DEVELOPMENT OF THE POSSIBILITY TO CLONE FOREIGN GENES INTO TWO OF THE MOST IMPORTANT INDUSTRIAL FUNGI : ASPERGILLUS ORYZAE USED FOR PRODUCTION OF CITRIC ACID AND PENICILLIUM CHRYSOGENUM USED FOR PRODUCTION OF PENICILLIN.
IN PARTICULAR, THE FEASIBILITY OF USING THE NITRATE CONTROL REGION FOR INDUCTION OF THE EXPRESSION OF THE INSERTED INTERLEUKOCIDIN GENE WOULD BE OF PARAMOUNT COMMERCIAL INTEREST.
A study was made fundamental and applied geneticaspects of filamentous fungi involving the development of genetic transformation and expression systems which are of central importance for the study and exploitation of filamentous fungi. Studies focussed on 2 selection systems which have a concomitant phenotype (ie, resistance to an antibiotic agent resulting in a specific growth defect). Certain acceptable fungi were programmed to synthesize and secrete heterologous proteins of commercial value in copious amounts.
Transformation systems were successfully generated for a variety of industrial fungi based on the nitrate reductase system (niaD). These include Aspergillus oryzae, A niger, Penicillium chrysogenum, Gibberella fujikuroi, Cephalosporium acremonium. Transformation based on the uracil system (pyrG) was also developed for A oryzae and A niger. Such results indicate that 2 reproducible reliable routes exist that introduce genes of commercial interest into filamentous fungi.
Deoxyribonucleic acid (DNA) constructs were also made in order to genetically programme fungi to express genes of commercial interest. Initial investigations focussed on the expression and secretion of human interleukin-6 (hIL-6). Such studies resulted in the production of this substance by filamentous fungi.
DEVELOPMENT OF A TRANSFORMATION AND EXPRESSION SYSTEM IN ASPERGILLUS ORYZAE AND PENICILLIUM CHRYSOGENUM FOR CLONING THE NIA A GENE FOR NITRATE ASSIMILATION.
IN PARTICULAR :
1. THE EQUIVALENT NIA A GENE FROM THE INDUSTRIAL STRAIN ASPERGILLUS ORYZAE WILL BE CLONED BY CROOS-HYBRIDISATION WITH THE AVAILABLE GENE FROM THE MODEL SPECIES ASPERGILLUS NIDULANS.
2. ATTEMPS WILL BE MADE TO INCREASE THE FREQUENCY OF TRANSFORMATION OF ASPERGILLUS ORYZAE BY VARYING THE PERIOD OF CELL WALL DIGESTION ON OTHER PARAMETERS AND BY INSERTION OF CERTAIN DNA SEQUENCES SUCH AS ANS 1 FROM ASPERGILLUS NIDULANS OR SEQUENCES FROM PUC 8.
3. THE CLONED GENE STRUCTURE WILL BE CHARACTERIZED BY S1 MAPPING AND BY NUCLEOTIDE SEQUENCING. THE POSITION AND STRUCTURE OF PROMOTOR REGION WILL BE DETERMINED.
Funding SchemeCSC - Cost-sharing contracts