Simultaneous <i>In Vivo</i> Assembly and Targeted Genome Integration of Gene Clusters in <i>Trichoderma reesei</i>

The saprophytic filamentous fungus <i>Trichoderma reesei</i> 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 <i>T. reesei</i>, are still in demand. Here, we developed a method for the simultaneous <i>in vivo</i> assembly and targeted genome integration of multiple DNA fragments (SATIMD) in <i>T. reesei</i>. 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 <i>T. reesei</i> 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 <i>T. reesei</i> but also can facilitate large-scale fungal strain genetic engineering.