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Sugar-based raw materials have been the main carbon source for industrial microbial fermentation. However, due to resource and cost factors, humans need to continuously explore the synthesis of bulk chemicals and fuels with new, cheaper raw materials and highly efficient recombinant microorganisms. Carbon-carbon gases (such as CO and CO2, etc.) are important carbon resources and have a wide range of sources, including exhaust gas from large-scale petrochemical and smelting enterprises, and synthesis gas produced from the gasification of carbon-containing substances such as coal, petroleum, natural gas, and biomass. Syngas) etc. Therefore, the construction of a carbon-fixing microorganism that can efficiently use a carbon gas and its conversion into a target product by fermentation will open up a new path for resolving global resources and energy issues, which is of great significance to the sustainable development of industry.
Clostridium ljungdahlii is a very few carbon-fixing microorganisms that are close to industrial applications. It can utilize CO and CO2 to efficiently synthesize ethanol, which has attracted widespread attention. However, its genetic operating system is not yet perfect, and its molecular components are particularly scarce, so it cannot be effectively designed and modified. Huang He and others from the Jiang Weihong Research Group of the Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, under the guidance of researcher Gu Yang, first screened and identified a strong heterologous promoter suitable for use in C. ljungdahlii for efficient driving. The expression of the Cas9 protein derived from Streptococcus pyogenes and the guide RNA in this bacterium was used to avoid the possibility of non-specific recombination using its endogenous promoter. On this basis, through tests on multiple key genes, it was proved that the system can effectively identify and cleave the target sequence, and then quickly complete the deletion of the target gene, which can be completed in five days and the efficiency can reach 50%-100%. In addition, the exogenous plasmids in the mutant strains can be rapidly eliminated by subculturing under selective pressure for subsequent gene manipulations.
The gene editing system successfully overcomes the deficiency of the existing genetic manipulation method of Clostridium ljungdahlii, provides great convenience for the research of this important industrial carbon-fixing bacterium, and lays a foundation for the subsequent establishment of more precise molecular manipulation techniques. The work was published in the ACS Synthetic Biology magazine on June 8 and entitled CRISPR/Cas9-based efficient genome editing in Clostridium ljungdahlii, an autotrophic gas-fermenting bacterium.
The above research was supported by the "863" project of the Ministry of Science and Technology, the key deployment projects of the Chinese Academy of Sciences and the Sino-British synthetic biology project.
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