Introduction: Since Aspergillus oryzae produces large quantity of enzymes, it is significant to establish a host-vector system to use this microorganism as a host for heterologous protein expression. In various filamentous fungi, the genes, which confer resistance to hygromycin B, aureobasidin, and G418, have been used as genetic markers for the gene manipulation. However, A. oryzae is resistant to these antibiotics. We have recently developed a transformation system for A. oryzae RIB40 by using bleomycin-resistance gene as a selective marker. In the present study, we generated ligD knock out strain, by using bleomycin resistance selection. And we generated exogenous or endogenous enzyme over-expresser by transformation with expression vector carrying bleomycin resistant gene. Methods: The ligD disruption cassette consisted of bleomycin-resistance expression cassette and 2kb of 5’ and 3’ franking sequence of ligD, the β -glucuronidase encoding gene uidA from E. coli over-expression vector and a polygalacturonase encoding gene pgaB from A. oryzae over-expression vector were introduced into the protoplasts from A. oryzae RIB40 by the polyethylene glycol method, respectively. Results: The disruption of the ligD locus in genomic DNA of transformants was confirmed by colony PCR and the Southern blot analysis. Activity of each enzyme was detected from cell free extract or culture filtrate of the each transformant selected by bleomycin resistance. Discussion: In the present study, we successfully generated a gene disruptant and over-expresser of two kinds of enzymes by using bleomycin resistance transformant selection system of A. oryzae.
Full conference title:
10th EUROPEAN CONFERENCE ON FUNGAL GENETICS
- ECFG 10th (2010)