Designing and Development of FRET-Based Nanosensor for Real Time Analysis of N-Acetyl-5-Neuraminic Acid in Living Cells

Ruphi Naz,Urooj Fatima,Walid H Soufan,Mohd Mohsin,Mostafa A Abdel-Maksoud,Mohammad K Okla,Ibrahim A Alaraidh,Altaf Ahmad

doi:10.3389/fnut.2021.621273

Abstract

N-acetyl-5-neuraminic acid (NeuAc) plays crucial role in improving the growth, brain development, brain health maintenance, and immunity enhancement of infants. Commercially, it is used in the production of antiviral drugs, infant milk formulas, cosmetics, dietary supplements, and pharmaceutical products. Because of the rapidly increasing demand, metabolic engineering approach has attracted increasing attention for NeuAc biosynthesis. However, knowledge of metabolite flux in biosynthetic pathways is one of the major challenges in the practice of metabolic engineering. So, an understanding of the flux of NeuAc is needed to determine its cellular level at real time. The analysis of the flux can only be performed using a tool that has the capacity to measure metabolite level in cells without affecting other metabolic processes. A Fluorescence Resonance Energy Transfer (FRET)-based genetically-encoded nanosensor has been generated in this study to monitor the level of NeuAc in prokaryotic and eukaryotic cells. Sialic acid periplasmic binding protein (SiaP) from Haemophilus influenzae was exploited as a sensory element for the generation of nanosensor. The enhanced cyan fluorescent protein (ECFP) and Venus were used as Fluroscence Resonance Energy Transfer (FRET) pair. The nanosensor, which was termed fluorescent indicator protein for sialic acid (FLIP-SA), was successfully transformed into, and expressed in Escherichia coli BL21 (DE3) cells. The expressed protein of the nanosensor was isolated and purified. The purified nanosensor protein was characterized to assess the affinity, specificity, and stability in the pH range. The developed nanosensor exhibited FRET change after addition to NeuAc. The developed nanosensor was highly specific, exhibited pH stability, and detected NeuAc levels in the nanomolar to milimolar range. FLIP-SA was successfully introduced in bacterial and yeast cells and reported the real-time intracellular levels of NeuAc non-invasively. The FLIP-SA is an excellent tool for the metabolic flux analysis of the NeuAc biosynthetic pathway and, thus, may help unravel the regulatory mechanism of the metabolic pathway of NeuAc. Furthermore, FLIP-SA can be used for the high-throughput screening of E. coli mutant libraries for varied NeuAc production levels.

Highlights

The N-acetyl-5-neuraminic acid (NeuAc) is the most common member of the sialic acid (SA) family [1, 2]
At the point where NeuAc ties to Sialic acid periplasmic binding protein (SiaP), the pocket comes closer and the two domains twist at a hinge region [28, 29] (Figure 1A)
The present results demonstrated that the addition of exogenous NeuAc to FLIP-SA-expressing bacterial cells leads to a response from the FLIP-SA sensor

Summary

Introduction

The N-acetyl-5-neuraminic acid (NeuAc) is the most common member of the sialic acid (SA) family [1, 2]. It is engaged in various metabolic functions in bacteria and animals. As NeuAc has a negative charge, it contributes to the biophysical properties of living systems This negative charge causes charge repulsion and prevents the undesirable contacts of cells in the blood circulation [5, 6]. Potent antiviral drugs such as zanamivir and oseltamivir have been synthesized from NeuAc [12, 13] Most importantly, it is an important component of the human milk oligosaccharide (HMO) family that helps in the improved development of infant [14]. The SA-containing HMO (sialyllactoses) is involved in the promotion of probiotic proliferation and in muscle and liver development in newborn babies, as well as in the optimization of brain metabolism [15]

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Discussion

Conclusion