A highly efficient genetic system for the identification of a harzianum B biosynthetic gene cluster in Trichoderma hypoxylon

Trichoderma hypoxylon is a fungicolous species which produces rich secondary metabolites. However, no genetic transformation method is available for further studies. Here, we developed a marker-less transformation system based on the complementation of an uridine/uracil biosynthetic gene by protoplast transformation. An uridine/uracil auxotrophic mutant of Δ hpyr4 was obtained by using a positive screening protocol with 5'-fluoroorotic acid as a selective reagent. To improve the homologous integration rates, the orthologues of ku70 and lig4 which play critical roles in non-homologous end-joining recombination were disrupted. The resulting hlig4 mutant showed remarkable transformation rates of 89 %, while no change was found in the hku70 deletion mutant compared with the WT strain. This suggests tha hlig4 play a key role in the non-homologous recombination in this strain. Using this system, the biosynthetic gene cluster of trichothecene ( ri ) harzianum B was identified by deletion of the htri5 in T. hypoxylon . Comparative genome analysis revealed that the trichothecene biosynthetic gene cluster in T. hypoxylon shared similar organizations with T. arundinaceum and T. brevicompactum , even though their encoded products are different in structures. Taken together, the highly efficient genetic system provides a convenient tool for studying the biosynthetic diversity and mining the novel natural product from the fungi.