Background Due to its ability to grow fast on CO2, CO and H2 at high temperatures and with high energy efficiency, the thermophilic acetogen Thermoanaerobacter kivui 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.

Hi-TARGET: a fast, efficient and versatile CRISPR type I-B genome editing tool for the thermophilic acetogen Thermoanaerobacter kivui

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2025年4月30日

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Angeliki Sitara,
Rémi Hocq,
Alexander Jiwei Lu,
Stefan Pflügl

摘要

Background

Due to its ability to grow fast on CO₂, CO and H₂ at high temperatures and with high energy efficiency, the thermophilic acetogen Thermoanaerobacter kivui 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 PolC^C629Y, 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.