A versatile toolkit for high throughput functional genomics with Trichoderma reesei

Aiming at high efficiency and high throughput methods, we present here a construction kit for gene knock out in T. reesei. We provide a primer database for gene deletion using the pyr4, amdS and hph selection markers. For high throughput generation of gene knock outs, we constructed vectors using yeast mediated recombination and then transformed a T. reesei strain deficient in non-homologous end joining (NHEJ) by spore electroporation. This NHEJ-defect was subsequently removed by crossing of mutants with a sexually competent strain derived from the parental strain, QM9414.Using this strategy and the materials provided, high throughput gene deletion in T. reesei becomes feasible. Moreover, with the application of sexual development, the NHEJ-defect can be removed efficiently and without the need for additional selection markers. The same advantages apply for the construction of multiple mutants by crossing of strains with different gene deletions, which is now possible with considerably less hands-on time and minimal screening effort compared to a transformation approach. Consequently this toolkit can considerably boost research towards efficient exploitation of the resources of T. reesei for cellulase expression and hence second generation biofuel production.The increasing awareness of the limited availability of fossil fuels along with the environmental problems caused by their application initiated considerable research efforts towards clean and sustainable biofuels [1-3]. Thereby, the cellulases required to degrade cellulosic plant materials into small building blocks, which can be metabolized by yeast or other microbes to ethanol or hydrocarbon biofuel precursors, respectively, are one major focus of investigation [4]. Trichoderma reesei (Hypocrea jecorina) is currently the most efficient producer of enzyme mixtures for degradation of plant materials [5]. The cellulases produced by this fungus are utilized for diverse industrial processes, from biobleaching of t