Development of Trichoderma reesei transformation technology with the aid of an episomal vector system

In conventional transformation procedures of filamentous fungi the transformation frequencies have been modest and only integrative vectors have been available. In modern fungal molecular biology, however, screening approaches with the help episomal vectors have advanced the progress of the field greatly. For example, in enzyme discovery or protein engineering it is beneficial to be able to screen for new enzymes or improved mutant enzymes in the ultimate production host of the enzyme. Episomal vector systems have been developed for a few fungal species including the Aspergilli, but Trichoderma reesei has suffered from a lack of such a system. We have developed T. reesei transformation technology for genetic and enzyme screening purposes. The transformation procedure was optimised to improve the transformation frequencies of any vectors. Telomeric episomal vectors were used, and with these we obtained transformation frequencies that are far above those obtained with integrative vectors and are feasible for genetic and enzyme screening purposes. Procedures for plasmid rescue, colony picking and microtiter plate cultivation were also developed. The use of the novel transformation system in enzyme and genetic screening experiments will be discussed.