Angeliki Sitara, Rémi Hocq, Alexander Jiwei Lu, Stefan Pflügl
{"title":"Hi-TARGET：一种快速、高效、多功能的CRISPR I-B型基因组编辑工具，用于嗜热厌氧菌kivui","authors":"Angeliki Sitara, Rémi Hocq, Alexander Jiwei Lu, Stefan Pflügl","doi":"10.1186/s13068-025-02647-0","DOIUrl":null,"url":null,"abstract":"<div><h3>Background</h3><p>Due to its ability to grow fast on CO<sub>2</sub>, CO and H<sub>2</sub> at high temperatures and with high energy efficiency, the thermophilic acetogen <i>Thermoanaerobacter</i> <i>kivui</i> could become an attractive host for industrial biotechnology. In a circular carbon economy, diversification and upgrading of C1 platform feedstocks into value-added products (e. g., ethanol, acetone and isopropanol) could become crucial. To that end, genetic and bioprocess engineering tools are required to facilitate the development of bioproduction scenarios. Currently, the genome editing tools available for <i>T. kivui</i> present some limitations in speed and efficiency, thus restricting the development of a powerful strain chassis for industrial applications.</p><h3>Results</h3><p>In this study, we developed the versatile genome editing tool Hi-TARGET, based on the endogenous CRISPR Type I-B system of <i>T. kivui</i>. Hi-TARGET demonstrated 100% efficiency for gene knock-out (from both purified plasmid and cloning mixture) and knock-in, and 49% efficiency for creating point mutations. Furthermore, we optimized the transformation and plating protocol and increased transformation efficiency by 245-fold to 1.96 × 10<sup>4</sup> ± 8.7 × 10<sup>3</sup> CFU μg<sup>−1</sup>. Subsequently, Hi-TARGET was used to demonstrate gene knock-outs (<i>pyrE</i>, <i>rexA</i>, <i>hrcA</i>), a knock-in (<i>ldh</i>::pFAST), a single nucleotide mutation corresponding to PolC<sup>C629Y</sup>, and knock-down of the fluorescent protein pFAST. Analysis of the ∆<i>rexA</i> deletion mutant created with Hi-TARGET revealed that the transcriptional repressor <i>rexA</i> is likely involved in the regulation of the expression of lactate dehydrogenase (<i>ldh</i>). Following genome engineering, an optimized curing procedure for edited strains was devised. In total, the time required from DNA to a clean, edited strain is 12 days, rendering Hi-TARGET a fast, robust and complete method for engineering <i>T. kivui</i>.</p><h3>Conclusions</h3><p>The CRISPR-based genome editing tool Hi-TARGET developed for <i>T. kivui</i> can be used for scarless deletion, insertion, point mutation and gene knock-down, thus fast-tracking the generation of industrially-relevant strains for the production of carbon-negative chemicals and fuels as well as facilitating studies of acetogen metabolism and physiology.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"18 1","pages":""},"PeriodicalIF":6.1000,"publicationDate":"2025-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-025-02647-0","citationCount":"0","resultStr":"{\"title\":\"Hi-TARGET: a fast, efficient and versatile CRISPR type I-B genome editing tool for the thermophilic acetogen Thermoanaerobacter kivui\",\"authors\":\"Angeliki Sitara, Rémi Hocq, Alexander Jiwei Lu, Stefan Pflügl\",\"doi\":\"10.1186/s13068-025-02647-0\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><h3>Background</h3><p>Due to its ability to grow fast on CO<sub>2</sub>, CO and H<sub>2</sub> at high temperatures and with high energy efficiency, the thermophilic acetogen <i>Thermoanaerobacter</i> <i>kivui</i> could become an attractive host for industrial biotechnology. In a circular carbon economy, diversification and upgrading of C1 platform feedstocks into value-added products (e. g., ethanol, acetone and isopropanol) could become crucial. To that end, genetic and bioprocess engineering tools are required to facilitate the development of bioproduction scenarios. Currently, the genome editing tools available for <i>T. kivui</i> present some limitations in speed and efficiency, thus restricting the development of a powerful strain chassis for industrial applications.</p><h3>Results</h3><p>In this study, we developed the versatile genome editing tool Hi-TARGET, based on the endogenous CRISPR Type I-B system of <i>T. kivui</i>. Hi-TARGET demonstrated 100% efficiency for gene knock-out (from both purified plasmid and cloning mixture) and knock-in, and 49% efficiency for creating point mutations. Furthermore, we optimized the transformation and plating protocol and increased transformation efficiency by 245-fold to 1.96 × 10<sup>4</sup> ± 8.7 × 10<sup>3</sup> CFU μg<sup>−1</sup>. Subsequently, Hi-TARGET was used to demonstrate gene knock-outs (<i>pyrE</i>, <i>rexA</i>, <i>hrcA</i>), a knock-in (<i>ldh</i>::pFAST), a single nucleotide mutation corresponding to PolC<sup>C629Y</sup>, and knock-down of the fluorescent protein pFAST. Analysis of the ∆<i>rexA</i> deletion mutant created with Hi-TARGET revealed that the transcriptional repressor <i>rexA</i> is likely involved in the regulation of the expression of lactate dehydrogenase (<i>ldh</i>). Following genome engineering, an optimized curing procedure for edited strains was devised. 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Hi-TARGET: a fast, efficient and versatile CRISPR type I-B genome editing tool for the thermophilic acetogen Thermoanaerobacter kivui
Background
Due to its ability to grow fast on CO2, CO and H2 at high temperatures and with high energy efficiency, the thermophilic acetogen Thermoanaerobacterkivui could become an attractive host for industrial biotechnology. In a circular carbon economy, diversification and upgrading of C1 platform feedstocks into value-added products (e. g., ethanol, acetone and isopropanol) could become crucial. To that end, genetic and bioprocess engineering tools are required to facilitate the development of bioproduction scenarios. Currently, the genome editing tools available for T. kivui present some limitations in speed and efficiency, thus restricting the development of a powerful strain chassis for industrial applications.
Results
In this study, we developed the versatile genome editing tool Hi-TARGET, based on the endogenous CRISPR Type I-B system of T. kivui. Hi-TARGET demonstrated 100% efficiency for gene knock-out (from both purified plasmid and cloning mixture) and knock-in, and 49% efficiency for creating point mutations. Furthermore, we optimized the transformation and plating protocol and increased transformation efficiency by 245-fold to 1.96 × 104 ± 8.7 × 103 CFU μg−1. Subsequently, Hi-TARGET was used to demonstrate gene knock-outs (pyrE, rexA, hrcA), a knock-in (ldh::pFAST), a single nucleotide mutation corresponding to PolCC629Y, and knock-down of the fluorescent protein pFAST. Analysis of the ∆rexA deletion mutant created with Hi-TARGET revealed that the transcriptional repressor rexA is likely involved in the regulation of the expression of lactate dehydrogenase (ldh). Following genome engineering, an optimized curing procedure for edited strains was devised. In total, the time required from DNA to a clean, edited strain is 12 days, rendering Hi-TARGET a fast, robust and complete method for engineering T. kivui.
Conclusions
The CRISPR-based genome editing tool Hi-TARGET developed for T. kivui can be used for scarless deletion, insertion, point mutation and gene knock-down, thus fast-tracking the generation of industrially-relevant strains for the production of carbon-negative chemicals and fuels as well as facilitating studies of acetogen metabolism and physiology.
期刊介绍：
Biotechnology for Biofuels is an open access peer-reviewed journal featuring high-quality studies describing technological and operational advances in the production of biofuels, chemicals and other bioproducts. The journal emphasizes understanding and advancing the application of biotechnology and synergistic operations to improve plants and biological conversion systems for the biological production of these products from biomass, intermediates derived from biomass, or CO2, as well as upstream or downstream operations that are integral to biological conversion of biomass.
Biotechnology for Biofuels focuses on the following areas:
• Development of terrestrial plant feedstocks
• Development of algal feedstocks
• Biomass pretreatment, fractionation and extraction for biological conversion
• Enzyme engineering, production and analysis
• Bacterial genetics, physiology and metabolic engineering
• Fungal/yeast genetics, physiology and metabolic engineering
• Fermentation, biocatalytic conversion and reaction dynamics
• Biological production of chemicals and bioproducts from biomass
• Anaerobic digestion, biohydrogen and bioelectricity
• Bioprocess integration, techno-economic analysis, modelling and policy
• Life cycle assessment and environmental impact analysis
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