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

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

doi:10.1186/s12951-016-0204-y

Abstract

BackgroundEngineering microorganisms in order to improve the metabolite flux needs a detailed knowledge of the concentrations and flux rates of metabolites and metabolic intermediates in vivo. Fluorescence resonance energy transfer (FRET) based genetically encoded nanosensors represent a promising tool for measuring the metabolite levels and corresponding rate changes in live cells. Here, we report the development of a series of FRET based genetically encoded nanosensor for real time measurement of lysine at cellular level, as the improvement of microbial strains for the production of l-lysine is of major interest in industrial biotechnology.ResultsThe lysine binding periplasmic protein (LAO) from Salmonella enterica serovar typhimurium LT2 strain was used as the reporter element for the sensor. The LAO was sandwiched between GFP variants i.e. cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Affinity, pH stability, specificity and metal ions effects was scrutinized for the in vitro characterization of this nanosensor, named as FLIPK. The FLIPK is specific to lysine and found to be stable with the pH within the physiological range. The calculated affinity (Kd) of FLIPK was 97 µM. For physiological applications, mutants with different binding affinities were also generated and investigated in vitro. The developed nanosensor efficiently monitored the intracellular level of lysine in bacterial as well as yeast cell.ConclusionThe developed novel lysine fluorescence resonance energy transfer sensors can be used for in vivo monitoring of lysine levels in prokaryotes as well as eukaryotes. The potential of these sensors is that they can be used as reporter tools in the development of metabolically engineered microbial strains or for real-time monitoring of intracellular lysine during fermentation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-016-0204-y) contains supplementary material, which is available to authorized users.

Highlights

Engineering microorganisms in order to improve the metabolite flux needs a detailed knowledge of the concentrations and flux rates of metabolites and metabolic intermediates in vivo
Construction of a lysine sensor The lysine binding periplasmic protein (LAO) from Salmonella enterica serovar typhimurium LT2 strain was used as the reporter element for the sensor
It was found that the LAO closed state reached a higher twisting angle in the presence of l-lysine than those found for l-arginine and l-histidine [20]

Summary

Introduction

Engineering microorganisms in order to improve the metabolite flux needs a detailed knowledge of the concentrations and flux rates of metabolites and metabolic intermediates in vivo. We report the development of a series of FRET based genetically encoded nanosensor for real time measurement of lysine at cellular level, as the improvement of microbial strains for the production of l-lysine is of major interest in industrial biotechnology. Ameen et al J Nanobiotechnol (2016) 14:49 animal growth and improving meat quality This action makes possible reduction of protein level of the diet, reducing nitrogen synthesis excretion, and can consistently reduce the cost of feed. For large scale production of lysine from microbes, metabolic engineering is being used for developing improved bacterial strain [5]. Major bottleneck problem in the metabolic engineering is the absence of a suitable tool for real time monitoring of flux of a metabolite in the biosynthetic pathway

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Conclusion