BACKGROUND: The c-type cytochrome of the CymA of Shewanella oneidensis MR-1 is essential for the anaerobic respiration of Shewanella sp. and transfers electrons from the inner membrane to various terminal electron acceptors, such as soluble redox shuttles and insoluble metal oxides. CymA is believed to be a passage to the outer membrane for dissipating the respiratory electron to the carbon electrode in a microbial fuel cell (MFC) with simultaneous electricity generation. While the deletion and heterologous expression of cymA in Escherichia coli have been studied, there are no reports of the overexpression and its effects on the corresponding bioelectrochemical performance in a MFC. RESULTS:The cymA gene was overexpressed in Shewanella oneidensis MR-1, and its upregulation was examined under aerobic, anaerobic, and MFC operating conditions by a reverse transcription-polymerase chain reaction (RT-PCR). Overexpression of the CymA protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The MFCs inoculated with the engineered strains of MR-1 achieved a higher maximum power of 0.13 mW and specific growth rate of 0.087 h −1 than those of the wild type MR-1 (0.11 mW and 0.043 h −1 , respectively). The higher electrochemical activity of the mutant strains demonstrated by cyclic voltammetry and linear sweep voltammetry, indicates that more respiratory electrons can be transferred to the electrodes through overexpression of the cymA gene of MR-1 in a MFC. CONCLUSION: Overexpression of CymA improves the bioelectrochemical performance of MFCs. This suggests that metabolic engineering of a membrane-associated redox protein, such as CymA, can further improve electricity generation of MFCs and produce an electrochemically enhanced bioprocess. Industry phosphate buffer (pH 7.0) and resuspended with the same buffer.The re-suspended cells were disrupted using a bead beater (Fastprep FP120, Fisher Scientific, Hampton, NH, USA) at a speed of 6.0 for 20 s per cycle for a total of 5 cycles. The disrupted cells

An attempt to alter protein surface charges through traditional protein engineering approaches often affects the native protein structure significantly and induces misfolding. This limitation is a major hindrance in modulating protein properties through surface charge variations. In this study, as a strategy to overcome such a limitation, we attempted to co-introduce stabilizing mutations that can neutralize the destabilizing effect of protein surface charge variation. Two sets of rational mutations were designed; one to increase the number of surface charged amino acids and the other to decrease the number of surface charged amino acids by mutating surface polar uncharged amino acids and charged amino acids, respectively. These two sets of mutations were introduced into Green Fluorescent Protein (GFP) together with or without stabilizing mutations. The co-introduction of stabilizing mutations along with mutations for surface charge modification allowed us to obtain functionally active protein variants (s-GFP(+15-17) and s-GFP(+5-6)). When the protein properties such as fluorescent activity, folding rate and kinetic stability were assessed, we found the possibility that the protein stability can be modulated independently of activity and folding by engineering protein surface charges. The aggregation properties of GFP could also be altered through the surface charge engineering.

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BackgroundInclusion bodies (IBs) were generally considered to be inactive protein deposits and did not hold any attractive values in biotechnological applications. Recently, some IBs of recombinant proteins were confirmed to show their functional properties such as enzyme activities, fluorescence, etc. Such biologically active IBs are not commonly formed, but they have great potentials in the fields of biocatalysis, material science and nanotechnology.ResultsIn this study, we characterized the IBs of DL4, a deletion variant of green fluorescent protein which forms active intracellular aggregates. The DL4 proteins expressed in Escherichia coli were exclusively deposited to IBs, and the IBs were estimated to be mostly composed of active proteins. The spectral properties and quantum yield of the DL4 variant in the active IBs were almost same with those of its native protein. Refolding and stability studies revealed that the deletion mutation in DL4 didn’t affect the folding efficiency of the protein, but destabilized its structure. Analyses specific for amyloid-like structures informed that the inner architecture of DL4 IBs might be amorphous rather than well-organized. The diameter of fluorescent DL4 IBs could be decreased up to 100–200 nm by reducing the expression time of the protein in vivo.ConclusionsTo our knowledge, DL4 is the first GFP variant that folds correctly but aggregates exclusively in vivo without any self-aggregating/assembling tags. The fluorescent DL4 IBs have potentials to be used as fluorescent biomaterials. This study also suggests that biologically active IBs can be achieved through engineering a target protein itself.

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