After reporting FAST in Thermoanaerobacter kivui as a protein fluorescent reporter for the first time ever in 2023, TU Wien’s Rémi Hocq and team have just disclosed Hi-TARGET, a CRISPR type I-B genome-editing tool for the thermophilic acetogen. Building up on their previous work, they implemented FAST to validate knock-downs and knock-ins.
Why anaerobic thermophiles?
FAST in anaerobic thermophiles – © 2019 LetsTalkScience
Anaerobic thermophilic bacteria are top-of-the-list candidates for turning one-carbon feedstock into biofuels or chemicals. Indeed, anaerobes typically display high carbon and energy efficiency. Beyond, thermophilic microbes offer additional benefits compared to mesophiles. Namely, they feature higher catalytic turnover rates, lower cooling/distillation costs and contamination risks.
Of those anaerobes, T. kivui is of utmost relevance. It indeed converts one-carbon feedstocks, e.g., carbon dioxide or any mixture CO2/[H]. And also, forward and reverse genetic approaches are readily applicable to T. kivui.
Now, metabolic engineering still remains challenging. The genetic toolbox is not so wide. And anaerobic lifestyle restricts the use of common fluorescent reporters such as GFP and conventional fluorescent proteins. Those indeed require oxygen for chromophore maturation.
Genetic engineering of Thermoanaerobacter kivui
In 2023, the team validated for the first time pFAST, the latest release of FAST, in T. kivui. After optimization, they found it highly functional even at 66°C growth temperature with cognate fluorogens, tfLime (green) and tfCoral (red). Finally, pFAST enabled simple, direct, quantitative and fast fluorescence measurements on a variety of expression plasmids. The authors implemented both flow cytometry and plate reading setups.
In 2025, the same team have disclosed the genome-editing method Hi-TARGET for T. kivui engineering based on its endogenous type I-B CRISPR system. After optimization, they found it fast, efficient, versatile. Indeed, 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. 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. This work builds up on the initial implementation of FAST in T. kivui hence demonstrating its versatility and robustness.
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 assays, 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.The authors
The Twinkle Factory offers commercial fluorogens for FAST, pFAST, greenFAST & redFAST, frFAST, nirFAST, and split versions, e.g., splitFAST. tfCarmine, a near-infrared fluorogen has recently completed the product range.
Read more about FAST and Thermoanaerobacter kivui
Research Square 2025 – Sitara, A., Hocq, R., Lu, A. J., & Pflügl, S. Hi-TARGET: A fast, efficient and versatile CRISPR type I-B genome editing tool for the thermophilic acetogen Thermoanaerobacter kivui
Front. Bioeng. Biotechnol. 2023 – Hocq, R., Bottone, S., Gautier, A., & Pflügl, S. A fluorescent reporter system for anaerobic thermophiles
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