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Outline

Bio-sensors are invaluable tools to easily visualize and track changes in the cell and its environment. Protein based bio-sensors have the advantage of being easily targeted to any part of the eukaryotic cell and . Thus they are important components of the Aspergillus arsenal, which is an array of parts and devices to make Aspergillus niger more amenable to work with. We envision the use of ATP and pH bio-sensors to monitor the micro-environment in the cell where a pathway of interest has been targeted, in our case, the lovastatin biosynthesis pathway.

Such bio-sensors can provide quantitative information that can prompt us to maybe direct pathways to proper cell compartments or change the growth conditions to increase the yields. The actual fluctuations of proton and ATP concentrations in live cells can be pursued corresponding to different growth conditions. Consequently, filling gaps in knowledge about the cell physiology which is also essential in the context of host engineering. For example, the ATP bio-sensor will be instrumental in exploring the metabolic capabilities of the single cell like phenotype of A. niger at 45'C.

Introduction

A great array of new and existing biological devices and circuits have been designed and streamlined for useful purposes. However, currently there is not enough emphasis on how these devices and circuits respond to changes in their physiological surroundings, such as the effect of changes in pH and the availability of ATP. Thus we tested bio-sensors that enable us to monitor the dynamic changes of pH and ATP in live cells. The ATP bio-sensor is based on the phenomenon of fluorescence resonance energy transfer (FRET) and the pH bio-sensor is based on changes in the ionic state of the recombinant red fluorescence protein (pHRed). By combining multicolour, pHRed (red fluorescence) and FRET (green fluorescence), we can simultaneously image intracellular pH and ATP, as changes in metabolism often correlate with changes in pH and ATP concentrations in the cell. These bio-sensors are useful to non-invasively quantify specific measurements in living cells.

Ratiometric pH biosensor

Regulation of intracellular pH is important for the metabolism of a functional cell as it is tightly regulated by the complex interactions between the consumption, production, transportation and buffering of H+ [3][4]. To maintain protein stability, enzyme and ion channel activity, growth and division, it is essential to retain pH within their physiological range of pH 7.5 [1]. In addition, it is important to maintain a proton gradient between the cytoplasm and mitochondria, which allow the formation of ATP to sustain their metabolism. Aspergillus niger is a GRAS organism that is among the favourites in producing organic acid as it can tolerate acidic environment. Since Aspergillus niger can produce high amount of organic acid, it would be interesting to investigate the fluctuation and difference in pH between the cytoplasm and mitochondria during the production of citric acid. This would be interesting to determine whether the pH, at a certain position in a filamentous fungi, is optimal for a certain reaction. This will allow us to direct and target a metabolic pathway into its appropriate compartments for optimal production. This will also allow us to elucidating the mechanism in how fast the homeostatic pH is being regulated when acid is production with extracellular acidic environment.

A novel approach in measuring intracellular pH in a non-invasive way has been developed by Mathew Tantama et. al. 2011, by using a recombinant red fluorescent protein (RFP), termed mKeima, had been genetically engineered into a S213A mutant, called pHRed. This will allow it to exhibit dual excitation peaks at 440 nm and 585 nm and an emission peak at 610 nm that respond ratiometrically well to pH changes [5]. The advantages of using a ratiometric method is that the estimation of pH is not affected by photobleaching, changes in focus of the microscope, variations in laser intensity, optical path length and illumination intensity. The excitation peak at 585 nm is caused by the anionic chromophore in an acidic condition whereas the excitation peak at 440 nm is caused by the protonated neutral chromophore in an alkaline condition.

Figure 1) How different pH levels affect the excitation peaks of pHRedA. A: pHRed in acidic condition has an excitation peak at 585 nm with an emission peak at 610 nm. B: pHRed in alkaline condition has an excitation peak at 440 nm with an emission peak 610 nm.

The pH can be calculated by measuring the fluorescence intensity ratio between F585 and F440 (F585/ F440); and compare it with a calibration curve, see FIG. pH responses to ratio intensity of F585/ F440 are not affected in different buffer concentration such as Na+, K+, Mg2+, Cl−, Ca2+, HCO3−, H2O2, DTT, and temperature (21°C to 37°C). Based on these findings, pHRed had proven to be useful in monitoring and analyzing the dynamic changes in pH.

Figure 2)[5]

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.

Figure 1) Working of the ATP FRET sensor [6]

FRET based ATP bio-sensors, dubbed as Ateams, have been previously developed and implemented to quantify in vivo ATP levels in HeLa and yeast cells [6], [7] . The sensor consist of mseCFP (a variant of CFP), the ATP sensing domain is a subunit of the Bacillus subtilis FoF1-ATP synthase and mVenus (a YFP variant). The YFP/CFP emission ratio (527 nm/475 nm) gives an estimation of the ATP concentration. This sensor has been found to be ATP specific and pH independent, although it is temperature sensitive with a higher ATP binding capacity at lower temperatures. The Ateams have been requested from the Imamura lab in Japan for cloning and expression into A. niger and E. coli.

pH biosensor

Approach

The outline of the approach is as follows:

1.Make 2 contructs for targeting pHRed to cytoplasmic and mitochondria.
2.Purify strepTagII-pHRed from sonificated E. coli. and make a calibration curve.
3.Transform Aspergilllus niger with cytoplasmic pHRed and mitochondrial pHRed
4.Measure in vivo pH during citric acid production by A. niger in time lapse using a fluorescence microscope

Research Methods

To utilize pHRed for in vivo measurements of pH inside a filamentous fungi during citric acid production, two constructs were made. With one targeting the cytoplasm and the other targeting the mitochondria by transforming fungi with a naked pHRed sensor and pHRed sensor fused with a mitochondrial signal peptide, respectively. The mitochondrial signal peptide has been proven to target the mitochondria of A. niger [2] . To measure pH, a calibration curve is established by purifying streptagII fused at N-terminus of pHRed proteins from sonificated E. coli, and is observed under a fluorescence microscope.

Results

After transformation of DH5α with 1µl of mixture (gibson assembly with pJet1.2 blunt vector), 7 colonies were picked and were cultured in 5 ml LB medium with ampicillin. Afterwards, plasmids were isolated using a miniprep kit (thermo scientific) according to their protocol using a homemade silicon column. Culture #1 till #3 were digested with pstI and notI; culture #4 till #7 were digested with nsiI and notI, see Figure 1.

Figure 1) lane 1, 3 and 4 contain pJet1.2 with the appropriate mit-pHRed (803 bp). Lane 5-7 contain the pJet1.2 with the appropriate pHRed (725 bp).

ATP biosensor

Aim

Implement a FRET based sensor that will measure ATP levels between compartments in growing A. 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 like phenotype of A. 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

Clone and express the Ateam bio-sensor (kindly donated by the Imamura lab in Japan) into A. niger directed to the cytolpasm and mitochodria - 1. Codon optimized for A. niger and 2. Not codon optimized.

Use sorbic acid in the growth media of A. niger to disturb the ATP pool inside the cell for testing the functionality of the Ateam sensor.

Express sensor in Escherichia coli and purify with the help of a Histidine tag for in-vitro characterization in a buffer mimicking A. niger physiological environment.

Research Methods

The initial target was to introduce the following versions of the ATP sensor into A. niger – 1. ATP sensitive and cytoplasmic localization; 2. ATP sensitive and mitochondrial localization; 3. ATP in-sensitive and cytoplasmic localization; 4. Codon optimized, ATP sensitive and cytoplasmic localization; 5. Codon optimized, ATP sensitive and cytoplasmic localization.

Also, it was aimed to express the ATP sensor in Escherchia coli and purify with the help of a N-terminal Histidine tag for in-vitro characterization of the sensor. The in-vitro characterization of the purified sensor involves exciting the CFP with 435 nm light and measuring the YFP/CFP emission ratio (at 527/475 nm respectively) at different ATP concentrations. The characterization of the sensor was carried out at room temperature in a buffer mimicking the in-vivo conditions in A. niger.