A fluorescent reporter system for anaerobic thermophiles

Frontiers in Bioengineering and Biotechnology

Bioengineering and Biotechnology

Rémi Hocq¹  Stefan Pflügl¹  Sara Bottone²  Arnaud Gautier² 

[1] Institute of Chemical, Environmental and Bioscience Engineering, Technische Universität Wien, Vienna, Austria;Christian Doppler Laboratory for Optimized Expression of Carbohydrate-Active Enzymes, Institute of Chemical, Environmental and Bioscience Engineering, Technische Universität Wien, Vienna, Austria;Laboratoire des Biomolécules (LBM), Centre National de la Recherche Scientifique (CNRS), Sorbonne Université, École Normale Supérieure, Université PSL, Paris, France;Institut Universitaire de France, Paris, France;

关键词: anaerobic thermophiles;    fluorescent reporter;    fluorescence-activating and absorption-shifting tag;    genetic tools;    acetogen;    Thermoanaerobacter;   

DOI  :  10.3389/fbioe.2023.1226889

 received in 2023-05-22, accepted in 2023-06-23,  发布年份 2023

来源: Frontiers

【 摘 要 】

Owing to their inherent capacity to make invisible biological processes visible and quantifiable, fluorescent reporter systems have numerous applications in biotechnology. For classical fluorescent protein systems (i.e., GFP and derivatives), chromophore maturation is O2-dependent, restricting their applications to aerobic organisms. In this work, we pioneered the use of the oxygen-independent system FAST (Fluorescence Activating and absorption Shifting tag) in the thermophilic anaerobe Thermoanaerobacter kivui. We developed a modular cloning system that was used to easily clone a library of FAST expression cassettes in an E. coli—Thermoanaerobacter shuttle plasmid. FAST-mediated fluorescence was then assessed in vivo in T. kivui, and we observed bright green and red fluorescence for cells grown at 55°C. Next, we took advantage of this functional reporter system to characterize a set of homologous and heterologous promoters by quantifying gene expression, expanding the T. kivui genetic toolbox. Low fluorescence at 66°C (Topt for T. kivui) was subsequently investigated at the single-cell level using flow cytometry and attributed to plasmid instability at higher temperatures. Adaptive laboratory evolution circumvented this issue and drastically enhanced fluorescence at 66°C. Whole plasmid sequencing revealed the evolved strain carried functional plasmids truncated at the Gram-positive origin of replication, that could however not be linked to the increased fluorescence displayed by the evolved strain. Collectively, our work demonstrates the applicability of the FAST fluorescent reporter systems to T. kivui, paving the way for further applications in thermophilic anaerobes.

【 授权许可】

Unknown   
Copyright © 2023 Hocq, Bottone, Gautier and Pflügl.