title: A novel platform for heterologous gene expression in Trichoderma reesei (Teleomorph Hypocrea jecorina).
abstract: 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.
authors.forename: Mikael Skaanning
authors.initials: MS
authors.lastname: Jørgensen
authors.forename: Dominique Aubert
authors.initials: DA
authors.lastname: Skovlund
authors.forename: Pia Francke
authors.initials: PF
authors.lastname: Johannesen
authors.affiliation: Department of Systems Biology, Center for Microbial Biotechnology, Technical University of Denmark, Building 223, DK-2800 Lyngby, Denmark. um@bio.dtu.dk.
authors.forename: Uffe H
authors.initials: UH
authors.lastname: Mortensen
mesh_terms: D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005800:Genes, Fungal; D005822:Genetic Vectors / Q000378:metabolism; D014242:Trichoderma / Q000235:genetics / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins