Custom-made transcriptional biosensors for metabolic engineering

Koch, Mathilde; Pandi, Amir; Borkowski, Olivier; Batista, Angelo Cardoso; Faulon, Jean-Loup

doi:10.1016/j.copbio.2019.02.016

Cited by 75 publications

(69 citation statements)

References 60 publications

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“…Next, by mining publicly available transcriptome and phenome datasets (Cherry et al, 2012;Regenberg et al, 2006), this study demonstrates successful selection criteria for identification of candidate essential genes, encoding a broad array of metabolic functions, which can be coupled to biosensors and thus enable selective growth based on small-molecule concentrations. With the increasing number of biosensors being developed, and demonstrations of fermentation-based manufacturing of valuable chemicals based on anabolic metabolism (Koch et al, 2019;Nielsen and Keasling, 2016), this combined approach should be applicable for stabilizing and optimizing many more bioprocesses in the future.…”

Section: Discussionmentioning

confidence: 99%

Regulatory control circuits for stabilizing long-term anabolic product formation in yeast

D’ambrosio

Dore

Blasi

et al. 2020

Preprint

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Engineering living cells for production of chemicals, enzymes and therapeutics can burden cells due to use of limited native co-factor availability and/or expression burdens, totalling a fitness deficit compared to parental cells encoded through long evolutionary trajectories to maximise fitness. Ultimately, this discrepancy puts a selective pressure against fitness-burdened engineered cells under prolonged bioprocesses, and potentially leads to complete eradication of highperforming engineered cells at the population level. Here we present the mutation landscapes of fitness-burdened yeast cells engineered for vanillin-β-glucoside production. Next, we design synthetic control circuits based on transcriptome analysis and biosensors responsive to vanillin-βglucoside pathway intermediates in order to stabilize vanillin-β-glucoside production over ~55 generations in sequential passage experiments. Furthermore, using biosensors with two different modes of action we identify control circuits linking vanillin-β-glucoside pathway flux to various essential cellular functions, and demonstrate control circuits robustness and 92% higher vanillin-βglucoside production, including 5-fold increase in total vanillin-β-glucoside pathway metabolite accumulation, in a fed-batch fermentation compared to vanillin-β-glucoside producing cells without control circuits.

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Section: Discussionmentioning

confidence: 99%

Regulatory control circuits for stabilizing long-term anabolic product formation in yeast

D’ambrosio

Dore

Blasi

et al. 2020

Preprint

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“…The development of new aTF-based biosensors is an important area of the synthetic biology field, as aTFs are versatile biological tools for a range of methodologies and applications [23,24]. Developing biosensors for aromatic molecules is especially important due to their utility in industry and their availability from biomass sources [62].…”

Section: Discussionmentioning

confidence: 99%

“…Although aTF biosensors have been broadly used, the repertoire of effectors with a known aTF is limited [7]. Therefore strategies to generate new biosensors for desired molecules have been pursued [23,24].…”

Section: Introductionmentioning

confidence: 99%

Directed evolution of the PcaV allosteric transcription factor to generate a biosensor for aromatic aldehydes

Machado

Currin

Dixon

2019

Preprint

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Background:Genetically encoded biosensors are useful tools for the detection of metabolites and industrially valuable molecules, and present many potential applications in biotechnology and biomedicine. However, the most common approach to develop biosensors relies on employing a limited set of naturally occurring allosteric transcript factors (aTFs). Therefore, altering the substrate specificity of aTFs towards the detection of new effectors is an important goal. Results:Here, the PcaV repressor, a member of the MarR aTF family, was used to develop a biosensor for the detection of hydroxyl-substituted benzoic acids, including protocatechuic acid (PCA). The PCA biosensor was further subjected to directed evolution to alter its substrate specificity towards vanillin and other closely related aromatic aldehydes, to generate the Van2 biosensor. Substrate recognition of Van2 was explored in vitro using a range of biochemical and biophysical analyses, and extensive in vivo genetic-phenotypic analysis was performed to determine the role of each amino acid change upon biosensor performance. Conclusions:This is the first study to report directed evolution of a member of the MarR aTF family, and demonstrates the plasticity of the PCA biosensor by altering its substrate specificity to generate a biosensor for aromatic aldehydes.

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“…TF‐based cell‐free biosensors have been used to identify specific small molecules, such as inorganic ions and organic molecules (bacterial quorum sensing molecules), and they are also used in high‐throughput screening and metabolic engineering. [ 48,60,61 ] Specific TFs or natural conformational TFs are selected by different analytes, such as TF in response to aromatic compounds (XylS‐AraC, XylR‐NtrC, and LysR), metal ions (MerR, ArsR, DtxR, Fur, and NikR), or antibiotics (TetR and MarR). [ 62 ] When analytes (ligands) are present in a cell‐free environment, the response of TFs is immediately obtained, and binding of ligands to their activated TFs or the release of ligand‐bound inhibitory TFs can lead to the reporter gene expression ( Figure 2 A).…”

Section: Principle and Workflow Of Cell‐free Biosensorsmentioning

confidence: 99%

Advances in Cell‐Free Biosensors: Principle, Mechanism, and Applications

Zhang

Guo

2020

Biotechnology Journal

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Synthetic biology has promoted the development of biosensors as tools for detecting trace substances. In the past, biosensors based on synthetic biology have been designed on living cells, but the development of cell biosensors has been greatly limited by defects such as genetically modified organism problem and the obstruction of cell membrane. However, the advent of cell‐free synthetic biology addresses these limitations. Biosensors based on the cell‐free protein synthesis system have the advantages of higher safety, higher sensitivity, and faster response time over cell biosensors, which make cell‐free biosensors have a broader application prospect. This review summarizes the workflow of various cell‐free biosensors, including the identification of analytes and signal output. The detection range of cell‐free biosensors is greatly enlarged by different recognition mechanisms and output methods. In addition, the review also discusses the applications of cell‐free biosensors in environmental monitoring and health diagnosis, as well as existing deficiencies and aspects that should be improved. In the future, through continuous improvement and optimization, the potential of cell‐free biosensors will be stimulated, and their application fields will be expanded.

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Custom-made transcriptional biosensors for metabolic engineering

Cited by 75 publications

References 60 publications

Regulatory control circuits for stabilizing long-term anabolic product formation in yeast

Regulatory control circuits for stabilizing long-term anabolic product formation in yeast

Directed evolution of the PcaV allosteric transcription factor to generate a biosensor for aromatic aldehydes

Advances in Cell‐Free Biosensors: Principle, Mechanism, and Applications

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