CONSTRUCTION OF NEW CLONING VECTORS IN INDUSTRIAL GRAM NEGATIVE SOIL BACTERIA. MANIPULATION OF PSEUDOMONADS CAPABLE OF SPECIFIC BIOCONVERSIONS. 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 (PSEUDOMONADS).
- LIGNOCELLULOSE BIODEGRADATION (PSEUDOMONAS, FLAVOBACTERIUM, ACINETOBACTER, KLEBSIELLA, AEROMONAS).
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.
IMPROVEMENT OF PSEUDOMONADS AS BIOCATALYSTS IN THE PRODUCTION OF FINE CHEMICALS AND IN DEGRADATION OF POLLUTANTS.
1. THE ALK GENES AND GENE PRODUCTS CONTROLLING GROWTH OF PSEUDOMONAS PUTIDA ON N-ALKANES WILL BE IDENTIFIED, MODIFIED AND (RE-) INTRODUCED INTO PSEUDOMONAS PUTIDA AND VARIOUS OTHER GRAM-NEGATIVE BACTERIA USING NEW BROAD HOST RANGE VECTORS.
MODIFICATIONS IN THE ALKANE PATHWAY WILL ALLOW ACCUMULATION OF PATHWAY INTERMEDIATES SUCH AS EPOXIDES AND (DI-) CARBOXYLIC ACIDS.
2. THE ALK SYSTEM WILL BE MUTAGENIZED TO ALTER SUBSTRATE SPECIFICITY.
3. THE ALTERNATIVE OXYGENASE SYSTEMS ISOLATED FROM OTHER GRAM NEGATIVE ORGANISMS WILL BE INTRODUCED IN PSEUDOMONAS OLEOVORANS.
4. VECTOR SYSTEMS WILL BE DEVELOPED AIMED AT INCREASING THE GENETIC STABILITY OF MANIPULATED PSEUDOMONADS.
Funding SchemeCSC - Cost-sharing contracts