Novel aspects to transformation and gene expression in Aspergillus

Species of the genus Aspergillus serve as model systems for transformation and gene expression studies in filamentous fungi. In this investigation, the plasmid vectors pP01/2α, with replicative potential in the basidiomycete Pleurotus ostreatus, were further analyzed. They were found to include a 1.15-kb sequence (P01A) from P1 bacteriophage. This sequence was apparently recognized by P. ostreatus as a surrogate replicon. Studies with Aspergillus niger and Aspergillus nidulans, however, gave no evidence for autonomous replication of transforming plasmid vectors derived from pP01α. A. niger was transformed to hygromycin B resistance, and A. nidulans to pyrimidine prototrophy. Integrative transformation of A. nidulans using polyethyleneglycol treatment of protoplasts was compared with transformation of intact conidia by means of microprojectile bombardment using a helium-driven biolistic device. Effects of conidial density, helium pressure, size of microprojectiles and other parameters on transformation frequency were investigated. Typically, several plasmid molecules per conidium were delivered by microprojectiles and integrated into high molecular weight DNA. Overall transformation efficiency and mitotic stability of transformants were compared for biolistics and chemical transformation. Established transformation protocols were used to introduce a plasmid into A. nidulans that was constructed in order to express a synthetic protein-based polymer gene under transcriptional control of the constitutive gpdA promoter of A. nidulans. The presence and expression of the (GVGVP)₁₂₁ gene was demonstrated in four stable transformants by Southern- and Northern-blot hybridization. An enriched protein fraction from one of the transformants exhibited polymer protein synthesis, albeit with low translational efficiency. Finally, co-transformation and reporter gene (GUS) studies were conducted to provide evidence that the A. nidulans gpdA promoter is up-regulated by osmotic stress.