• Journal of Microbiology and Biotechnology
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• v.33 no.10
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• pp.1257-1267
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• 2023

Although rational genetic engineering is nowadays the favored method for microbial strain improvement, building up mutant libraries based on directed evolution for improvement is still in many cases the better option. In this regard, the demand for precise and efficient screening methods for mutants with high performance has stimulated the development of biosensor-based high-throughput screening strategies. Genetically encoded biosensors provide powerful tools to couple the desired phenotype to a detectable signal, such as fluorescence and growth rate. Herein, we review recent advances in engineering several classes of biosensors and their applications in directed evolution. Furthermore, we compare and discuss the screening advantages and limitations of two-component biosensors, transcription-factor-based biosensors, and RNA-based biosensors. Engineering these biosensors has focused mainly on modifying the expression level or structure of the biosensor components to optimize the dynamic range, specificity, and detection range. Finally, the applications of biosensors in the evolution of proteins, metabolic pathways, and genome-scale metabolic networks are described. This review provides potential guidance in the design of biosensors and their applications in improving the bioproduction of microbial cell factories through directed evolution.

• Journal of Microbiology and Biotechnology
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• v.33 no.4
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• pp.552-558
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• 2023

Levulinic acid (LA) is a valuable chemical used in fuel additives, fragrances, and polymers. In this study, we proposed possible biosynthetic pathways for LA production from lignin and poly(ethylene terephthalate). We also created a genetically encoded biosensor responsive to LA, which can be used for screening and evolving the LA biosynthesis pathway genes, by employing an LvaR transcriptional regulator of Pseudomonas putida KT2440 to express a fluorescent reporter gene. The LvaR regulator senses LA as a cognate ligand. The LA biosensor was first examined in an Escherichia coli strain and was found to be non-functional. When the host of the LA biosensor was switched from E. coli to P. putida KT2440, the LA biosensor showed a linear correlation between fluorescence intensity and LA concentration in the range of 0.156-10 mM LA. In addition, we determined that 0.156 mM LA was the limit of LA detection in P. putida KT2440 harboring an LA-responsive biosensor. The maximal fluorescence increase was 12.3-fold in the presence of 10 mM LA compared to that in the absence of LA. The individual cell responses to LA concentrations reflected the population-averaged responses, which enabled high-throughput screening of enzymes and metabolic pathways involved in LA biosynthesis and sustainable production of LA in engineered microbes.

• Molecules and Cells
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• v.39 no.1
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• pp.46-52
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• 2016

It is increasingly apparent that nature evolved peroxiredoxins not only as $H_2O_2$ scavengers but also as highly sensitive $H_2O_2$ sensors and signal transducers. Here we ask whether the $H_2O_2$ sensing role of Prx can be exploited to develop probes that allow to monitor intracellular $H_2O_2$ levels with unprecedented sensitivity. Indeed, simple gel shift assays visualizing the oxidation of endogenous 2-Cys peroxiredoxins have already been used to detect subtle changes in intracellular $H_2O_2$ concentration. The challenge however is to create a genetically encoded probe that offers real-time measurements of $H_2O_2$ levels in intact cells via the Prx oxidation state. We discuss potential design strategies for Prx-based probes based on either the redoxsensitive fluorophore roGFP or the conformation-sensitive fluorophore cpYFP. Furthermore, we outline the structural and chemical complexities which need to be addressed when using Prx as a sensing moiety for $H_2O_2$ probes. We suggest experimental strategies to investigate the influence of these complexities on probe behavior. In doing so, we hope to stimulate the development of Prx-based probes which may spearhead the further study of cellular $H_2O_2$ homeostasis and Prx signaling.

• Molecules and Cells
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• v.41 no.9
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• pp.809-817
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• 2018

Discovery of the naturally evolved fluorescent proteins and their genetically engineered biosensors have enormously contributed to current bio-imaging techniques. These reporters to trace dynamic changes of intracellular protein activities have continuously transformed according to the various demands in biological studies. Along with that, light-inducible optogenetic technologies have offered scientists to perturb, control and analyze the function of intracellular machineries in spatiotemporal manner. In this review, we present an overview of the molecular strategies that have been exploited for producing genetically encoded protein reporters and various optogenetic modules. Finally, in particular, we discuss the current efforts for combined use of these reporters and optogenetic modules as a powerful tactic for the control and imaging of signaling events in cells and tissues.

• International Journal of Oral Biology
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• v.38 no.4
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• pp.169-173
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• 2013

HyPer is the genetically encoded biosensor of intracellular hydrogen peroxide ($H_2O_2$), the most stable of the reactive oxygen species (ROS) generated by living cells. HyPer has a high sensitivity and specificity for detecting intracellular $H_2O_2$ by confocal laser microscopy. However, it was not known whether high speed ratiometric imaging of $H_2O_2$ by HyPer is possible. We thus investigated the sensitivity of HyPer in detecting changes to the intracellular $H_2O_2$ levels in HEK293 and PC12 cells using a microfluorometer imaging system. Increase in the HyPer ratio were clearly evident on stimulations of more than $100{\mu}M$ $H_2O_2$ and fast changes in the HyPer ratio were observed on ratiometric fluorescent images after $H_2O_2$ treatment. These results suggest that HyPer is a potent biosensor of the fast temporal production of intracellular $H_2O_2$.

• Journal of Life Science
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• v.33 no.2
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• pp.191-198
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• 2023

As a member of the focal adhesion complex of the plasma membrane, Src is a nonreceptor tyrosine kinase that controls cell adhesion and motility. However, how Src activity is regulated in the plasma membrane microdomain in response to components of the extracellular matrix (ECM) remains unclear. This study compared and investigated the activity of Src in response to three representative ECM proteins: collagen type 1, fibronectin, and laminin. Genetically encoded FRET-based Src biosensors for plasma membrane subdomains were used. FRET-based biosensors allow the real-time analysis of protein activity in living cells based on their high spatiotemporal resolution. The results showed that Src activity was maintained at a high level under all ECM conditions of the lipid raft, and there was no significant difference between the ECM conditions. In contrast, Src activity was maintained at a low level in the non-lipid raft membrane. In addition, the Src activity of lipid rafts remained significantly higher than that of non-lipid raft regions under the same ECM conditions. In conclusion, this study demonstrates that Src activity can be controlled differently by lipid rafts and non-lipid raft microdomains.

• Journal of Life Science
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• v.32 no.2
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• pp.148-154
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• 2022

Focal adhesion kinase (FAK) is known to regulate cell adhesion, migration, and mechanotransduction in focal adhesions (FAs). However, studies on how FAK activity is regulated in the plasma membrane microdomains according to the composition of extracellular matrix (ECM) proteins are still lacking. A genetically encoded fluorescence resonance energy transfer (FRET)-based biosensor can provide useful information on the activity of intracellular signals with high spatiotemporal resolution. In this study, we analyzed the FAK activities in lipid raft (detergent-resistant membrane) and non-lipid raft (non-detergent-resistant membrane) microdomains using FRET-based membrane targeting FAK biosensors (FAK-Lyn and FAK-KRas biosensors) under four different ECM protein compositions: glass, type 1 collagen, fibronectin, and laminin. Interestingly, FAK activity in response to laminin in a lipid raft microdomain was lower than that in other ECM conditions. Cells subjected to fibronectin showed higher FAK activity in a lipid raft microdomain than that in a non-lipid raft microdomain. Therefore, this study demonstrates that the FAK activity can be distinctively regulated according to the ECM type and the environment of the plasma membrane microdomains.