FRET-Based Genetically Encoded Nanosensor for Real-Time Monitoring of the Flux of α-Tocopherol in Living Cells.

Vitamin E plays an exemplary role in living organisms. α-Tocopherol is the most superior and active form of naturally occurring vitamin E that meets the requirements of human beings as it possesses the α-tocopherol transfer protein (α-TTP). α-Tocopherol deficiency can lead to severe anemia, certain cancers, several neurodegenerative and cardiovascular diseases, and most importantly male infertility. As a result of the depletion of its natural sources, researchers have tried to employ metabolic engineering to enhance α-tocopherol production to meet the human consumption demand. However, the metabolic engineering approach relies on the metabolic flux of a metabolite in its biosynthetic pathway. Analysis of the metabolic flux of a metabolite needs a method that can monitor the α-tocopherol level in living cells. This study was undertaken to construct a FRET (fluorescence resonance energy transfer)-based nanosensor for monitoring the α-tocopherol flux in prokaryotic and eukaryotic living cells. The human α-TTP was sandwiched between a pair of FRET fluorophores to construct the nanosensor, which was denoted as FLIP-α (the fluorescence indicator for α-tocopherol). FLIP-α showed excellence in monitoring the α-tocopherol flux with high specificity. The sensor was examined for its pH stability for physiological applications, where it shows no pH hindrance to its activity. The calculated affinity of this nanosensor was 100 µM. It monitored the real-time flux of α-tocopherol in bacterial and yeast cells, proving its biocompatibility in monitoring the α-tocopherol dynamics in living cells. Being noninvasive, FLIP-α provides high temporal and spatial resolutions, which holds an indispensable significance in bioimaging metabolic pathways that are highly compartmentalized.

Designing and construction of genetically encoded FRET-based nanosensor for qualitative analysis of digoxin.

Nanobiotechnological improvements defined on the utilization of biological materials and principles have enormously partaken to revolutionize physical, chemical, and biological sciences. However, the exploration of plant nanobiotechnology is still in its outset. The search for novel tools to monitor plant biomolecules is an emerging issue for the nanobiotechnologists. Given this, a genetically encoded FRET-based nanobiosensor has been developed to monitor the popular plant cardiac glycoside - digoxin, which is used as the most common prescription drug for heart-related illnesses across the world. Digoxin is sourced from the leaves of the foxglove plant (Digitalis purpurea L.) and has a significant demand in the medical sector. Moreover, with the rising popularity of the herbal formulations in the global market, attention towards the authentication and quality control of the herbal drugs is important. Furthermore, digoxin has a very narrow therapeutic range, i.e., 0.6 nM - 2.6 nM. Therefore, strict monitoring of blood digoxin levels is necessary. Besides, previously used techniques for drug authentication and quantification of small-molecule drugs in blood samples are not the best choice available. The nanobiosensor is based on the FRET (Fluorescence Resonance Energy Transfer) mechanism, and it is constructed in such a way that it gives a changed FRET output in the presence of digoxin. Two fluorophores, enhanced cyan fluorescent protein (ECFP) and Venus, were attached on either end of the sensory domain - human nuclear receptor ROR-gamma (RORγt). The developed nanobiosensor was named as fluorescent indicator protein for digoxin, (FLIP-digoxin). The ligand binding affinity of FLIP-digoxin was calculated as 425 µM. Affinity mutants of the FLIP-digoxin were also generated to measure digoxin in wide concentration ranges. This sensor offers high-throughput qualitative analysis of digoxin in Digitalis preparations procured from local drug stores. It confirms the authenticity of the preparations through the detection of digoxin. The FLIP-1n was also able to monitor digoxin concentration in serum samples in lesser than 5 min. The nanobiosensor was found pH stable, digoxin-specific, non- interfered by the biological serum species and can perform high throughput screening of the Digitalis powder, infusion and tincture preparations.

Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor.

(+)-Catechin is an important antioxidant of green tea (Camelia sinensis (L.) O. Kuntze). Catechin is known for its positive role in anticancerous activity, extracellular matrix degradation, cell death regulation, diabetes, and other related disorders. As a result of enormous interest in and great demand for catechin, its biosynthesis using metabolic engineering has become the subject of concentrated research with the aim of enhancing (+)-catechin production. Metabolic flux is an essential concept in the practice of metabolic engineering as it helps in the identification of the regulatory element of a biosynthetic pathway. In the present study, an attempt was made to analyze the metabolic flux of the (+)-catechin biosynthesis pathway in order to decipher the regulatory element of this pathway. Firstly, a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor (FLIP-Cat, fluorescence indicator protein for (+)-catechin) was developed for real-time monitoring of (+)-catechin flux. In vitro characterization of the purified protein of the nanosensor showed that the nanosensor was pH stable and (+)-catechin specific. Its calculated Kd was 139 µM. The nanosensor also performed real-time monitoring of (+)-catechin in bacterial cells. In the second step of this study, an entire (+)-catechin biosynthesis pathway was constructed and expressed in E. coli in two sets of plasmid constructs: pET26b-PT7-rbs-PAL-PT7-rbs-4CL-PT7-rbs-CHS-PT7-rbs-CHI and pET26b-T7-rbs-F3H-PT7-rbs- DFR-PT7-rbs-LCR. The E. coli harboring the FLIP-Cat was transformed with these plasmid constructs. The metabolic flux analysis of (+)-catechin was carried out using the FLIP-Cat. The FLIP-Cat successfully monitored the flux of catechin after adding tyrosine, 4-coumaric acid, 4-coumaroyl CoA, naringenin chalcone, naringenin, dihydroquercetin, and leucocyanidin, individually, with the bacterial cells expressing the nanosensor as well as the genes of the (+)-catechin biosynthesis pathway. Dihydroflavonol reductase (DFR) was identified as the main regulatory element of the (+)-catechin biosynthesis pathway. Information about this regulatory element of the (+)-catechin biosynthesis pathway can be used for manipulating the (+)-catechin biosynthesis pathway using a metabolic engineering approach to enhance production of (+)-catechin.