Design, Optimization and Application of Small Molecule Biosensor in Metabolic Engineering

Liu, Yang; Liu, Ye; Wang, Meng

doi:10.3389/fmicb.2017.02012

Cited by 91 publications

(56 citation statements)

References 79 publications

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“…Biosensor development based on transcription factors has recently seen rapid development (Rogers et al , ; Lehning et al , ; Liu et al , ; Carpenter et al , ). Yet, three aspects of our work are worth to be highlighted: As no endogenous FBP‐binding transcription factor is known in yeast, we had to transfer the B. subtilis transcription factor CggR into yeast.…”

Section: Discussionmentioning

confidence: 99%

“…Through single-cell flow cytometry and time-lapse fluorescence microscopy experiments, we demonstrated the applicability of the sensor to reveal differences in glycolytic flux between single cells. Biosensor development based on transcription factors has recently seen rapid development (Rogers et al, 2016;Lehning et al, 2017;Liu et al, 2017;Carpenter et al, 2018). Yet, three aspects of our work are worth to be highlighted: As no endogenous FBPbinding transcription factor is known in yeast, we had to transfer the B. subtilis transcription factor CggR into yeast.…”

Section: Discussionmentioning

confidence: 99%

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Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor

Monteiro

Hubmann

Takhaveev

et al. 2019

Molecular Systems Biology

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Metabolic heterogeneity between individual cells of a population harbors significant challenges for fundamental and applied research. Identifying metabolic heterogeneity and investigating its emergence require tools to zoom into metabolism of individual cells. While methods exist to measure metabolite levels in single cells, we lack capability to measure metabolic flux, i.e., the ultimate functional output of metabolic activity, on the single‐cell level. Here, combining promoter engineering, computational protein design, biochemical methods, proteomics, and metabolomics, we developed a biosensor to measure glycolytic flux in single yeast cells. Therefore, drawing on the robust cell‐intrinsic correlation between glycolytic flux and levels of fructose‐1,6‐bisphosphate (FBP), we transplanted the B. subtilis FBP‐binding transcription factor CggR into yeast. With the developed biosensor, we robustly identified cell subpopulations with different FBP levels in mixed cultures, when subjected to flow cytometry and microscopy. Employing microfluidics, we were also able to assess the temporal FBP/glycolytic flux dynamics during the cell cycle. We anticipate that our biosensor will become a valuable tool to identify and study metabolic heterogeneity in cell populations.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor

Monteiro

Hubmann

Takhaveev

et al. 2019

Molecular Systems Biology

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“…Between these, Spinach, Spinach2 and Broccoli aptamers have been widely used to build ligand-responsive biosensors for live-cell Riboswitches are also suitable for the engineering of more complex regulations through the implementation of biochemical logic gates and feedback systems [49][50][51]. RNA-based biosensors are also extensively used for the engineering of metabolic pathways to optimise the production of small molecules such as industrial and fuel compounds [52], or for the sensing of intracellular behaviours and environmental conditions [53].…”

Section: Natural and Synthetic Riboswitchesmentioning

confidence: 99%

Engineering CRISPR guide RNA riboswitches for in vivo applications

Galizi

Jaramillo

2019

Current Opinion in Biotechnology

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NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/. Publisher's statement:Please refer to the repository item page, publisher's statement section, for further information.For more information, please contact the WRAP Team at: wrap@warwick.ac.uk.instead of 3′ of the target site (precisely TTTV for Cpf1, NGG for SpCas9), has intrinsic RNase activity for the processing of multiple crRNAs from a single transcript [9,10] and makes staggered cuts (5'′ overhang) whilst Cas9 makes "blunt"'blunt' cuts in the genome [7]. CRISPR-associated endonuclease and related gRNAs can work heterologously in most species to produce mutagenesis, gene knockin/knockout and chromosome deletion/translocations. If the protein is engineered by knocking out each of its two nickase domains to generate the catalytically inert proteins 'dCas9' and 'dCpf1', the sgRNA:dCas9 (or sgRNA:dCpf1) complex will remain bound to its double-stranded DNA CRISPR-based genome editing provides a simple and scalable toolbox for a variety of therapeutic and biotechnology applications. Whilst the fundamental properties of CRISPR proved easily transferable from the native prokaryotic hosts to eukaryotic and multicellular organisms, the tight control of the CRISPR-editing activity remains a major challenge. Here we summarise recent developments of CRISPR and riboswitch technologies and recommend novel functionalised synthetic-gRNA (sgRNA) designs to achieve inducible and spatiotemporal regulation of CRISPR-based genetic editors in response to cellular or extracellular stimuli. We believe that future advances of these tools will have major implications for both basic and applied research, spanning from fundamental genetic studies and synthetic biology to genetic editing and gene therapy.(dsDNA) substrate [2]. As many other DNA-binding proteins, such as the popular zinc fingers, sgRNA:dCas9 or dCpf1 can work as a transcriptional repressor for CRISPR interfering (CRISPRi) without further engineering [11,12] or by adding transcriptional repression domains such as the Krüppel associated box (KRAB) [13,14]. Deactivated CRISPR-associated endonucleases can be engineered to function as transcriptional activators or epigenetic modifiers other than being utilized for high-resolution genome imagining [15] and DNA/RNA pull-down [16]. Hereafter we will refer to all these variants as CRISPR-editors. At least in theory, it is possible to reprogram CRISPR specificity to recognizse any target region flanking a short PAM sequence by simply providing different gRNA-spacers. The PAM therefore represents the sole CRISPR-targetability constraint, which has been shown possible to broaden by mutating the PAM interacting domain of the protein [17,18]. These findings empowered over the recent years a vast range of applications from basic biology to biotechnology and medicine. Nonetheless, several challenges are still hampering the broad applicability, efficiency, specificity and safety of CRISPR-editing systems (Figure 1). Between these, the preci...

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“…These results demonstrate that our flux sensor can be also used with microscopy, and is thus suitable for discrimination of individual cells with regards to their glycolytic flux levels even within clonal cell populations. Biosensor development based on transcription factors has recently seen rapid development 31,[51][52][53] . Three aspects of our work are worth to be highlighted: As no endogenous FBP-binding transcription factor is known in yeast, we had to transfer the B.…”

Section: Proof Of Concept: Quantification Of Glycolytic Flux In Coeximentioning

confidence: 99%

Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor

Monteiro

Hubmann

Norder

et al. 2019

Preprint

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Metabolic heterogeneity between individual cells of a population harbors offers significantchallenges for fundamental and applied research. Identifying metabolic heterogeneity and investigating its emergence requires tools to zoom into metabolism of individual cells.While methods exist to measure metabolite levels in single cells, we lack capability to measure metabolic flux, i.e. the ultimate functional output of metabolic activity, on the single-cell level. Here, combining promoter engineering, computational protein design, biochemical methods, proteomics and metabolomics, we developed a biosensor to measure glycolytic flux in single yeast cells, by drawing on the robust cell-intrinsic correlation between glycolytic flux and levels of fructose-1,6-bisphosphate (FBP), and by transplanting the B. subtilis FBP-binding transcription factor CggR into yeast. As proof of principle, using fluorescence microscopy, we applied the sensor to identify metabolic subpopulations in yeast cultures. We anticipate that our biosensor will become a valuable tool to identify and study metabolic heterogeneity in cell populations. perform flux-dependent regulation 29,30 . Biosensors for such metabolites, such as recently accomplished for E. coli 31 , would in principle allow for measurement of metabolic fluxes in single cells, in combination with microscopy or flow cytometry.Here, drawing on glycolytic flux-signalling metabolite fructose-1,6-bisphosphate (FBP) levels in yeast 30 and using the B. subtilis FBP-binding transcription factor CggR 32,33 , we developed a biosensor, which allows to measure glycolytic flux in single yeast cells. To this end, we used computational protein design, biochemical, proteome and metabolome analyses, for (i) the development of a synthetic yeast promoter regulated by the bacterial transcriptional factor CggR, (ii) the engineering of the transcription factors' FBP binding site towards increasing the sensor's dynamic range, and (iii) the establishment of growthindependent CggR expression levels. We demonstrate the applicability of the biosensor for flow cytometry and time-lapse fluorescence microscopy. We envision that the biosensor will open new avenues for both fundamental and applied metabolic research, not only for monitoring glycolytic flux, but also for engineering control circuits with glycolytic flux as input variable. Results Design of biosensor conceptFor our biosensor, we exploited the fact that the level of the glycolytic intermediate fructose-1,6-biphosphate (FBP) in yeast 30,34 , similar to other organisms 27 , strongly correlates with the glycolysis flux 28,34,35 , and that changing FBP levels exert fluxdependent regulation. In B. subtilis, for instance, FBP binds to the transcription factor (TF) CggR 33 , which when bound to its target DNA forms a tetrameric assembly of two dimers, through which transcription gets inhibited 36 . Upon binding of FBP to the CggR-DNA complex, the dimer-dimer contacts of CggR are disrupted, and the promoter is derepressed 37 .Here, we aimed to transplant the B...

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Design, Optimization and Application of Small Molecule Biosensor in Metabolic Engineering

Cited by 91 publications

References 79 publications

Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor

Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor

Engineering CRISPR guide RNA riboswitches for in vivo applications

Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor

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