Designing, construction and characterization of genetically encoded FRET-based nanosensor for real time monitoring of lysine flux in living cells

Ameen, Seema; Ahmad, Mohammad; Mohsin, Mohd; Qureshi, M. Irfan; Ibrahim, Mohamed M.; Abdin, M. Z.; Ahmad, Altaf

doi:10.1186/s12951-016-0204-y

Cited by 49 publications

(29 citation statements)

References 35 publications

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“…By contrast, the sensor with the cpLAO-BP showed a typical sigmoidal binding isotherm with a high sensitivity, which referred to a FRET-ratio shift of ΔR = 0.47 (28%, Figure 2). Similar sensors described in the literature displayed a FRET-ratio shift of 57% [21] or of ΔR = 0.28 (56%) [43], respectively.…”

Section: Resultsmentioning

confidence: 59%

A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis

Steffen

Otten

Engelmann

et al. 2016

Sensors

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Background: The fast development of microbial production strains for basic and fine chemicals is increasingly carried out in small scale cultivation systems to allow for higher throughput. Such parallelized systems create a need for new rapid online detection systems to quantify the respective target compound. In this regard, biosensors, especially genetically encoded Förster resonance energy transfer (FRET)-based biosensors, offer tremendous opportunities. As a proof-of-concept, we have created a toolbox of FRET-based biosensors for the ratiometric determination of l-lysine in fermentation broth. Methods: The sensor toolbox was constructed based on a sensor that consists of an optimized central lysine-/arginine-/ornithine-binding protein (LAO-BP) flanked by two fluorescent proteins (enhanced cyan fluorescent protein (ECFP), Citrine). Further sensor variants with altered affinity and sensitivity were obtained by circular permutation of the binding protein as well as the introduction of flexible and rigid linkers between the fluorescent proteins and the LAO-BP, respectively. Results: The sensor prototype was applied to monitor the extracellular l-lysine concentration of the l-lysine producing Corynebacterium glutamicum (C. glutamicum) strain DM1933 in a BioLector® microscale cultivation device. The results matched well with data obtained by HPLC analysis and the Ninhydrin assay, demonstrating the high potential of FRET-based biosensors for high-throughput microbial bioprocess optimization.

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Section: Resultsmentioning

confidence: 59%

A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis

Steffen

Otten

Engelmann

et al. 2016

Sensors

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“…We expect that this study will help to develop many more FRET-based genetically encoded nanosensors based on steady-state fluorescence anisotropy. Up to now, a diverse range of FRET-based genetically encoded nanosensors have been designed and constructed by exploring different sets of fluorescent proteins and different ligand binding periplasmic proteins to acquire live-cell imaging of bacterial cells for the ideal representation of the nanosensors [30][31][32]. The FLIP-SP will be useful for studying sulfate uptake, translocation, and regulatory mechanisms controlling compartmental SO 4 2− homeostasis.…”

Section: Discussionmentioning

confidence: 99%

A Non-Invasive Tool for Real-Time Measurement of Sulfate in Living Cells

Fatima

Okla

Mohsin

et al. 2020

IJMS

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Sulfur (S) is an essential element for all forms of life. It is involved in numerous essential processes because S is considered as the primary source of one of the essential amino acids, methionine, which plays an important role in biological events. For the control and regulation of sulfate in a metabolic network through fluxomics, a non-invasive tool is highly desirable that opens the door to monitor the level of the sulfate in real time and space in living cells without fractionation of the cells or tissue. Here, we engineered a FRET (fluorescence resonance energy transfer) based sensor for sulfate, which is genetically-encoded and named as FLIP-SP (Fluorescent indicator protein for sulfate). The FLIP-SP can measure the level of the sulfate in live cells. This sensor was constructed by the fusion of fluorescent proteins at the N-and C-terminus of sulfate binding protein (sbp). The FLIP-SP is highly specific to sulfate, and showed pH stability. Real-time monitoring of the level of sulfate in prokaryotic and eukaryotic cells showed sensor bio-compatibility with living cells. We expect that this sulfate sensor offers a valuable strategy in the understanding of the regulation of the flux of sulfate in the metabolic network.

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“…This permits GEII to reliably detect even small changes in isoleucine concentration under high noise conditions. It must be noted that such large dynamic ranges are relatively uncommon for PBP-based sensors, with a number of sensors exhibiting only moderate FRET ratio changes (∆ FRET-RATIO < 0.3) [25][26][27]. The explanation of the impressive dynamic range of GEII is two-fold.…”

Section: Spectral Analysis Of Nanosensormentioning

confidence: 99%

Real-Time Optical Detection of Isoleucine in Living Cells through a Genetically-Encoded Nanosensor

Singh

Sharma

Alqarawi

et al. 2019

Sensors

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Isoleucine is one of the branched chain amino acids that plays a major role in the energy metabolism of human beings and animals. However, detailed investigation of specific receptors for isoleucine has not been carried out because of the non-availability of a tool that can monitor the metabolic flux of this amino acid in live cells. This study presents a novel genetically-encoded nanosensor for real-time monitoring of isoleucine in living cells. This nanosensor was developed by sandwiching a periplasmic binding protein (LivJ) of E. coli between a fluorescent protein pair, ECFP (Enhanced Cyan Fluorescent Protein), and Venus. The sensor, named GEII (Genetically Encoded Isoleucine Indicator), was pH stable, isoleucine-specific, and had a binding affinity (Kd) of 63 ± 6 μM. The GEII successfully performed real-time monitoring of isoleucine in bacterial and yeast cells, thereby, establishing its bio-compatibility in monitoring isoleucine in living cells. As a further enhancement, in silico random mutagenesis was carried out to identify a set of viable mutations, which were subsequently experimentally verified to create a library of affinity mutants with a significantly expanded operating range (96 nM–1493 μM). In addition to its applicability in understanding the underlying functions of receptors of isoleucine in metabolic regulation, the GEII can also be used for metabolic engineering of bacteria for enhanced production of isoleucine in animal feed industries.

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Designing, construction and characterization of genetically encoded FRET-based nanosensor for real time monitoring of lysine flux in living cells

Cited by 49 publications

References 35 publications

A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis

A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis

A Non-Invasive Tool for Real-Time Measurement of Sulfate in Living Cells

Real-Time Optical Detection of Isoleucine in Living Cells through a Genetically-Encoded Nanosensor

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