Biomass Energy Technology Research Centre, Key Laboratory of Development and Application of Rural Renewable Energy (Ministry of Agriculture and Rural Affairs), Biogas Institute of Ministry of Agriculture and Rural Affairs, Section 4-13, Renmin Rd. South, Chengdu, 610041, China.
Graduate School of Chinese Academy of Agricultural Science, Beijing, 100081, China.
Appl Microbiol Biotechnol. 2023 Dec;107(23):7151-7163. doi: 10.1007/s00253-023-12783-y. Epub 2023 Sep 20.
Zymomonas mobilis is regarded as a potential chassis for the production of platform chemicals. Genome editing using the CRISPR-Cas system could meet the need for gene modification in metabolic engineering. However, the low curing efficiency of CRISPR editing plasmid is a common bottleneck in Z. mobilis. In this study, we utilized a theophylline-dependent riboswitch to regulate the expression of the replicase gene of the editing plasmid, thereby promoting the elimination of exogeneous plasmid. The riboswitch D (RSD) with rigorous regulatory ability was identified as the optimal candidate by comparing the transformation efficiency of four theophylline riboswitch-based backbone editing plasmids, and the optimal theophylline concentration for inducing RSD was determined to be 2 mM. A highly effective method for eliminating the editing plasmid, cells with RSD-based editing plasmid which were cultured in liquid and solid RM media in alternating passages at 37 °C without shaking, was established by testing the curing efficiency of backbone editing plasmids pMini and pMini-RSD in RM medium with or without theophylline at 30 °C or 37 °C. Finally, the RSD-based editing plasmid was applied to genome editing, resulting in an increase of more than 10% in plasmid elimination efficiency compared to that of pMini-based editing plasmid. KEY POINTS: • An effective strategy for curing CRISPR editing plasmid has been established in Z. mobilis. • Elimination efficiency of the CRISPR editing plasmid was enhanced by 10% to 20% under the regulation of theophylline-dependent riboswitch RSD.
运动发酵单胞菌被认为是生产平台化学品的潜在底盘。使用 CRISPR-Cas 系统进行基因组编辑可以满足代谢工程中基因修饰的需求。然而，CRISPR 编辑质粒的低修复效率是运动发酵单胞菌中的一个常见瓶颈。在本研究中，我们利用茶碱依赖性核糖开关来调节编辑质粒的复制酶基因的表达，从而促进外源质粒的消除。通过比较四种基于茶碱核糖开关的骨架编辑质粒的转化效率，鉴定出具有严格调控能力的核糖开关 D（RSD）作为最佳候选物，并确定了诱导 RSD 的最佳茶碱浓度为 2 mM。通过在 37°C 下不摇动地在 RM 液体和固体培养基中交替传代培养带有 RSD 编辑质粒的细胞，建立了一种高效消除编辑质粒的方法。在 30°C 或 37°C 时，在含有或不含有茶碱的 RM 培养基中测试骨架编辑质粒 pMini 和 pMini-RSD 的修复效率，确定了 RSD 为基础的编辑质粒在 RM 培养基中的修复效率。最后，将 RSD 为基础的编辑质粒应用于基因组编辑，与基于 pMini 的编辑质粒相比，质粒消除效率提高了 10%以上。关键点：• 在运动发酵单胞菌中建立了一种有效的 CRISPR 编辑质粒修复策略。• 通过茶碱依赖性核糖开关 RSD 的调节，CRISPR 编辑质粒的消除效率提高了 10%到 20%。
