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

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 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.