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Background  The industrially applied filamentous fungus  Trichoderma reesei  has received substantial interest due to its highly efficient synthesis apparatus of cellulytic enzymes. However, the production of heterologous enzymes in  T. reesei  still remains low mainly due to lack of tools for genetic engineering.    Results  In this study we present new genetic tools for  T. reesei  to further expand its use in industrial production. We have developed an expression platform where genes are inserted into a versatile expression vector via highly efficient uracil-excision cloning and subsequently inserted into a defined position in the  T. reesei  genome ensuring that enzyme production from different transformants can be directly compared. The  ade2  locus was selected as integration site since  ade2  mutants develop red pigment that facilitates easy and rapid detection of correctly targeted transformants. In addition, our system includes a  tku70  disruption to increase gene targeting efficiency and a new bidirectional marker,  pyr2 , for iterative gene targeting. The dual selection system, color and prototrophism, ensures that correct transformants containing the desired gene inserted into the defined expression site can be selected with an efficiency approaching 100%.    Conclusions  The new genetic tools we have developed are suitable for high-throughput integration of genes into the genome of  T. reesei  and can easily be combined with techniques for generation of defined mutants. Moreover, the usability of the novel expression system with  ade2  as integration site was confirmed by expression of a  Thermomyces lanuginosus  lipase.

A novel platform for heterologous gene expression in Trichoderma reesei (Teleomorph Hypocrea jecorina)