| Summary: | Microbial fuel cell (MFC) is a promising technology for energy harvest from biomass. However, the reported performances of MFCs with wild-type (WT) and biologically modified Shewanella oneidensis MR-1 still require further improvement towards substantial electron extraction from biomass. Several crucial challenges need to be addressed. Firstly, to achieve flexible operation of MFCs as well as to develop biosensors and biocomputing, switchable electron transfer according to a synthetic genetic circuit in the S. oneidensis cell is desired. Secondly, poor biofilm formation and low physiological concentration of flavins as the electron shuttles or cofacotrs of S. oneidensis limit its extracellular electron transfer (EET) efficiency and the MFC performances. Thirdly, sugar-fed MFCs are essential for energy harvest from biomass. However, S. oneidensis is unable to utilize sugars in current systems. In this PhD work, synthetic biology approaches were applied to engineer the EET pathway of S. oneidensis to address these challenges. In the first work, for demonstration of switchable electrodes controlled by Boolean logic gates, a genetic AND logic gate based on a synthetic quorum-sensing (QS) module was constructed in a S. oneidensis mtrA knockout mutant. The genetic circuit contained an isopropyl β-D-1-thiogalactopyranoside (IPTG) responding module and a N-(3-oxo-hexanoyl)-L-homoserine lacton (3-oxo-C6-HSL) signaling module. These modules then controlled the downstream expression of mtrA gene. The presence of two input signals activated the expression of the periplasmic decaheme cytochrome MtrA to regenerate the extracellular electron transfer conduit, enabling the AND-gated MFC. Flavin-mediated electron transfer is the rate-limiting step of EET in S. oneidensis due to the low concentration of secreted flavins. In the second work, a synthetic flavin biosynthesis pathway from Bacillus subtilis was heterologously expressed in S. oneidensis, resulting in ~25.7 times’ increase in secreted flavin concentration (from 0.98 to 26.15 μM total flavins). This synthetic flavin module enabled enhanced bidirectional EET rate of S. oneidensis, in which its maximum power output in MFC increased ~13.2 times (from 16.4 to 233.0 mW/m2), and the inward current increased ~15.5 times (from 0.16 to 2.55 A/m2). To further develop sugar-fed MFC with efficient EET rate, a synthetic microbial consortium composed of genetically modified E. coli and S. oneidensis was constructed focusing on rationally tuning the structure of the electrode-associated microbial community to favor rapid electron transfer. A synthetic riboflavin pathway originated from B. subtilis was incorporated into the fermenter E. coli, and a putrescine disruption mutant S. oneidensis CP2-1-S1 with altered surface property from hydrophilic to hydrophobic was adopted as the electricigen. Assisted by the overproduced flavins (increased from 3.3 to 115.2 µM by the recombinant E. coli) and strong hydrophobic interaction between S. oneidensis and the carbon electrode, the percentage of S. oneidensis in the electrode-attached microbial community was increased from 48.2% to 98.2%. Additionally, the cell number of immobilized S. oneidensis was also elevated by ~3.0 times. Xylose-fed MFC inoculated with the fully engineered microbial consortium generated a maximum power density of 728.6 mW/m2, which is 6.8 times higher than that inoculated with WT co-culture (92.8 mW/m2), and is the highest value among current reported MFC work with biologically modified S. oneidensis.
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