Paper Authors and Title

Chiara Del Greco (Atlantic Technological University), Mary Heneghan, Christopher Golden, “Establishing a transformation system for the Thermophilic Fungus Rasamsonia Emersonii


Filamentous fungi are an invaluable tool in biotechnology for the manufacture of recombinant proteins.  It is therefore an essential criterion to be able to transform fungi with recombinant DNA.  In this study a series of parameters were optimised to carry out Protoplast-Mediated transformation of the thermophilic fungus Rasamsonia emersonii with the pTH-eGFP plasmid.

Two plasmid minipreps were performed and analysed using gel electrophoresis.  Plasmid identity was confirmed by restriction digestion with EcoRI, HindIII and KpnI.  DNA yield and purity was determined via NanoDrop analysis.  Results indicated low DNA purity in both samples obtained.

Studies were carried out to determine the incubation conditions required to identify initial stages of spore germination.  Results demonstrated that incubating Rasamsonia emersonii spores at 45°C for 5 hours, followed by decreasing the temperature to 37°C overnight yielded the optimum time point for the addition of protoplast enzyme solution.

An enzyme solution (7U/mL Cellulase, 0.1U/mL Chitinase, 0.07U/mL Protease) was tested under varying parameters to identify protoplast generation conditions.  These included fungal tissue (spores, mycelia, germinating spores), enzyme digestion temperatures (25°C, 37°C, 25°C-37°C) and protoplast regeneration media (4% SDA, 0.7M KCl, 1% Starch, 0.8M Mannitol).  No protoplasts were generated during the course of this study.  This suggests that further optimisation is needed with a focus on the enzyme solution looking to optimise buffer osmolarity and enzyme concentration.  The fungus failed to grow on the regeneration media indicating that this particular media is not suitable for recovery of Rasamsonia emersonii protoplasts.