Department of Chemical and Biological Engineering, 4140 Biorenewables Research Laboratory, Iowa State University, Ames, IA, 50011, USA.
NSF Engineering Research Center for Biorenewable Chemicals (CBiRC), 4140 Biorenewables Research Laboratory, Iowa State University, Ames, IA, 50011, USA.
Biotechnol J. 2018 Sep;13(9):e1700598. doi: 10.1002/biot.201700598. Epub 2018 Jul 29.
Scheffersomyces stipitis, renowned for its native xylose-utilizing capacity, has recently demonstrated its potential in producing health-promoting shikimate pathway derivatives. However, its broader application is hampered by the low transformation efficiency and the lack of genetic engineering tools to enable sophisticated genomic manipulations. S. stipitis employs the predominant non-homologous end joining (NHEJ) mechanism for repairing DNA double-strand breaks (DSB), which is less desired due to its incompetence in achieving precise genome editing. Using CRISPR technology, here a ku70Δku80Δ deficient strain in which homologous recombination (HR)-based genome editing appeared dominant for the first time in S. stipitis is constructed. To build all essential tools for efficiently manipulating this highly promising nonconventional microbial host, the gene knockdown tool is also established, and repression efficiency is improved by incorporating a transcriptional repressor Mxi1 into the CRISPR-dCas9 platform. All these results are obtained with the improved transformation efficiency, which is 191-fold higher than that obtained with the traditional parameters used in yeast transformation. This work paves the way for advancing a new microbial chassis and provides a guideline for developing efficient CRISPR tools in other nonconventional yeasts.
产朊假丝酵母以其天然利用木糖的能力而闻名，最近已被证明在生产促进健康的莽草酸途径衍生物方面具有潜力。然而，由于转化效率低，缺乏基因工程工具来进行复杂的基因组操作，其更广泛的应用受到了阻碍。产朊假丝酵母采用主要的非同源末端连接（NHEJ）机制来修复 DNA 双链断裂（DSB），由于其在实现精确基因组编辑方面的能力不足，因此这种机制不太理想。在这里，我们使用 CRISPR 技术构建了一个 ku70Δku80Δ 缺陷菌株，这是首次在产朊假丝酵母中发现同源重组（HR）为基础的基因组编辑占主导地位。为了构建用于有效操作这种极具前景的非常规微生物宿主的所有必要工具，还建立了基因敲低工具，并通过将转录抑制剂 Mxi1 整合到 CRISPR-dCas9 平台中，提高了抑制效率。所有这些结果都是在提高转化效率的情况下获得的，其效率比传统酵母转化参数提高了 191 倍。这项工作为推进新的微生物底盘奠定了基础，并为开发其他非常规酵母中高效的 CRISPR 工具提供了指导。
