CONSTRUCTION OF NEW CLONING VECTORS IN INDUSTRIAL GRAM NEGATIVE SOIL BACTERIA. THE LACK OF USEFUL VECTORS IS PRESENTLY A BOTTLENECK TO THE PROGRESS OF GENETIC ENGINEERING OF THE GRAM NEGATIVE SOIL BACTERIA USED FOR :
- INDUSTRIAL FERMENTATIONS (PSEUDOMONAS, METHYLOTROPHS),
- POLLUTION CONTROL (PSEUDOMONAS, XANTHOBACTER, ALCALIGENES, ACINETOBACTER),
- LIGNOCELLULOSE BIODEGRADATION AND THE CARBON CYCLE OF THE EARTH (PSEUDOMONAS, FLAVOBACTERIUM, ACINETOBACTER, KLEBSIELLA, ACROMONES),
- SYMBIOTIC NITROGEN CENTER FIXATION (RHIZOBIUM) AND PLANT GENETIC ENGINEERING (AGROBACTERIUM).
A major bottleneck to the application of pseudomonads in biotechnology was the lack of suitable plasmids that would enable the introduction of cloned deoxyribonucleic acid (DNA) into the proper bacterium and subsequent production of the desired enzymatic activity.
In order to contribute to the rectification of this problem the following research was carried out:
development of new specific purpose plasmids, applicable to a wide range of Gram negative bacteria;
analysis of key enzymes in the conversion of detergents, lignin monomers, aliphatic and aromatic hydrocarbons;
the application of modified pseudomonads to the production of fine chemicals.
With the plasmids constructed enzymes can be produced in a variety of Gram negative bacteria in response to temperature increase or the addition of trace amounts of chemicals (eg alkane sulphates or alkylsulphates). 4 of these enzyme systems were investigated in detail: vanillate demethoxylase and alkylsulphatase are key enzymes in the use of environmental pollutants (lignindetergents), and the alkane hydroxylases and xylene hydroxylases both convert bulk chemicals (aliphatic and aromatic hydrocarbons) into fine chemicals (alcohols and epoxides). Genetically modified Pseudomonas strains were constructed that produce elevated amounts of each of these 4 enzyme systems, enabling their biochemical characterization and facilitating their future application to biotechnology processes.
CONSTRUCTION OF REGULATED HIGH EXPRESSION VECTORS FOR STABLE CLONING IN GRAM NEGATIVE SOIL BACTERIA.
IN PARTICULAR: STRONG PROMOTOR SEQUENCIES FROM SEVERAL GRAM NEGATIVE BACTERIA OF INDUSTRIAL INTEREST WILL BE ISOLATED AND SEQUENCED.
Funding SchemeCSC - Cost-sharing contracts