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It is of great significance to utilize CO2 as feedstock to synthesize biobased products, particularly single cell protein (SCP) as the alternative food and feed. Bioelectrochemical system (BES) driven by clean electric energy has been regarded as a promising way for Cupriavidus necator to produce SCP from CO2 directly. At present, the key problem of culturing C. necator in BES is that reactive oxygen species (ROS) generated in cathode chamber are harmful to bacterial growth. Therefore, it is necessary to find a solution to mitigate the negative effect of ROS. In this study, we constructed a number of C. necator strains displayed with superoxide dismutase (SOD), which allowed the decomposition of superoxide anion radical. The effects of promoters and signal peptides on the cell surface displayed SOD were analyzed. The proteins displayed on the surface were further verified by the fluorescence experiment. Finally, the growth of C. necator CMS incorporating a pBAD-SOD-E-tag-IgAβ plasmid could achieve 4.9 ± 1.0 of OD600 by 7 days, equivalent to 1.7 ± 0.3 g/L dry cell weight (DCW), and the production rate was 0.24 ± 0.04 g/L/d DCW, around 2.7-fold increase than the original C. necator CMS (1.8 ± 0.3 of OD600). This study can provide an effective and novel strategy of cultivating strains for the production of CO2-derived SCP or other chemicals in BES. [ABSTRACT FROM AUTHOR]

Marine microbial ecosystems can be viewed as a huge ocean-battery charged by solar energy. It provides a model for fabricating bio-solar cell, a bioelectrochemical system that converts light into electricity. Here, we fabricate a bio-solar cell consisting of a four-species microbial community by mimicking the ecological structure of marine microbial ecosystems. We demonstrate such ecological structure consisting of primary producer, primary degrader, and ultimate consumers is essential for achieving high power density and stability. Furthermore, the four-species microbial community is assembled into a spatial-temporally compacted cell using conductive hydrogel as a sediment-like anaerobic matrix, forming a miniaturized bionic ocean-battery. This battery directly converts light into electricity with a maximum power of 380 μW and stably operates for over one month. Reproducing the photoelectric conversion function of marine microbial ecosystems in this bionic battery overcomes the sluggish and network-like electron transfer, showing the biotechnological potential of synthetic microbial ecology. Marine ecosystems are dominated by microbial communities. Inspired by the photoelectric conversion feature of the marine ecosystems, the authors design a four-species microbial community to mimic primary producer, primary degrader, and ultimate consumers, and show its ability to convert light into electricity for over one month. [ABSTRACT FROM AUTHOR]

Background: Diaphorase (DI) has received wide attention as the key anodic enzyme mediating the electron transfer and electric energy generation in enzymatic biofuel cells (EBFCs). Lowering the anodic pH may be a useful strategy for constructing high-performance in EBFCs. However, most DI suffered from the poor activity at low pHs. Therefore, it is necessary to modify the activity and its acidic tolerance to further improve the performance of the EBFC. Results: This paper attempts to improve the enzyme activity of DI originated from Geobacillus stearothermophilus under acidic conditions through directed evolution. Three rounds of random mutagenesis by error-prone PCR of the GsDI gene followed by high-throughput screening allowed the identification of the mutant 3–8 (H37Q, S73T, F105L, S68T, G61S, D74V) exhibiting a 4- or 7-fold increase in the catalytic activity at pH 5.4 or 4.5 compared to that of the wild type. And the pH stability of mutant 3–8 was significantly better than that of wild type and showed a 1.3 times higher in the stability at pH 5.4. The EBFC anode equipped with 0.5 mg of mutant 3–8 achieved a maximum current of 40 μA at pH 5.4, much higher than that with the same loading of the wild type enzyme. Conclusion: The GsDI has been improved in the specific activity and pH stability by directed evolution which leads to the improvement of the EBFC performance. Also, the enlarged catalytic channel of mutant and decreased B-factor may be beneficial for the activity and stability. These results suggest that this engineered DI will be a useful candidate for the construction of enhanced EBFCs. [ABSTRACT FROM AUTHOR]

Since cellulose accessibility has become more recognized as the major substrate characteristic limiting hydrolysis rates and glucan digestibilities, cellulose solvent-based lignocellulose pretreatments have gained attention. In this study, we employed cellulose solvent- and organic solvent-based lignocellulose fractionation using two cellulose solvents: concentrated phosphoric acid [~85 % (w/w) HPO] and an ionic liquid Butyl-3-methylimidazolium chloride ([BMIM]Cl). Enzymatic glucan digestibilities of concentrated phosphoric acid- and [BMIM]Cl-pretreated corn stover were 96 and 55 % after 72 h at five filter paper units of cellulase per gram of glucan, respectively. Regenerated amorphous cellulose by concentrated phosphoric acid and [BMIM]Cl had digestibilities of 100 and 92 %, respectively. Our results suggested that differences in enzymatic glucan digestibilities of concentrated phosphoric acid- and [BMIM]Cl-pretreated corn stover were attributed to combinatory factors. These results provide insights into mechanisms of cellulose solvent-based pretreatment and effects of residual cellulose solvents and lignin on enzymatic cellulose hydrolysis. [ABSTRACT FROM AUTHOR]

Clostridium thermocellum cellodextrin phosphorylase ( CtCDP), a single-module protein without an apparent carbohydrate-binding module, has reported activities on soluble cellodextrin with a degree of polymerization (DP) from two to five. In this study, CtCDP was first discovered to have weak activities on weakly water-soluble celloheptaose and insoluble regenerated amorphous cellulose (RAC). To enhance its activity on solid cellulosic materials, four cellulose binding modules, e.g., CBM3 (type A) from C. thermocellum CbhA, CBM4-2 (type B) from Rhodothermus marinus Xyn10A, CBM6 (type B) from Cellvibrio mixtus Cel5B, and CBM9-2 (type C) from Thermotoga maritima Xyn10A, were fused to the C terminus of CtCDP. Fusion of any selected CBM with CtCDP did not influence its kinetic parameters on cellobiose but affected the binding and catalytic properties on celloheptaose and RAC differently. Among them, addition of CBM9 to CtCDP resulted in a 2.7-fold increase of catalytic efficiency for degrading celloheptaose. CtCDP-CBM9 exhibited enhanced specific activities over 20% on the short-chain RAC (DP = 14) and more than 50% on the long-chain RAC (DP = 164). The chimeric protein CtCDP-CBM9 would be the first step to construct a cellulose phosphorylase for in vitro hydrogen production from cellulose by synthetic pathway biotransformation (SyPaB). [ABSTRACT FROM AUTHOR]

Engineering the coenzyme specificity of redox enzymes plays an important role in metabolic engineering, synthetic biology, and biocatalysis, but it has rarely been applied to bioelectrochemistry. Here we develop a rational design strategy to change the coenzyme specificity of 6-phosphogluconate dehydrogenase (6PGDH) from a hyperthermophilic bacterium Thermotoga maritima from its natural coenzyme NADP+ to NAD+. Through amino acid-sequence alignment of NADP+- and NAD+-preferred 6PGDH enzymes and computer-aided substrate-coenzyme docking, the key amino acid residues responsible for binding the phosphate group of NADP+ were identified. Four mutants were obtained via site-directed mutagenesis. The best mutant N32E/R33I/T34I exhibited a ~6.4 × 104-fold reversal of the coenzyme selectivity from NADP+ to NAD+. The maximum power density and current density of the biobattery catalyzed by the mutant were 0.135 mW cm−2 and 0.255 mA cm−2, ~25% higher than those obtained from the wide-type 6PGDH-based biobattery at the room temperature. By using this 6PGDH mutant, the optimal temperature of running the biobattery was as high as 65 °C, leading to a high power density of 1.75 mW cm−2. This study demonstrates coenzyme engineering of a hyperthermophilic 6PGDH and its application to high-temperature biobatteries. [ABSTRACT FROM AUTHOR]