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Metabolic engineering: Approaches Towards Improved Stress Tolerance in Microorganisms and Plants

Holmberg, Niklas (1996)
Abstract
By using metabolic engineering it is now possible to introduce new biosynthetic pathways or redirect already existing ones in a wide range of pro- and eukaryotic organisms. This thesis mainly addresses some approaches using metabolic engineering directed towards enhancing the stress tolerance in microorganisms and higher plants.



The main approach employed in plants was to introduce the betA gene encoding bacterial choline dehydrogenase (CDH) which catalyses the bioconversion of choline to the potent osmoprotectant glycine betaine. Transgenic tobacco and potato expressing CDH exhibited enhanced sodium chloride and freezing stress tolerance, respectively.



A bifunctional enzyme was constructed of two key... (More)
By using metabolic engineering it is now possible to introduce new biosynthetic pathways or redirect already existing ones in a wide range of pro- and eukaryotic organisms. This thesis mainly addresses some approaches using metabolic engineering directed towards enhancing the stress tolerance in microorganisms and higher plants.



The main approach employed in plants was to introduce the betA gene encoding bacterial choline dehydrogenase (CDH) which catalyses the bioconversion of choline to the potent osmoprotectant glycine betaine. Transgenic tobacco and potato expressing CDH exhibited enhanced sodium chloride and freezing stress tolerance, respectively.



A bifunctional enzyme was constructed of two key enzymes of the E. coli proline biosynthesis pathway by in frame gene fusion. When grown in salt containing media proline auxothrophic bacteria harbouring plasmids expressing the bifunctional enzyme displayed shorter generation times and increased free proline concentrations. Moreover, a mechanism was proposed where the proximity of the enzymes reduces the break down of a labile intermediate.



Analogues of an antifreeze protein found in the winter flounder were constructed as chimeric proteins with the Spa domain of Staphylococcal protein A. The constructions were expressed in E.coli where they conferred enhanced sodium chloride and freezing tolerance. In order to further investigate these characteristics a randomly mutated library of chimeric antifreeze protein analogues was created in E. coli and screened for improved salt tolerance. Furthermore, as the selected salt tolerant clones also conferred improved freezing tolerance a connection between salt and freezing tolerance was proposed.



A plant expression system for the Vitreoscilla hemoglobin (VHb) was constructed and transformed into tobacco. Transgenic tobacco expressing VHb exhibited faster germination and flowering, and altered production of certain secondary metabolites. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Prof. D. Hanson, Andrew, Horticultural Sciences Dept., University of Florida, USA
publishing date
type
Thesis
publication status
published
subject
keywords
Vitreoscilla hemoglobin, Transgenic, Solanum tuberosum, Salt tolerance, Nicotiana tabaccum, Metabolic Engineering, Freezing tolerance, Choline dehydrogenase, Antifreeze protein, Bifunctional enzyme, Biochemistry, Metabolism, Biokemi, metabolism, Biotechnology, Bioteknik
pages
144 pages
publisher
Niklas Holmberg, Dept. of Pure and Applied Biochemistry, Chemical Centre, P.O. Box 124, S-221 00 Lund, SWEDEN
defense location
Lecture Hall C, Chemical Centre, University of Lund
defense date
1996-12-10 10:15
external identifiers
  • Other:LUTKHD/TKBK-1041/1-144/1996
ISBN
91-628-2261
language
English
LU publication?
no
id
756b2dc1-c682-4c5b-9204-656cbe262556 (old id 28867)
date added to LUP
2007-06-14 09:19:16
date last changed
2016-09-19 08:45:14
@phdthesis{756b2dc1-c682-4c5b-9204-656cbe262556,
  abstract     = {By using metabolic engineering it is now possible to introduce new biosynthetic pathways or redirect already existing ones in a wide range of pro- and eukaryotic organisms. This thesis mainly addresses some approaches using metabolic engineering directed towards enhancing the stress tolerance in microorganisms and higher plants.<br/><br>
<br/><br>
The main approach employed in plants was to introduce the betA gene encoding bacterial choline dehydrogenase (CDH) which catalyses the bioconversion of choline to the potent osmoprotectant glycine betaine. Transgenic tobacco and potato expressing CDH exhibited enhanced sodium chloride and freezing stress tolerance, respectively.<br/><br>
<br/><br>
A bifunctional enzyme was constructed of two key enzymes of the E. coli proline biosynthesis pathway by in frame gene fusion. When grown in salt containing media proline auxothrophic bacteria harbouring plasmids expressing the bifunctional enzyme displayed shorter generation times and increased free proline concentrations. Moreover, a mechanism was proposed where the proximity of the enzymes reduces the break down of a labile intermediate.<br/><br>
<br/><br>
Analogues of an antifreeze protein found in the winter flounder were constructed as chimeric proteins with the Spa domain of Staphylococcal protein A. The constructions were expressed in E.coli where they conferred enhanced sodium chloride and freezing tolerance. In order to further investigate these characteristics a randomly mutated library of chimeric antifreeze protein analogues was created in E. coli and screened for improved salt tolerance. Furthermore, as the selected salt tolerant clones also conferred improved freezing tolerance a connection between salt and freezing tolerance was proposed.<br/><br>
<br/><br>
A plant expression system for the Vitreoscilla hemoglobin (VHb) was constructed and transformed into tobacco. Transgenic tobacco expressing VHb exhibited faster germination and flowering, and altered production of certain secondary metabolites.},
  author       = {Holmberg, Niklas},
  isbn         = {91-628-2261},
  keyword      = {Vitreoscilla hemoglobin,Transgenic,Solanum tuberosum,Salt tolerance,Nicotiana tabaccum,Metabolic Engineering,Freezing tolerance,Choline dehydrogenase,Antifreeze protein,Bifunctional enzyme,Biochemistry,Metabolism,Biokemi,metabolism,Biotechnology,Bioteknik},
  language     = {eng},
  pages        = {144},
  publisher    = {Niklas Holmberg, Dept. of Pure and Applied Biochemistry, Chemical Centre, P.O. Box 124, S-221 00 Lund, SWEDEN},
  title        = {Metabolic engineering: Approaches Towards Improved Stress Tolerance in Microorganisms and Plants},
  year         = {1996},
}