Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
ACS Synth Biol. 2024 Jul 19;13(7):2105-2114. doi: 10.1021/acssynbio.4c00157. Epub 2024 Jun 13.
The budding yeast has emerged as a promising microbial chassis in industrial biotechnology. However, a lack of efficient molecular genetic manipulation tools and strategies has hindered the development of as a biomanufacturing platform. In this study, we developed and applied a CRISPR/Cas9-based genome editing method to . Single-gene editing efficiency was increased to 80% by disrupting the nonhomologous end-joining-related gene and performing a series of process optimizations. Subsequently, the CRISPR/Cas9 system was explored based on different sgRNA delivery modes for simultaneous multigene editing. With the aid of the color indicator, the editing efficiencies of two and three genes reached 73.3 and 36%, respectively, in the Δ strain. Furthermore, the CRISPR/Cas9 system was used for multisite integration to enhance lactase production and combinatorial knockout of and to characterize the extracellular secretion of lactase in . Generally, genome editing is a powerful tool for constructing cell factories for protein and chemical production.
出芽酵母已成为工业生物技术中有前途的微生物底盘。然而，缺乏有效的分子遗传操作工具和策略阻碍了作为生物制造平台的发展。在这项研究中，我们开发并应用了基于 CRISPR/Cas9 的基因组编辑方法来。通过破坏非同源末端连接相关基因并进行一系列工艺优化，将单基因编辑效率提高到 80%。随后，基于不同 sgRNA 传递模式探索了 CRISPR/Cas9 系统，以进行同时的多基因编辑。借助显色指示剂，在Δ菌株中，两个和三个基因的编辑效率分别达到了 73.3%和 36%。此外，CRISPR/Cas9 系统用于多位点整合以提高乳糖酶的产量，并对和进行组合敲除，以研究乳糖酶在中的细胞外分泌。总的来说，基因组编辑是构建用于蛋白质和化学品生产的工程细胞工厂的有力工具。
