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

Flaiz, Maximilian; Baur, Tina; Gaibler, Jana; Kröly, Christian; Dürre, Peter

doi:10.1021/acssynbio.1c00554

Cited by 15 publications

(20 citation statements)

References 58 publications

(91 reference statements)

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“…Further factors to contribute to culture heterogeneity are cell morphogenesis or sporulation, both of which are especially apparent in clostridial cultures (Jones et al 2008;Tracy et al 2010). Most recently, Flaiz et al (2022) also demonstrated heterogeneity of C. saccharoperbutylacetonicum cultures expressing feg in a P tcdB -dependent manner with tcdR controlled by P bgaL . Again, culture heterogeneity was observed throughout all growth phases, although in this case, the number of fluorescent cells was higher compared to the present study (Flaiz et al 2022).…”

Section: Discussionmentioning

confidence: 99%

Production of propionate using metabolically engineered strains of Clostridium saccharoperbutylacetonicum

Baur

Wentzel

Dürre

2022

Appl Microbiol Biotechnol

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The carboxylic acid propionate is a valuable platform chemical with applications in various fields. The biological production of this acid has become of great interest as it can be considered a sustainable alternative to petrochemical synthesis. In this work, Clostridium saccharoperbutylacetonicum was metabolically engineered to produce propionate via the acrylate pathway. In total, the established synthetic pathway comprised eight genes encoding the enzymes catalyzing the conversion of pyruvate to propionate. These included the propionate CoA-transferase, the lactoyl-CoA dehydratase, and the acryloyl-CoA reductase from Anaerotignum neopropionicum as well as a D-lactate dehydrogenase from Leuconostoc mesenteroides subsp. mesenteroides. Due to difficulties in assembling all genes on one plasmid under the control of standard promoters, the PtcdB-tcdR promoter system from Clostridium difficile was integrated into a two-plasmid system carrying the acrylate pathway genes. Several promoters were analyzed for their activity in C. saccharoperbutylacetonicum using the fluorescence-activating and absorption-shifting tag (FAST) as a fluorescent reporter to identify suitable candidates to drive tcdR expression. After selecting the lactose-inducible PbgaL promoter, engineered C. saccharoperbutylacetonicum strains produced 0.7 mM propionate upon induction of gene expression. The low productivity was suspected to be a consequence of a metabolic imbalance leading to acryloyl-CoA accumulation in the cells. To even out the proposed imbalance, the propionate-synthesis operons were rearranged, thereby increasing the propionate concentration by almost four-fold. This study is the first one to report recombinant propionate production using a clostridial host strain that has opened a new path towards bio-based propionate to be improved further in subsequent work. Key points • Determination of promoter activities in C. saccharoperbutylacetonicum using FAST. • Implementation of propionate production in C. saccharoperbutylacetonicum. • Elevation of propionate production by 375% to a concentration of 3 mM.

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Section: Discussionmentioning

confidence: 99%

Production of propionate using metabolically engineered strains of Clostridium saccharoperbutylacetonicum

Baur

Wentzel

Dürre

2022

Appl Microbiol Biotechnol

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“…For example, FAST:HMBR fluorescence of anaerobic Clostridium organisms was monitored aerobically using confocal microscopy, microplate reader measurements and flow cytometry measurements (21). Another recent study applied flow cytometry (aerobic) to detect fluorescence of anaerobic, acetogenic bacteria (29). Other studies avoid oxygen when detecting FAST:fluorogen fluorescence of anaerobic organisms, e.g., by placing a fluorescence microscope or microplate reader into anaerobic chambers (14, 30).…”

Section: Discussionmentioning

confidence: 99%

“…Another recent study applied flow cytometry (aerobic) to detect fluorescence of anaerobic, acetogenic bacteria (29). Other studies avoid oxygen when detecting FAST:fluorogen fluorescence of anaerobic organisms, e.g., by placing a fluorescence microscope or microplate reader into anaerobic chambers (14,30).…”

Section: Aerobic Vs Anaerobic Detection Of Fast:fluorogen Fluorescencementioning

confidence: 99%

Application of the fluorescence-activating and absorption-shifting tag (FAST) for flow cytometry in methanogenic archaea

Adlung

Scheller²

2022

Preprint

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Methane-producing archaea play a crucial role in the global carbon cycle and are used for biotechnological fuel production. Methanogenic model organisms such as Methanococcus maripaludis and Methanosarcina acetivorans are biochemically characterized and can be genetically engineered using a variety of molecular tools. Methanogens’ anaerobic lifestyle and autofluorescence, however, restrict the use of common fluorescent reporter proteins (e.g., GFP and derivatives) which require oxygen for chromophore maturation. Here, we employ the tandem activation and absorption-shifting tag protein 2 (tdFAST2) which is fluorescent when the cell-permeable fluorescent ligand (fluorogen) 4-hydroxy-3,5-dimethoxybenzylidene rhodanine (HBR-3,5DOM) is present. tdFAST2 expression in M. acetivorans and M. maripaludis is not cytotoxic and tdFAST2:HBR-3,5DOM fluorescence can be clearly distinguished from the autofluorescence. In flow cytometry experiments, mixed methanogen cultures can be clearly distinguished which allows high-throughput investigations of dynamics within single and mixed cultures.

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“…This behavior had been previously described only in fixed embryo or in live embryos analyzed by label-free nonlinear microscopy, but these techniques did not give access to the individual phases of the cell cycle. GreenFAST and redFAST were recently used as fluorescent reporters for investigating the coculture dynamics of metabolically engineered Clostridium saccharoperbutylacetonicum strains under anaerobic conditions …”

Section: The Growing Family Of Fast Reportersmentioning

confidence: 99%

“…GreenFAST and redFAST were recently used as fluorescent reporters for investigating the coculture dynamics of metabolically engineered Clostridium saccharoperbutylacetonicum strains under anaerobic conditions. 40 ■ FAST-BASED SENSORS Topological variants of FAST were developed to design fluorescent chemogenetic biosensors (Figure 5). The possibility to condition fluorogen binding, and thus fluorescence, to a given recognition event or cellular signal enabled the development of sensors for various applications in biological research.…”

Section: ■ the Growing Family Of Fast Reportersmentioning

confidence: 99%

Fluorescence-Activating and Absorption-Shifting Tags for Advanced Imaging and Biosensing

Gautier

2022

Acc. Chem. Res.

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Metrics & More Article Recommendations CONSPECTUS: Fluorescent labels and biosensors play central roles in biological and medical research. Targeted to specific biomolecules or cells, they allow noninvasive imaging of the machinery that govern cells and organisms in real time. Recently, chemogenetic reporters made of organic dyes specifically anchored to genetic tags have challenged the paradigm of fully genetically encoded fluorescent proteins. Combining the advantage of synthetic fluorophores with the targeting selectivity of genetically encoded tags, these chemogenetic reporters open new exciting prospects for studying cell biochemistry and biology.In this Account, we present the growing toolbox of fluorescence-activating and absorption-shifting tags (FASTs), small monomeric proteins of 14 kDa (125 amino acids residues) that can be used as markers to monitor gene expression and protein localization in live cells and organisms. Engineered by directed protein evolution from the photoactive yellow protein (PYP) from the bacterium Halorhodospira halophila, prototypical FAST binds and stabilizes the fluorescent state of live-cell compatible hydroxybenzylidene rhodanine chromophores. This class of chromophores are normally dark when free in solution or in cells because they dissipate light energy through nonradiative processes. The protein cavity of FAST allows the stabilization of the deprotonated state of the chromophore and blocks the chromophore into a planar conformation, which leads to highly fluorescent protein-chromophore assemblies. The use of such fluorogenic dyes (also called fluorogens) enables the imaging of FAST fusion proteins in cells with high contrast without the need to remove unbound ligands through separate washing steps. Fluorogens with various spectral properties exist nowadays allowing investigators to adjust the spectral properties of FAST to their experimental conditions. Molecular engineering allowed furthermore to generate membrane-impermeant fluorogens for the selective labeling of cell-surface proteins. Over the years, we generated a collection of FAST variants with expanded spectral properties or fluorogen selectivity using a concerted strategy involving molecular engineering and directed protein evolution. Moreover, protein engineering allowed us to adapt FASTs for the design of fluorescent biosensors. Circular permutation enabled the generation of FAST variants with increased conformational flexibility for the design of biosensors in which fluorogen binding is conditioned to the recognition of a given analyte. Bisection of FASTs into two complementary fragments allowed us furthermore to create split variants with reversible complementation that allow the detection and imaging of dynamic protein−protein interactions. We provide, here, a general overview of the current state of development of these different systems and their applications for advanced live cell imaging and biosensing and discuss potential future directions.

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

Cited by 15 publications

References 58 publications

Production of propionate using metabolically engineered strains of Clostridium saccharoperbutylacetonicum

Production of propionate using metabolically engineered strains of Clostridium saccharoperbutylacetonicum

Application of the fluorescence-activating and absorption-shifting tag (FAST) for flow cytometry in methanogenic archaea

Fluorescence-Activating and Absorption-Shifting Tags for Advanced Imaging and Biosensing

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