increasing the local oxygen concentration under oxygen-limiting conditions. Furthermore, VHb may increase the level and activity of terminal oxidases, preferably o, thus enhancing respiration and thereby ATP formation, or alternatively act as an alternative terminal oxidase itself. VHb has been shown to have numerous positive effects on diverse cellular processes which are affected by the availability of oxygen or subjected to oxygen-poor conditions. Heterologous VHb expression has led to higher cell density and faster growth, higher yield of recombinant proteins or higher production of secondary metabolites, altered by-product formation, and enhanced bioremediation of toxic wastes.<br/><br>
<br/><br>
This thesis focuses on development of novel, improved forms of VHb, a set of point mutated VHb forms and a VHb-VHb fusion protein, and on the characterization of their growth properties in Escherichia coli. We have thus demonstrated that error-prone PCR can be used to generate VHb mutants able to further promote growth rates in microaerobic E. coli cultures. In addition, these mutants produce higher levels of active ribosomes in their host cells. The VHb-VHb fusion protein also enhances E. coli cell growth under fully aerobic conditions. In order to delineate the metabolic consequences of VHb expression, transcript profiling using high-density DNA arrays has been carried out. These data have revealed that previously obtained metabolic results using e.g. NMR methodologies, are supported by global gene expression measurements.},
  author       = {Andersson, Charlotte},
  isbn         = {91-628-5483-6},
  keyword      = {Biochemical technology,Biokemisk teknik,gene expression analysis,transcript profiling,microarray,DNA chips,microaerobic,hypoxia,oxygen-limited,bacterial hemoglobin,Escherichia coli,Vitreoscilla hemoglobin (VHb)},
  language     = {eng},
  pages        = {220},
  publisher    = {ARRAY(0x83eb638)},
  title        = {Vitreoscilla hemoglobin. In vitro mutagenesis and transcript profiling to improve its biotechnological usefulness},
  year         = {2002},
}