Simultaneous In Vivo Assembly and Targeted Genome Integration of Gene Clusters in Trichoderma reesei

The saprophytic filamentous fungus Trichoderma reesei represents one of the most prolific cellulase producers and also has the potential to be developed into a tractable fungal host for biosynthesizing secondary metabolite products. To expedite the genetic engineering of filamentous fungi, efficient DNA assembly processes that can facilitate the transfer of large-sized DNA to fungal hosts, including T. reesei, are still in demand. Here, we developed a method for the simultaneous in vivo assembly and targeted genome integration of multiple DNA fragments (SATIMD) in T. reesei. While efficient orderly DNA end fusions were achieved by homologous recombination (HR) with various lengths of sequence overlaps (100-500 bp), the assembled DNA was also precisely integrated into a specific locus when combined with CRISPR/Cas9-mediated genome cutting. Specifically, we have used this method to achieve the assembly and functional expression of T. reesei key transcriptional activator Xyr1 for cellulase genes. Moreover, fusions and targeted integration of up to 10 different DNA fragments comprising the 32.7 kb sorbicillinoids biosynthetic gene cluster via a single-step transformation was demonstrated. We envision that SATIMD is a powerful tool not only useful for direct large heterologous gene cluster assembly in T. reesei but also can facilitate large-scale fungal strain genetic engineering.