Establishment of Green- and Red-Fluorescent Reporter Proteins Based on the Fluorescence-Activating and Absorption-Shifting Tag for Use in Acetogenic and Solventogenic Anaerobes

Anaerobic bacteria are promising biocatalysts to produce industrially relevant products from nonfood feedstocks. Several anaerobes are genetically accessible, and various molecular tools for metabolic engineering are available. Still, the use of bright fluorescent reporters, which are commonly used in molecular biological approaches is limited under anaerobic conditions. Therefore, the establishment of different anaerobic fluorescent reporter proteins is of great interest. Here, we present the establishment of the green-and red-fluorescent reporter proteins greenFAST and redFAST for use in different solventogenic and acetogenic bacteria. Green fluorescence of greenFAST was bright in Clostridium saccharoperbutylacetonicum, Clostridium acetobutylicum, Acetobacterium woodii, and Eubacterium limosum, while only C. saccharoperbutylacetonicum showed bright red fluorescence when producing redFAST. We used both reporter proteins in C. saccharoperbutylacetonicum for multicolor approaches. These include the investigation of the co-culture dynamics of metabolically engineered strains. Moreover, we established a tightly regulated inducible two-plasmid system and used greenFAST and redFAST to track the coexistence and interaction of both plasmids under anaerobic conditions in C. saccharoperbutylacetonicum. The establishment of greenFAST and redFAST as fluorescent reporters opens the door for further multicolor approaches to investigate cell dynamics, gene expression, or protein localization under anaerobic conditions.