State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China.
Biotechnol Lett. 2024 Dec;46(6):1319-1332. doi: 10.1007/s10529-024-03519-7. Epub 2024 Jul 31.
Non-pathogenic mycobacteria, including Mycolicibacterium neoaurum, can directly utilize phytosterols for large-scale industrial production of steroid medicine intermediates due to their natural steroid metabolism pathway. The targeted genetic modification of M. neoaurum is conducive to the selection of high-yield engineering bacteria with high-value-added product, such as Pregnadien-20-carboxylic acid (PDC), which is an important precursor for synthesizing some corticosteroids. Based on heterologous type II CRISPR/sth1Cas9 system, a simple strategy was developed to genetic engineer M. neoaurum genome. Here, a customizable plasmid tool pMSC9 was constructed from pMV261 with integration of sth1Cas9 protein and corresponding sgRNA scaffold. Subsequently, the pMSC9 was inserted with spacer sequences corresponding to different targeted genes, generating editing plasmids, and then transformed into M. neoaurum. As a result, the targeted genes were introduced with DNA double stand breaks (DSBs) by CRISPR/sth1Cas9 system and then repaired by innate non-homologous end-joining (NHEJ) mechanism. Finally, editing plasmids were cured from correctly edited M. neoaurum mutants by means of no resistance cultivation, and the resulting mutant deleting the one target gene was used as the host to which another target gene could be deleted via the same process. This study demonstrated that the CRISPR/sth1Cas9 tool allowed M. neoaurum strains to be rapidly edited. And the editing mode of CRISPR/sth1Cas9 system indicated that this tool was an important supplement to the gene editing toolbox of M. neoaurum.
非致病分枝杆菌，包括新金色分枝杆菌，由于其天然的甾体代谢途径，可以直接利用植物甾醇进行甾体药物中间体的大规模工业生产。对新金色分枝杆菌进行靶向基因修饰有利于选择具有高附加值产品的高产工程菌，如孕烯-20-羧酸（PDC），它是合成某些皮质激素的重要前体。基于异源 II 型 CRISPR/sth1Cas9 系统，开发了一种简单的基因工程新金色分枝杆菌基因组的策略。在此，构建了一个可定制的质粒工具 pMSC9，它来源于 pMV261，整合了 sth1Cas9 蛋白和相应的 sgRNA 支架。随后，将 pMSC9 插入与不同靶向基因对应的间隔序列，生成编辑质粒，然后转化到新金色分枝杆菌中。结果，CRISPR/sth1Cas9 系统通过 DNA 双链断裂（DSBs）将靶向基因引入，然后通过内源性非同源末端连接（NHEJ）机制进行修复。最后，通过无抗性培养从正确编辑的新金色分枝杆菌突变体中去除编辑质粒，将产生的缺失一个目标基因的突变体用作宿主，通过相同的过程可以删除另一个目标基因。这项研究表明，CRISPR/sth1Cas9 工具允许快速编辑新金色分枝杆菌菌株。并且 CRISPR/sth1Cas9 系统的编辑模式表明，该工具是新金色分枝杆菌基因编辑工具箱的重要补充。
