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DEVELOPMENT OF GENE MEDIATED TRANSFER AND SELECTION SYSTEM FOR FILAMENTOUS FUNGI

Objective

DEVELOPMENT OF A GENE-TRANSFER AND GENE-EXPRESSION SYSTEM INTO TWO OF THE MOST IMPORTANT INDUSTRIAL FUNGI: SUCH AS ASPERGILLUS NIGER AND ASPERGILLUS ORYZAE WHICH ARE USED FOR PRODUCTION OF CITRIC ACID AND EXTRACELLULAR ENZYMES. IN PARTICULAR, THE FEASIBILITY OF USING ASPERGILLUS EXPRESSION SIGNALS FOR EXPRESSION OF THE INSERTED HUMAN INTERLEUKIN 6 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:
1) A GENE MEDIATED TRANSFER SYSTEM FOR ASPERGILLUS ORYZAE ACCORDING TO A STRATEGY WHICH HAS BEEN APPLIED SUCCESSFULLY FOR ASPERGILLYS NIDULANS;
2) SYSTEMS TO EXPRESS COMMERCIALLY IMPORTANT EXTRACELLULAR PROTEINS IN ASPERGILLUS SPECIES.
IN PARTICULAR:
A. HOMOLOGOUS GENE CLONING SYSTEM WILL BE DEVELOPED WHICH WOULD PERMIT THE ISOLATION, IDENTIFICATION AND ANALYSIS OF FUNGAL GENES OF INDUSTRIAL IMPORTANCE.
B. VECTOR WILL BE CONSTRUCTED FOR EXPRESSION OF CHIMAERIC GENES UNDER CONTROL OF ASPERGILLUS EXPRESSION SIGNALS.
C. THE SYSTEM WILL BE EXPLOITED FOR CLONING COMMERCIALLY IMPORTANT GENES.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

ORGANISATIE VOOR TNO
Address
Medical Biological Laboratory (Tno) P.o. Box 45
2280 AA Rijswijk
Netherlands

Participants (2)

LABORATORY OF MOLECULAR BIOLOGY
Belgium
Address

Gent
UNIVERSITY OF ST ANDREWS
United Kingdom
Address
College Gate, 66 North Street
KY16 9AH St Andrews