Zhang Jun-Zhe, Li Yu-Zhen, Xi Zhi-Ning, Zhang Yue, Liu Zi-Yong, Ma Xiao-Qing, Li Fu-Li
State Key Laboratory of Photoelectric Conversion and Utilization of Solar Energy, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China.
Shandong C1 Refinery Engineering Research Center, Qingdao New Energy Shandong Laboratory, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China.
Appl Environ Microbiol. 2025 Apr 23;91(4):e0218324. doi: 10.1128/aem.02183-24. Epub 2025 Mar 26.
, as a model acetogen strain, represents a novel platform for biotechnological production for CO fixation. The genome of harbors two gene loci associated with glycyl radical enzyme-associated microcompartments (GRMs), which are predicted to play essential roles in choline and 1,2-propanediol (1,2-PD) metabolism. This study validated the functions of these GRM loci and identified two inducible promoters, of which P was induced by choline, while P was induced by 1,2-PD. Subsequently, the highly expressed P and tightly controlled P were applied to improve CRISPR/Cas9 gene editing tools. Specifically, P was used to develop a highly efficient gene knockout tool based on an all-in-one plasmid, achieving 100% deletion efficiency for multiple genes, including , , , and . On the other hand, the gene was integrated downstream of P into the genome. The integrated efficiently mediated gene editing in by introducing plasmids containing a gRNA cassette along with the relevant homology arms. This was exemplified by the construction of the strain, where the 2,3-butanediol dehydrogenase gene was replaced with a pyruvate decarboxylase gene from and the 3-HB Syn KI strain, in which an artificial 3-hydroxybutyric acid synthesis pathway was inserted into the genome. This study highlights the effectiveness and convenience of the inducible CRISPR/Cas9 gene editing systems, thereby enriching the CRISPR/Cas toolkit in acetogens.
A CRISPR/Cas9 genetic tool controlled by a constitutive promoter has been developed for precise gene deletion in . However, its efficiency was hindered by the toxicity resulting from the constitutive expression of cas9 and the large plasmids, leading to a low overall success rate. Inducible promoters, which allow for the transcription of target genes to be switched on and off in the presence or absence of inducers, have a broad range of applications. In this study, we identify two inducible promoters and apply them to enhance the CRISPR/Cas9 tools. The improved CRISPR/Cas9 tools facilitate gene editing with high efficiency, potentially playing significant roles in advancing genetic research and metabolic engineering of .
作为一种典型的产乙酸菌菌株，是用于CO固定生物技术生产的新型平台。的基因组含有两个与甘氨酰自由基酶相关微区室（GRMs）相关的基因座，预计它们在胆碱和1,2 - 丙二醇（1,2 - PD）代谢中起重要作用。本研究验证了这些GRM基因座的功能，并鉴定了两个诱导型启动子，其中P由胆碱诱导，而P由1,2 - PD诱导。随后，将高表达的P和严格控制的P应用于改进CRISPR/Cas9基因编辑工具。具体而言，P用于开发基于一体化质粒的高效基因敲除工具，对多个基因（包括、、和）实现了100%的缺失效率。另一方面，基因整合到P下游的基因组中。整合的通过引入含有gRNA盒以及相关同源臂的质粒有效地介导了中的基因编辑。这在构建菌株和3 - HB Syn KI菌株中得到了体现，在菌株中，2,3 - 丁二醇脱氢酶基因被来自的丙酮酸脱羧酶基因取代，在3 - HB Syn KI菌株中，人工3 - 羟基丁酸合成途径被插入到基因组中。本研究突出了诱导型CRISPR/Cas9基因编辑系统的有效性和便利性，从而丰富了产乙酸菌中的CRISPR/Cas工具集。
已开发出一种由组成型启动子控制的CRISPR/Cas9基因工具，用于中的精确基因缺失。然而，其效率受到cas9组成型表达和大质粒产生的毒性的阻碍，导致总体成功率较低。诱导型启动子允许在有或没有诱导剂的情况下打开或关闭靶基因的转录，具有广泛的应用。在本研究中，我们鉴定了两个诱导型启动子，并将它们应用于增强CRISPR/Cas9工具。改进后的CRISPR/Cas9工具便于高效进行基因编辑，可能在推进的遗传研究和代谢工程中发挥重要作用。
