Team:TJU/Background


Background


1 Microbial fuel cells (MFCs)


A microbial fuel cell(MFC) is an electrochemical device which converts the chemical energy of fuel to electrical energy by the catalytic actions of microorganisms.[1]


Compared with energy from fossil fuel, such as oil and coal, MFC is of plethora advantages over other kinds of energy generators, e.g. wide sources of reactant, no emissions of environmental polluting gases (such as SOx, NOx, CO2 and CO), high conversion in theory, mild reaction condition, as a result, lack of sonic pollution, and so forth.[2]


Benefited from those merits, MFCs have a wide range of potential application. For example, MFCs are suitable for powering electrochemical sensors and small telemetry systems to transmit obtained signals to remote receivers while traditional batteries cannot because of restricted lifetime and frequent changes and recharges. Additionally, realistic energetically autonomous would probably be equipped with MFCs that utilize different fuels like sugar, fruit, dead insects, grass and weed. Such systems can solely power itself by MFCs to perform some behavior including motion, sensing, computing and communication. [3]



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2 Typical exoelectrogens of MFCs


Several types of biofuel cells including microbial fuel cell and enzymatic biofuel cell have been well documented in the literature. Various microbial or biochemical fuel cells have been developed using Desulfovibrio desulfuricans, Proteous vulgaris, Escherichia coli, Pseudomonas species and redox enzymes as biocatalysts.[4]


Of all the MFCs above, Shewanella species are best known for extracellular electron transfer(EET). Shewanella, a facultative anaerobe, has been widely used as a model anode biocatalyst in MFCs due to its easiness of cultivation, adaptability to aerobic and anaerobic environment and both respiratory and electron transfer versatility.They are able to exist in various kinds of environments and couple the oxidation of organic substrates such as riboflavins to the reduction of a wide variety of electron acceptors. When the electron acceptor are electrodes, their characteristic of transferring electrons from the cell surface to conductive materials shows great potential to improve the efficiency of bioelectrochemical systems. As a result, Shewanella has become the most widely used bacteria in MFCs.[5]


3 Microbial consortia


Microorganisms rarely live in isolated niches. More than 99% of microorganisms in environments cannot be successfully cultured by traditional cultivation technologies; one reason is that the maintenance of viability of these microbes may need supplementary metabolites or other signaling chemicals provided by other microbes in the ecosystems and communities. [6]


3.1 Interaction modes

Engineering cell-cell interactions and communications is at the central point of engineering synthetic communities. The interactions between microorganisms are ubiquitous and play a central role in determining the fate and evolutionary dynamics of individual organisms in microbial consortia, as well as system properties such as stability and dynamics of the entire communities.[6]


Also, delineation of interaction modes between microorganisms in natural niches could facilitate engineering novel microbial consortia and their traits. The interactions within the microbial ecological communities may have a null (0, or neutral), positive (+, beneficial or win), or negative (-, or detrimental or loss) impact on the partner microorganisms involved. Thus, the binary interaction outcomes could be classified into six different categories of pairwise interaction modes , i.e., neutralism(0/0), commensalism (+/0), amensalism (-/0), mutualism (+/+), competition (-/-), and parasitism or predation (+/-). [6]


3.2 Advantageous relationships of microbial consortia

The win-win relationship of symbiotic association is mutualism (+/+), in which both partner microorganisms derive benefits from one another., e.g., Stable co-culture in the same bioreactor was achieved by designing a mutualistic relationship between the two species in which a metabolic intermediate produced by E. coli was used and functionalized by yeast. This synthetic consortium produced 33 mg/L oxygenated taxanes, including a monoacetylated dioxygenated taxane.[7]


Commensalism (+/0) is a relationship in which one partner derives benefits from the other, while the other partner is not affected (neither harmful nor beneficial) by the association, e.g., an artificial microbial consortium consisting of Ketogulonicigenium vulgare and Bacillus megaterium for vitamin C production.[6]


Inspired by the concept of co-culture system, MFCs populated by mixed microbial communities have garnered much attention owing to their stability, robustness due to nutrient adaptability, stress resistance and general tendency to produce higher current density than those obtained using pure cultures. e.g., a mutualistic system established by the fermentative bacterium Escherichia coli and the dissimilatory metal-reducing bacterium Shewanella oneidensis in a bioelectrochemical device is reported with formate as the key metabolism.[8]



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3.2 Advantageous relationships of microbial consortia

Cytoplasmic proteolysis is an indispensable process for proper function of a cell. Degradation of many intracellular proteins is initiated by ATP-dependent proteinases, which are involved in the regulation of the level of proteins with short half-lives. In addition, they remove many damaged and abnormal proteins and thus play also an important role during stress.


ATP-dependent proteinases are large multi-subunit assemblies composed of proteolytic core domains and ATPase-containing regulatory domains on a single polypeptide chain or on distinct subunits, which can act as molecular chaperones. There are four main groupsof these proteinases in bacteria including Lon, Clp family, HslUV and FtsH.[9]


4 Intracellular protein degradation


4.1Theory of protein degradation in bacteria

Modifications that target proteins for proteolysis, and thereby affect protein stability, have been extensively studied. These modifications can serve as quality control features that regulate protein synthesis, remove defective proteins, recycle amino acids and inactivate proteins.


Bacteria co-translationally tag stalled or interrupted translational products with the SsrA peptide. This 11-aminoacid signal, targets the defective translation product to cellular proteases, including ClpA/XP, Lon and etc, The ssrA signal is encoded by a small, stable RNA containing an alanyltRNA domain and mRNA domain encoding the open reading frame of the tag (tmRNA). Upon encountering stalled or incomplete translation, the tmRNA and accessory factors are recruited to the ribosome to rescue the idle translation complex by replacing it with the tmRNA encoding the ssrA tag. The ssrA mRNA serves as the template for the addition of the ssrA peptide. The ssrA tag is added co-translationally to the carboxy-terminus (C-terminus) of incomplete proteins to target them for.[10]


4.2 Mf-lon

Interestingly, the ssrA tags encoded by the tmRNA molecules in most Mycoplasma are very different from those found in other bacteria. Most Mycoplasma genomes encode only two AAA+ proteases, Lon and FtsH, and have lost the genes for ClpXP, ClpAP. In the absence of ClpXP, the unusual Mycoplasma ssrA tag could serve as a degradation signal for the endogenous Lon or FtsH proteases.[11]


It has been shown that the ssrA tag sequence of M. florum (mf-ssrA) is efficiently recognized by the M. florum Lon protease (mf-Lon). Appending this tag to the C terminus of native or denatured proteins resulted in their rapid proteolysis by mf-Lon. Furthermore, mf-Lon did not degrade proteins bearing the E. coli ssrA tag (ec-ssrA), and E. coli Lon (ec-Lon) did not efficiently degrade proteins bearing the mf-ssrA tag. [11]



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Exogenous control of protein biosynthesis through transcriptional and translational regulation has been well established, but robust and tunable control of protein degradation in bacteria remains elusive. Controlled protein degradation would provide us with the ability to probe protein function without disrupting the transcriptional and translational regulation that control the expression of its cognate gene and to develop more complex synthetic gene circuits.[12]


As mentioned above, protein degradation in bacteria occurs in part through the tmRNA system with ssrA peptide to direct proteins to the endogenous ClpXP and ClpAP proteases for rapid degradation in E. coli. Variants of the E. coli ssrA tag (ec-ssrA) are commonly used to modify the degradation rate of attached proteins in both bacteria and eukaryotes, but these tags do not provide inducible control of degradation.[12]


So a synthetic degradation system based on the Gram-positive M. florum tmRNA system does not rely on host degradation and can function in a wide range of bacteria. M. florum ssrA tag (mf-ssrA) is degraded by its endogenous Lon protease (mf-Lon) but not by E. coli Lon or ClpXP, and mf-Lon does not recognize or degrade E.coli-ssrA, providing a protease and cognate degradation tag with orthogonal functionality in E. coli.[12]



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5 Acid inducible promoter -P170


P170 is a strongly acid-inducible promoter from Lactococcus lactis. P170 contains an extended -10 sequence (contained the TGN motif immediately upstream of the -10 region) and boxes ACD but no canonical -35 sequence. The lack of -35 sequence could explain their low basal activity at neutral pH. The higher basal activity of P170 could result from the extension of its -10 motif.


The novel 14 bp regulatory DNA region centered at around -41.5 and composed of three tetranucleotide sequences, boxes A, C and D. Boxes A and C are involved in promoter activity, with box C being more important than box A. Box D and the correct position of boxes ACD (centred at around -41.5) are essential to pH response. That these boxes constitute the DNA determinant of the acid induction and they were renamed more explicitly ‘ACiD-box’.


High level of P170 activity required both RcfB and acidic conditions. A trans-acting protein, RcfB, is involved in basal activity of P170 and is essential for their pH induction. The protein RcfB belongs to the Crp-Fnr family of transcription regulators, which, upon activation, bind a DNA recognition motif within a promoter region and activates transcription. When the cells are exposed to acid environments, the RcfB activation by an ‘acid’ signal allows its binding to the ACiD-box, resulting in transcription activation. [13]


References


[1]Guerrero-Rangel N, Garza R D L, Garza-Garcia Y, et al. Comparative Study of Three Cathodic Electron Acceptors on the Performance of Medatiorless Microbial Fuel Cell[J]. International Journal of Electrical & Power Engineering, 2010, (1):27-31
[2]Rahimnejad M, Adhami A, Darvari S, et al. Microbial fuel cell as new technology for bioelectricity generation: A review[J]. AEJ - Alexandria Engineering Journal, 2015, 88.
[3]Du Z, Li H, Gu T. A state of the art review on microbial fuel cells: A promising technology for wastewater treatment and bioenergy[J]. Biotechnology Advances, 2007, 25(5):464–482.
[4]Kim H J, Park H S, Hyun M S, et al. A mediator-less microbial fuel cell using a metal reducing bacterium, Shewanella putrefaciens[J]. Enzyme and Microbial Technology, 2002, 30(2): 145-152 [5]Saffarini D, Brockman K, Beliaev A, et al. Shewanella oneidensis and Extracellular Electron Transfer to Metal Oxides[M] Bacteria-Metal Interactions. Springer International Publishing, 2015: 21-40.
[6]Song H, Ding M Z, Jia X Q, et al. Synthetic microbial consortia: from systematic analysis to construction and applications[J]. Chemical Society Reviews, 2014, 43(20): 6954-6981.
[7] Zhou K, Qiao K, Edgar S, et al. Distributing a metabolic pathway among a microbial consortium enhances production of natural products.[J]. Nature Biotechnology, 2015, 33:377-383.
[8] Wang VB, Sivakumar K, Yang L, et al. Metabolite-enabled mutualistic interaction between Shewanella oneidensis and Escherichia coli in a co-culture using an electrode as electron acceptor.[J]. Scientific Reports, 2015, 5.
[9] Sauer R T, Baker T A. AAA+ proteases: ATP-fueled machines of protein destruction[J]. Annual review of biochemistry, 2011, 80: 587-612.
[10]Burns K E, Darwin K H. Pupylation versus ubiquitylation: tagging for proteasome‐dependent degradation[J]. Cellular microbiology, 2010, 12(4): 424-431.
[11]Gur E, Sauer R T. Evolution of the ssrA degradation tag in Mycoplasma: specificity switch to a different protease[J]. Proceedings of the National Academy of Sciences, 2008, 105(42): 16113-16118.
[12]Cameron D E, Collins J J. Tunable protein degradation in bacteria[J]. Nature biotechnology, 2014, 32(12): 1276-1281.
[13] Madsen, Søren M, Hindré, Thomas, Le Pennec, Jean‐Paul, et al. Two acid‐inducible promoters from Lactococcus lactis require the cis-acting ACiD‐box and the transcription regulator RcfB[J]. Molecular Microbiology, 2005, 56(3):735-746.

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