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Content archived on 2024-04-15

DEVELOPMENT OF A GENE MEDIATED TRANSFER AND SELECTION SYSTEM FOR FILAMENTOUS FUNGI

Objective

FILAMENTOUS FUNGI ARE GOOD CANDIDATES TO STUDY HETEROLOGOUS GENE EXPRESSION FOR SEVERAL REASONS :
- THEY CAN GROW ON INEXPENSIVE MEDIA.
- MANY OF THEM ARE NOT TOXIC TO MAN.
- TRANSFORMATION SYSTEMS ARE BEING DEVELOPED.
THEY ARE CAPABLE OF SECRETING LARGE AMOUNTS OF PROTEINS (E.G. CELLULASES, PROTEASES, AMYLASES, ETC).
A CRUCIAL STEP TOWARDS OBTAINING EFFICIENT HETEROLOGOUS GENE EXPRESSION IS ENGINEERING OF SUITABLE SIGNALS FOR INITIATION (PROMOTERS) AND TERMINATION (TERMINATORS) OF TRANSCRIPTION. THE DEVELOPMENT OF A HETEROLOGOUS GENE EXPRESSION SECRETION SYSTEM WILL EVENTUALLY FACILITATE THE PURIFICATION OF THE DESIRED PROTEIN, AND PREVENT POSSIBLE TOXIC INTERACTIONS WITH THE CELLULAR METABOLISM.
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.
DEVELOPMENTS OF A GENE TRANSFER SYSTEM FOR ASPERGILLUS SPECIES AS A CRITICAL STEP TOWARDS THE EVALUATION OF THESE ORGANISMS AS A HOST FOR HETEROLOGOUS PROTEIN SECRETION. THE CONTRIBUTION OF DR. FIERS TO THE JOINT PROJECT WILL BE TO ISOLATE TRANSCRIPTION SIGNALS AND TO CONSTRUCT RECOMBINANT PLASMIDS WHICH CONTAIN HETEROLOGOUS GENES IN EXPRESSION CASSETTES. THE PROJECT WILL ESPECIALLY FOCUS ON APPROACHES FOR THE ISOLATION OF TRANSCRIPTION PROMOTERS AND TERMINATORS FOR FILAMENTOUS FUNGI (ASPERGILLUS ORYZAE, ASPERGILLUS NIGER, TRICHODERMA REESEI).

Topic(s)

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Coordinator

RIJKSUNIVERSITEIT GENT
EU contribution
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Address
SINT-PIETERSNIEUWSTRAAT 25
9000 GENT
Belgium

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Participants (2)