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Introduction

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. Furthermore, the combination of pH-sensor with ATP-sensor will us to investigate in how the levels of ATP and H+ correlate together.

pHRed sensor

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 fluorescent protein. By genetically engineer RFP mKeima into a S213A mutant, it can exhibits dual excitation peaks that respond ratiometrically well to pH changes [5] . This mutated mKeima is called pHRed, which has proven useful for monitoring and analyzing pH.

Strategy and Approach

To utilize pHRed for in vivo measurements of pH inside a filamentous fungi during citric acid production, two constructs were made. With one targeting into the cytoplasm and the other targeting into the mitochondrial by transforming fungi with a naked pHRed sensor and pHRed sensor fused with mitochondrial signal peptide, respectively. The mitochondrial signal peptide has been proven to target into the mitochondria of Asp. 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.

Research Aims

1.Make 2 contructs for targeting pHRed to cytoplasmic and mitochondrial compartments.
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 Asp. Niger in time lapse using 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).

References

1.Bagar, T., K. Altenbach, et al. (2009). "Live-Cell imaging and measurement of intracellular pH in filamentous fungi using a genetically encoded ratiometric probe." Eukaryot Cell 8(5): 703-712.
2.Blumhoff, M. L., M. G. Steiger, et al. (2013). "Targeting enzymes to the right compartment: Metabolic engineering for itaconic acid production by Aspergillus niger." Metabolic Engineering 19(0): 26-32.
3.Felle, H. H. (1996). "Control of cytoplasmic pH under anoxic conditions and its implication for plasma membrane proton transport in Medicago sativa root hairs." Journal of Experimental Botany 47(7): 967-973.
4.Madshus, I. H. (1988). "Regulation of intracellular pH in eukaryotic cells." Biochem J 250(1): 1-8.
5.Tantama, M., Y. P. Hung, et al. (2011). "Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor." J Am Chem Soc 133(26): 10034-10037.