Eubacterium strain engineering with FAST is a continued and fruitful focus of Dr. Maximilian Flaiz of Wageningen University and Dr. Franck Bengelsdorf of Ulm University. Indeed, Flaiz et al. have just published “Refining and illuminating acetogenic Eubacterium strains for reclassification and metabolic engineering” in Microbial Cell Factories 2024.
The genus Eubacterium includes several acetogenic strains capable of fermenting C1-substrates into valuable biocommodities. Of those, Eubacterium limosum and closely related strains are furthermore capable to produce butyrate. Apart from its well-elucidated metabolism, E. limosum is also genetically accessible. As a result, it is a most promising candidate to be an industrial biocatalyst. The genetic toolbox already includes various selection markers and promoters, plasmid-based gene expression, as well as CRISPR-Cas and CRISPRi gene editing tools.
Plasmid electroporation is a key step towards engineered strains. The authors of this article were able to rapidly engineer a large variety of strains using a harmonized electroporation protocol. By employing FAST of The Twinkle Factory, they indeed detect fluorescent cells by applying flow cytometry. This screening method is easy, fast, and provides insights about successfully transformed cells at a single-cell level. Namely, this allowed them to assign eleven different strains of Eubacterium to distinct clades, E. limosum, E. callanderi and E. maltosivorans.
Anaerobic bacteria are expected to be pivotal in the future SmartCarbon world. Indeed, several anaerobes are already genetically accessible. Various molecular tools are available for metabolic engineering. Still, the use of bright fluorescent reporters, commonly used in molecular biological approaches, is limited under anaerobic conditions. This is what FAST of The Twinkle Factory is good at!
The Twinkle Factory offers commercial fluorogens for FAST and derivatives, splitFAST, greenFAST & redFAST, frFAST. Since recently, the range of reagents also includes fluorogenic molecular glues for CATCHFIRE.
More reading on strain engineering with FAST
- Microb. Cell Factories 2024 – Refining and illuminating acetogenic Eubacterium strains for reclassification and metabolic engineering
- ACS Synth. Biol. 2022 – Establishment of Green- and Red-Fluorescent Reporter Proteins Based on the Fluorescence-Activating and Absorption-Shifting Tag for Use in Acetogenic and Solventogenic Anaerobes
- Nat. Commun. 2021 – Auxin-mediated protein depletion for metabolic engineering in terpene-producing yeast
- Biotechnol. Biofuels 2021 – Production of the Biocommodities Butanol and Acetone from Methanol with Fluorescent FAST-tagged Proteins using Metabolically Engineered Strains of Eubacterium Limosum
- mBio 2020 – Interspecies Microbial Fusion and Large-Scale Exchange of Cytoplasmic Proteins and RNA in a Syntrophic Clostridium Coculture
- Appl. Environ. Microbiol. 2019 – A Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System for Clostridium Organisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST)