Vector pTAex3 was modified for use in characterising fungal polyketide synthases (PKS) and hybrid polyketide synthase-non-ribosomal peptide synthases (PKS-NRPS) in an arginine auxotroph of Aspergillus oryzae. In pTAex3GS the strong, starch-inducible amyB expression cassette was modified for GATEWAY LR recombination, and in further derivatives the argB selectable marker was replaced with basta- (bar) or bleomycin- (ble) resistance genes to allow co- or sequential transformation. Tailoring enzymes convert PKS/PKS-NRPS products to final secondary metabolites; tenellin production in Beauveria bassiana requires an enoyl reductase and two cytochrome P450s in addition to a PKS-NRPS encoded by tenS. We produced tenellin in A. oryzae by introducing all four genes in amyB expression cassettes on three separate plasmids. To simplify plasmid construction for whole-pathway expression pTAex3GS was converted to a yeast-E. coli shuttle vector. The A. nidulans argB gene functions well in A. oryzae, so promoters from three other arginine biosynthesis genes (carbomylphosphate synthase (P1), arginosuccinate synthase (P2) and arginosuccinate lyase (P3) were inserted by homologous recombination in yeast. The resultant pTAYAGSarg3P was further modified, replacing argB with bar and ble markers. Yeast recombination simultaneously placed the three tenellin tailoring genes downstream of the arg promoters, creating pTAYAGSarg3genes, and introduction of tenS by GATEWAY recombination reconstructed the whole tenellin synthesis pathway in pTAYAargTENELLIN, which was introduced into A. oryzae. While the easy construction principle has been proven, tenellin synthesis is not yet a likely outcome because in tests of the arg promoters only P2 gave an acceptable level of eGFP expression.
Full conference title:
10th EUROPEAN CONFERENCE ON FUNGAL GENETICS
- ECFG 10th (2010)