Fang Yu, Meng Xiangfeng, Liu Lin, Li Zhongye, Jia Kaili, Liu Weifeng
State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University, No. 72 Binhai Road, Qingdao 266237, P. R. China.
ACS Synth Biol. 2025 Feb 21;14(2):575-584. doi: 10.1021/acssynbio.4c00810. Epub 2025 Feb 6.
The saprophytic filamentous fungus 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 , are still in demand. Here, we developed a method for the simultaneous assembly and targeted genome integration of multiple DNA fragments (SATIMD) in . 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 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 but also can facilitate large-scale fungal strain genetic engineering.
腐生丝状真菌是最丰富的纤维素酶产生菌之一，也有潜力被开发成用于生物合成次级代谢产物的易处理真菌宿主。为了加速丝状真菌的基因工程，仍然需要能够促进将大尺寸DNA转移到真菌宿主的高效DNA组装方法，包括……在此，我们开发了一种在……中同时组装和靶向基因组整合多个DNA片段的方法（SATIMD）。通过具有各种长度序列重叠（100 - 500 bp）的同源重组（HR）实现了高效有序的DNA末端融合，当与CRISPR/Cas9介导的基因组切割相结合时，组装的DNA也能精确整合到特定位点。具体而言，我们已使用此方法实现了纤维素酶基因关键转录激活因子Xyr1的组装和功能表达。此外，还证明了通过一步转化对包含32.7 kb山梨素生物合成基因簇的多达10个不同DNA片段进行融合和靶向整合。我们设想SATIMD是一种强大的工具，不仅可用于在……中直接进行大型异源基因簇组装，还能促进大规模真菌菌株基因工程。
