Blueprints for Biosensors: Design, Limitations, and Applications

Carpenter, Alexander C.; Paulsen, Ian T.; Williams, Thomas

doi:10.3390/genes9080375

Cited by 111 publications

(99 citation statements)

References 106 publications

(220 reference statements)

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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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“…Biosensors can be classified into three types: riboswitch, ligand‐inducible allosteric protein, and specific transcription factor coupled with a reporter gene. [ 31 ]…”

Section: Transporter Mining and Characterizationmentioning

confidence: 99%

Transporter Engineering for Microbial Manufacturing

Zhu

Zhou

Wang

et al. 2020

Biotechnology Journal

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Microbes play an important role in biotransformation and biosynthesis of biofuels, natural products, and polymers. Therefore, microbial manufacturing has been widely used in medicine, industry, and agriculture. However, common strategies including enzyme engineering, pathway optimization, and host engineering are generally inadequate to obtain an efficient microbial production system. Transporter engineering provides an alternative strategy to promote the transmembrane transfer of substrates, intermediates, and final products in microbial cells and thus enhances production by alleviating feedback inhibition and cytotoxicity caused by final products. According to the current studies in transport engineering, native transporters usually have low expression and poor transportation ability, resulting in inefficient transport processes and microbial production. In this review, current approaches for transporter mining, characterization, and verification are comprehensively summarized. Practical approaches to enhance the transport system in engineered cells, such as balancing transporter overexpression and cell growth, and evolution of native transporters are discussed. Furthermore, the applications of transporter engineering in microbial manufacturing, including enhancement of substrate utilization, concentration of metabolic flux to the target pathway, and acceleration of efflux and recovery of products, demonstrate its outstanding advantages and promising prospects.

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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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Blueprints for Biosensors: Design, Limitations, and Applications

Cited by 111 publications

References 106 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

Transporter Engineering for Microbial Manufacturing

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

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