Background The interest in using methanol as a substrate to cultivate acetogens increased in recent years since it can be sustainably produced from syngas and has the additional benefit of reducing greenhouse gas emissions. Eubacterium limosum is one of the few acetogens that can utilize methanol, is genetically accessible and, therefore, a promising candidate for the recombinant production of biocommodities from this C1 carbon source. Although several genetic tools are already available for certain acetogens including E. limosum, the use of brightly fluorescent reporter proteins is still limited. Results In this study, we expanded the genetic toolbox of E. limosum by implementing the fluorescence-activating and absorption shifting tag (FAST) as a fluorescent reporter protein. Recombinant E. limosum strains that expressed the gene encoding FAST in an inducible and constitutive manner were constructed. Cultivation of these recombinant strains resulted in brightly fluorescent cells even under anaerobic conditions. Moreover, we produced the biocommodities butanol and acetone from methanol with recombinant E. limosum strains. Therefore, we used E.limosum cultures that produced FAST-tagged fusion proteins of the bifunctional acetaldehyde/alcohol dehydrogenase or the acetoacetate decarboxylase, respectively, and determined the fluorescence intensity and product concentrations during growth. Conclusions The addition of FAST as an oxygen-independent fluorescent reporter protein expands the genetic toolbox of E. limosum. Moreover, our results show that FAST-tagged fusion proteins can be constructed without negatively impacting the stability, functionality, and productivity of the resulting enzyme. Finally, butanol and acetone can be produced from methanol using recombinant E.limosum strains expressing genes encoding fluorescent FAST-tagged fusion proteins.

Caproicibacter fermentans gen. nov., sp. nov., a new caproateproducing bacterium and emended description of the genus Caproiciproducens Abstract A strictly anaerobic bacterial strain designated EA1 T was isolated from an enrichment culture inoculated with biogas reactor content. Cells of strain EA1 T are spore-forming rods (1-3×0.4-0.8 µm) and stain Gram-negative, albeit they possess a Grampositive type of cell-wall ultrastructure. Growth of strain EA1 T was observed at 30 and 37 °C and within a pH range of pH 5-9. The major components recovered in the fatty acid fraction were C 14:0 , C 16:0 , C 16:0 DMA (dimethyl acetal) and C 16:1 ω7c. Strain EA1 T fermented several mono-and disaccharides. Metabolic end products from fructose were acetate, butyrate, caproate and lactate. Furthermore, ethanol, CO 2 and H 2 were identified as products. The genome consists of a chromosome (3.9 Mbp) with 3797 predicted protein-encoding genes and a G+C content of 51.25 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain EA1 T represents a novel taxon within the family Oscillospiraceae. The most closely related type strains of EA1 T , based on 16S rRNA gene sequence identity, are Caproiciproducens galactitolivorans BS-1 T (94.9 %), [Clostridium] leptum DSM 753 T (93.8 %), [Clostridium] sporosphaeroides DSM 1294 T (91.7 %) and Ruminococcus bromii ATCC 27255 T (91.0 %). Further phenotypic characteristics of strain EA1 T differentiate it from related, validly described bacterial species. Strain EA1 T represents a novel genus and novel species within the family Oscillospiraceae. The proposed name is Caproicibacter fermentans gen. nov., sp. nov. The type strain is EA1 T (DSM 107079 T =JCM 33110 T ).

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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.

Background: The interest in using methanol as a substrate to cultivate acetogens increased in recent years since it can be sustainably produced from syngas and has the additional benefit of reducing greenhouse gas emissions. Eubacterium limosum is one of the few acetogens that can utilize methanol, is genetically accessible and, therefore, a promising candidate for the recombinant production of biocommodities from this C1 carbon source. Although several genetic tools are already available for certain acetogens including E. limosum, the use of brightly fluorescent reporter proteins is still limited.Results: In this study, we expanded the genetic toolbox of E. limosum by implementing the fluorescence-activating and absorption shifting tag (FAST) as a fluorescent reporter protein. Recombinant E. limosum strains that expressed the gene encoding FAST in an inducible and constitutive manner were constructed. Cultivation of these recombinant strains resulted in brightly fluorescent cells even under anaerobic conditions. Moreover, we produced the biocommodities butanol and acetone from methanol with recombinant E. limosum strains. Therefore, we used E. limosum cultures that produced FAST-tagged fusion proteins of the bifunctional acetaldehyde/alcohol dehydrogenase or the acetoacetate decarboxylase, respectively, and determined the fluorescence intensity and product yields during growth.Conclusions: The addition of FAST as an oxygen-independent fluorescent reporter protein expands the genetic toolbox of E. limosum. Moreover, our results show that FAST-tagged fusion proteins can be constructed without negatively impacting the stability, functionality, and productivity of the resulting enzyme. Finally, butanol and acetone can be produced from methanol using recombinant E. limosum strains expressing genes encoding fluorescent FAST-tagged fusion proteins.