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Abstract

Economics is a vital aspect of any system, be it the world, a country, a person or even a microbe. Fungi are being used extensively by the biotechnology industry to produce useful compounds like drugs, organic acids, enzymes, fuels, etc. Thus efficient use of cellular resources is of prime importance in these industrial workhorses. Now the energy currency of any cell is Adenosine tri-phosphate (ATP) molecule and visualizing the dynamics of ATP levels in living cells has been a challenge. To this end we aim to produce a Fluorescence resonance energy transfer (FRET) based sensor for live cell ATP measurements in Aspergillus niger. With this we hope to measure ATP levels between compartments in growing Aspergillus niger during major metabolic shifts to understand the energy management processes in the cell.

Introduction

Fluorescence resonance energy transfer (FRET) is a phenomenon widely exploited by bio-sensors to monitor concentrations and temporal fluctuations of metabolites and ions at cellular and sub-cellular level. FRET works by excitation of a fluorescent molecule (donor) by a light of particular wavelength, which consequently transfers this energy to an adjacent fluorescent molecule (acceptor) that in-turn emits light. This phenomenon is very sensitive to the distance between the donor and acceptor fluorophore groups. Thus, fusing fluorescent proteins with a sensing domain that undergoes big conformational changes upon binding of the sensory molecule confers the possibility for generating an assortment of custom-made genetically encoded biosensors. These are useful tools to non-invasively quantify metabolites in living cells.

description

FRET based sensors have been recently developed to quantify ATP levels in vivo in HeLa and yeast cells (1, 2). They consist of mseCFP, the ATP sensing domain and mVenus (YFP variant). The YFP/CFP emission ratio gives an estimation of the ATP concentration.

Aim

Construct a fluorescent protein based sensor that will measure ATP levels between compartments in growing Aspergillus niger during metabolic shifts.

Applications

An ATP bio-sensor can lead to visualization of the ATP levels in live Aspergillus cells. Inquiry into whether the single cell phenotype of Aspergillus niger still retains its metabolic activity can be carried out. It would be interesting to quantify the ATP levels in different organelles and investigate where and why such ATP levels exist. Also, the differences in cellular ATP under various growth environments can be studied. The differences in ATP levels for cells at the hyphal tip and those in the inner mycelium can be validated.

Approach

Four versions of the ATP sensor were introduced into A. niger – 1. ATP sensitive and cytoplasmic localization, 2. ATP sensitive and mitochondrial localization, 3. ATP in-sensitive and cytoplasmic localization, 4. A codon optimized version, ATP sensitive and cytoplasmic localization. The ATP sensor was expressed in Escherchia coli and purified by attaching an N-terminal Histidine tag. The purified sensor was characterized in vitro and the YFP/CFP emission ratio (at 527/475 nm respectively) to different ATP concentrations was tested at room temperature in a buffer mimicking the in-vivo conditions in A. niger.