SVIBOR - Project code: 1-08-058

MINISTRY OF SCIENCE AND TECHNOLOGY

Strossmayerov trg 4, HR - 10000 ZAGREB
tel.: +385 1 459 44 44, fax: +385 1 459 44 69
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Project code: 1-08-058


GENE EXPRESSION IN MYCELIAL MICROORGANISMS


Main researcher: PIGAC, JASENKA (37185)


Assistants
Type of research: basic
Duration from: 01/01/91. to 12/31/95.
Papers on project (total): 23
Papers on project quoted in Current Contents: 6
Institution name: Pliva - Istraživački institut, Zagreb (60)
Department/Institute: Biosynthesis and Biotechnology
Address: Prilaz baruna Filipovića 89
City: 10000 - Zagreb, Croatia
Communication
Phone: 385 (0)1 18-15-87
Fax: 385 (0)1 57-47-19
Summary: The available knowledge and results of long term studies of Streptomyces and Aspergillus in our laboratories as well as the application of the recent recombinant DNA methods and techniques have enabled these investigations. For the first time a rapid and reliable method for transformation of Streptomyces by electroporation has been published and used in our laboratories. That method has enabled introduction of various vectors as well as integration and expression of haemoglobin (VHb) gene from Vitreoscilla in S. rimosus R6 chromosome. The construction of an S. rimosus R6 strain lacking some enzymes (AT, KS, CLF) in OTC-polyketide synthase (PKS) is in progress. In parallel, actinorhodin-PKS genes were introduced into a bifunctional plasmid. Construction of R6 AT, KS, CLF-deleted strain and its transformation with plasmids harbouring PKS genes of other polyketide antibiotics are expected to give novel polyketide molecules. From the industrial Aspergillus niger strain the glucoamilase gene was isolated and cloned into the different A. niger strains. Transformants were analysed for the level of glucoamylase in respect to the glucoamylase gene copy number. The human proinsulin gene has been also synthesised by means of overlapping, complementary oligonucleotides in combination with PCR and cloned into two types of fungal expression vectors. Expression of the human proinsulin gene in various transformants was determined by insulin radioimmune assay.

Keywords: Streptomyces rimosus, Aspergillus niger, gene expression, homologous and heterologous products, oxytetracycline,polyketide antibiotics, glucoamylase, human insulin

Research goals: Streptomyces and Aspergilli are mycelial microorganisms similar in distinct differentiation, release of metabolites into medium, heterokaryon formation etc, and known as antibiotic and enzyme producers in biotechnology. The aim of this project is to study gene expression of a primary (glucoamylase) and a secondary (polyketide antibiotic, oxytetracycline) metabolite in mycelial microorganisms. Further investigations will comprise the expression of heterologous products in these microorganisms (new polyketide antibiotic, pharmaceutical protein). The total sequence of OTC-genes, and availability of various vectors (autonomous, integrative, bifunctional) enable introduction and expression of homologous and heterologous genes in these important microorganisms. The organisation of OTC-genes of mutants blocked in OTC-biosynthesis in respect to the parental R6 strain will be revealed. At least some of the mutants lacking the whole OTC-gene cluster could serve as hosts for introduction of foreign genes. An efficient and reliable method for transformation of mycelial fragments instead of protoplasts by electroporation will be elaborated and applied to R6 and 4018 strains. At least one heterologous gene will be introduced possibly by integration into R6 chromosome to enhance OTC yield. Site-directed mutagenesis of OTC-PKS genes, construction of various plasmids will enable construction of strains adequate for biosynthesis of new polyketide molecules. The aim of this research are the new insight on the glucoamylase gene regulation and expression, influence of gene copy number on glucoamylase gene expression, as well as the fungal strain potential for homologous and heterologous protein expression. To investigate glucoamylase expression in Aspergillus niger, various co/transformants will be analysed for the glucoamylase levels in respect to its gene copy number. Chemical and biochemical methods will be used to construct human proinsulin gene which will be under control of regulatory sequences of the glucoamylase gene. Certain modifications will be introduced into the synthetic human proinsulin gene to study the eventually positive effect on human insulin expression and secretion. The knowledge achieved on gene expression during this study is important for the development of new biotechnology both in PLIVA and in Croatia. According to many predictions, new biotechnology should become a dominant technology in the nearest future.

COOPERATION - PROJECTS


  1. Name of project: 1-08-021 Struktura složenost i stabilnost genoma streptomiceta
    Name of institution: Prehrambeno-biotehnološki fakultet
    City: 10000 - Zagreb, Croatia

  2. Name of project: ALIS LINK No.007 Molecular genetics of oxytetracycline biosynthesis from Streptomyces rimosus R6, the oxytetracycline producer
    Name of institution: University of Glasgow
    City: 441 - Glasgow, UK

COOPERATION - INSTITUTIONS


  1. Name of institution: Prehrambeno-biotehnološki fakultet
    Type of institution: Economical/Production
    City: 10000 - Zagreb, Croatia

  2. Name of institution: University of Glasgow
    Type of institution: Economical/Production
    City: 441 - Glasgow, UK
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