Enzymatic Electrosynthesis of Glycine from CO2 and NH3.

Enzymatic electrosynthesis has gained more and more interest as an emerging green synthesis platform, particularly for the fixation of CO2 . However, the simultaneous utilization of CO2 and a nitrogenous molecule for the enzymatic electrosynthesis of value-added products has never been reported. In this study, we constructed an in vitro multienzymatic cascade based on the reductive glycine pathway and demonstrated an enzymatic electrocatalytic system that allowed the simultaneous conversion of CO2 and NH3 as the sole carbon and nitrogen sources to synthesize glycine. Through effective coupling and the optimization of electrochemical cofactor regeneration and the multienzymatic cascade reaction, 0.81 mM glycine was yielded with a highest reaction rate of 8.69 mg L-1 h-1 and faradaic efficiency of 96.8 %. These results imply a promising alternative for enzymatic CO2 electroreduction and expand its products to nitrogenous chemicals.

Installing a Green Engine To Drive an Enzyme Cascade: A Light-Powered In Vitro Biosystem for Poly(3-hydroxybutyrate) Synthesis.

Many existing in vitro biosystems harness power from the chemical energy contained in substrates and co-substrates, and light or electric energy provided from abiotic parts, leading to a compromise in atom economy, incompatibility between biological and abiotic parts, and most importantly, incapability to spatiotemporally co-regenerate ATP and NADPH. In this study, we developed a light-powered in vitro biosystem for poly(3-hydroxybutyrate) (PHB) synthesis using natural thylakoid membranes (TMs) to regenerate ATP and NADPH for a five-enzyme cascade. Through effective coupling of cofactor regeneration and mass conversion, 20 mM PHB was yielded from 50 mM sodium acetate with a molar conversion efficiency of carbon of 80.0 % and a light-energy conversion efficiency of 3.04 %, which are much higher than the efficiencies of similar in vitro PHB synthesis biosystems. This suggests the promise of installing TMs as a green engine to drive more enzyme cascades.

Tackling the Challenges of Enzymatic (Bio)Fuel Cells.

The ever-increasing demands for clean and sustainable energy sources combined with rapid advances in biointegrated portable or implantable electronic devices have stimulated intensive research activities in enzymatic (bio)fuel cells (EFCs). The use of renewable biocatalysts, the utilization of abundant green, safe, and high energy density fuels, together with the capability of working at modest and biocompatible conditions make EFCs promising as next generation alternative power sources. However, the main challenges (low energy density, relatively low power density, poor operational stability, and limited voltage output) hinder future applications of EFCs. This review aims at exploring the underlying mechanism of EFCs and providing possible practical strategies, methodologies and insights to tackle these issues. First, this review summarizes approaches in achieving high energy densities in EFCs, particularly, employing enzyme cascades for the deep/complete oxidation of fuels. Second, strategies for increasing power densities in EFCs, including increasing enzyme activities, facilitating electron transfers, employing nanomaterials, and designing more efficient enzyme-electrode interfaces, are described. The potential of EFCs/(super)capacitor combination is discussed. Third, the review evaluates a range of strategies for improving the stability of EFCs, including the use of different enzyme immobilization approaches, tuning enzyme properties, designing protective matrixes, and using microbial surface displaying enzymes. Fourth, approaches for the improvement of the cell voltage of EFCs are highlighted. Finally, future developments and a prospective on EFCs are envisioned.

Efficient secretory production of large-size heterologous enzymes in Bacillus subtilis: A secretory partner and directed evolution.

Secretory production of recombinant proteins provides a simple approach to the production and purification of target proteins in the enzyme industry. We developed a combined strategy for the secretory production of three large-size heterologous enzymes with a special focus on 83-kDa isoamylase (IA) from an archaeon Sulfolobus tokodaii in a bacterium Bacillus subtilis. First, a secretory protein of the B. subtilis family 5 glycoside hydrolase endoglucanase (Cel5) was used as a fusion partner, along with the NprB signal peptide, to facilitate secretory production of IA. This secretory partner strategy was effective for the secretion of two other large enzymes: family 9 glycoside hydrolase from Clostridium phytofermentas and cellodextrin phosphorylase from Clostridium thermocellum. Second, the secretion of Cel5-IA was improved by directed evolution with two novel double-layer Petri-dish-based high-throughput screening (HTS) methods. The high-sensitivity HTS relied on the detection of high-activity Cel5 on the carboxymethylcellulose/Congo-red assay. The second modest-sensitivity HTS focused on the detection of low-activity IA on the amylodextrin-I2 assay. After six rounds of HTS, a secretory Cel5-IA level was increased to 234 mg/L, 155 times the wild-type IA with the NprB signal peptide only. This combinatory strategy could be useful to enhance the secretory production of large-size heterologous proteins in B. subtilis.

A hybrid system integrating xylose dehydrogenase and NAD+ coupled with PtNPs@MWCNTs composite for the real-time biosensing of xylose.

The wide application of xylose in the food, beverage, and pharmaceutical industries, as well as in the booming field of biorefinery, raises the demand for a rapid, accurate, and real-time xylose-sensing technique to rival the conventional methods based on chromatography, spectroscopy, and electrochemical analysis using non-specific enzymes or abiotic catalysts. Herein, a hybrid system comprising polyethylene glycerol swing-arm-tethered NAD+ and xylose dehydrogenase (XDH), coupled with platinum nanoparticles deposited on carbon nanotubes (PtNPs@MWCNTs), was constructed for the real-time sensing of xylose. The use of the PtNPs@MWCNTs composite enhanced the sensitivity of the electric response and reduced the oxidation potential of NADH significantly. Further, the NAD+ immobilization allowed an increase in its microenvironment concentration and facilitated cofactor regeneration. The screen-printed electrode cast with the hybrid system showed a wide xylose detection range of 0.5 to 10 mM or 3.33 to 66.61 mM, and a low detection limit of 0.01 mM or 3.33 mM (S/N = 3), when connected to a potentiostat or a homemade portable biosensor, respectively. The biosensor also exhibited excellent working stability as it retained 82% of its initial performance after 30 days. The analysis of various xylose-containing samples further revealed the merits of our portable xylose biosensor in real-time sensing, including its rapid response, inexpensive instrumentation, and high selectivity, suggesting its great potential in practical applications.

Coevolution of both Thermostability and Activity of Polyphosphate Glucokinase from Thermobifida fusca YX.

Thermostability and specific activity of enzymes are two of the most important properties for industrial biocatalysts. Here, we developed a petri dish-based double-layer high-throughput screening (HTS) strategy for rapid identification of desired mutants of polyphosphate glucokinase (PPGK) from a thermophilic actinobacterium, Thermobifida fusca YX, with both enhanced thermostability and activity. Escherichia coli colonies representing a PPGK mutant library were grown on the first-layer Phytagel-based plates, which can remain solid for 1 h, even at heat treatment temperatures of more than 100°C. The second layer that was poured on the first layer contained agarose, substrates, glucose 6-phosphate dehydrogenase (G6PDH), the redox dye tetranitroblue tetrazolium (TNBT), and phenazine methosulfate. G6PDH was able to oxidize the product from the PPGK-catalyzed reaction and generate NADH, which can be easily examined by a TNBT-based colorimetric assay. The best mutant obtained after four rounds of directed evolution had a 7,200-fold longer half-life at 55°C, 19.8°C higher midpoint of unfolding temperature (Tm ), and a nearly 3-fold enhancement in specific activities compared to those of the wild-type PPGK. The best mutant was used to produce 9.98 g/liter myo-inositol from 10 g/liter glucose, with a theoretical yield of 99.8%, along with two other hyperthermophilic enzymes at 70°C. This PPGK mutant featuring both great thermostability and high activity would be useful for ATP-free production of glucose 6-phosphate or its derived products.IMPORTANCE Polyphosphate glucokinase (PPGK) is an enzyme that transfers a terminal phosphate group from polyphosphate to glucose, producing glucose 6-phosphate. A petri dish-based double-layer high-throughput screening strategy was developed by using ultrathermostable Phytagel as the first layer instead of agar or agarose, followed by a redox dye-based assay for rapid identification of ultrathermostable PPGK mutants. The best mutant featuring both great thermostability and high activity could produce glucose 6-phosphate from glucose and polyphosphate without in vitro ATP regeneration.

Construction of Enzyme-Cofactor/Mediator Conjugates for Enhanced in Vitro Bioelectricity Generation.

Cofactor-dependent oxidoreduction and electron transfer play an important role in in vitro bioelectricity generation and many other enzyme biocatalysis reactions. To facilitate such electron generation and transfer, several approaches based on the coimmobilization of cofactors and oxidoreductases have been demonstrated. Herein, a convenient and immobilization-free approach of constructing enzyme-cofactor and enzyme-mediator conjugates was developed. The in vitro bioelectricity generation reactions via enzymatic fuel cells were evaluated. The cells equipped by the conjugates exhibited significantly improved power output and stability in contrast to those mediated by unconjugated enzymes. These results may bring a new avenue in constructing efficient in vitro electron transfer chains for various biocatalysis applications.

In vitro metabolic engineering of bioelectricity generation by the complete oxidation of glucose.

The direct generation of electricity from the most abundant renewable sugar, glucose, is an appealing alternative to the production of liquid biofuels and biohydrogen. However, enzyme-catalyzed bioelectricity generation from glucose suffers from low yields due to the incomplete oxidation of the six-carbon compound glucose via one or few enzymes. Here, we demonstrate a synthetic ATP- and CoA-free 12-enzyme pathway to implement the complete oxidation of glucose in vitro. This pathway is comprised of glucose phosphorylation via polyphosphate glucokinase, NADH generation catalyzed by glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), electron transfer from NADH to the anode, and glucose 6-phosphate regeneration via the non-oxidative pentose phosphate pathway and gluconeogenesis. The faraday efficiency from glucose to electrons via this pathway was as high as 98.8%, suggesting the generation of nearly 24 electrons per molecule of glucose. The generated current density was greatly increased from 2.8 to 6.9mAcm-2 by replacing a low-activity G6PDH with a high-activity G6PDH and introducing a new enzyme, 6-phosphogluconolactonase, between G6PDH and 6PGDH. These results suggest the great potential of high-yield bioelectricity generation through in vitro metabolic engineering.

Composition-dependent metallic glass alloys correlate atomic mobility with collective glass surface dynamics.

Glassy metallic alloys are richly tunable model systems for surface glassy dynamics. Here we study the correlation between atomic mobility, and the hopping rate of surface regions (clusters) that rearrange collectively on a minute to hour time scale. Increasing the proportion of low-mobility copper atoms in La-Ni-Al-Cu alloys reduces the cluster hopping rate, thus establishing a microscopic connection between atomic mobility and dynamics of collective rearrangements at a glass surface made from freshly exposed bulk glass. One composition, La60Ni15Al15Cu10, has a surface resistant to re-crystallization after three heating cycles. When thermally cycled, surface clusters grow in size from about 5 glass-forming units to about 8 glass-forming units, evidence of surface aging without crystal formation, although its bulk clearly forms larger crystalline domains. Such kinetically stable glass surfaces may be of use in applications where glassy coatings stable against heating are needed.

A wearable and flexible lactic-acid/O2 biofuel cell with an enhanced air-breathing biocathode.

The performance of biocathode in an enzymatic biofuel cell (EBFC) in the real application is somehow overlooked. Herein, a wearable and flexible lactic-acid/O2 EBFC enhanced with an air-breathing biocathode is designed to solve the limitation of biocathode that arises from the low solubility and slow mass transfer of the dissolved oxygen. To improve the oxygen supply efficiency for the air-breathing biocathode, a superhydrophobic base electrode creating an efficient air-solid-liquid triphase interface is developed. The designed EBFC with an 'island-bridge' configuration is integrated by assembling the current collectors of air-breathing biocathode and bioanode on a commercial laminating film (LF) screen-printed with a noninterfering circuit. It is found that the biocathode/bioanode area ratio should exceed 9:1 so that the designed EBFC (1A//9C) can achieve the optimal performance. This EBFC delivers an open circuit voltage of ca. 0.75 V and outputs a maximum power density of ca. 1.78 mW cm-2. In addition, a scaled-up EBFC (total bioanode area: 1.5 cm2) successfully powers a self-developed low-power device of heartrate in the pulse operation mode when applied on a volunteer's arm.

Construction of a bioelectrocatalytic system with bacterial surface displayed enzyme-nanomaterial hybrids.

To take advantage of the high specificity of enzymatic catalysis along with the high efficiency of electrochemical cofactor regeneration, a bacterial surface displayed enzyme-nanomaterial hybrid bioelectrocatalytic system is herein developed. A cofactor-dependent xylose reductase, capable of reducing xylose to xylitol, is displayed on the surface of Bacillus subtilis, followed by the attachment of copper nanomaterials via the binding of His-tagged enzyme with the nickel ion. This hybrid system can regenerate NADPH with a highest efficiency of 71.6% in 4 h without the usage of extra electron mediators, and 2.35 mM of xylitol can be synthesized after a series of optimization processes. This work opens up new possibilities for the construction and application of bioelectrocatalytic systems with enzyme-nanomaterial hybrids.

Kinetic study of electron transfer process in methyl orange decolorization by shewanella in MFCs with covalent organic frameworks modified anode.

As a new electrode material for electrochemical systems, covalent organic framework (COF) materials have been gradually applied to bioelectrochemical systems. In our previous study, the COFBTA-DPPD-rGO composite was synthesized via Schiff-base coupling between benzene-1,3,5-tricarbaldehyde (BTA) and 3,8-diamino-6-phenylphenanthridine (DPPD) on reduced graphene oxide (rGO) at room temperature. Here, COFBTA-DPPD-rGO modified MFC anode was used to assist microorganisms to decolorize methyl orange (MO), and the properties of MFCs were studied. The results showed that compared to the unmodified electrode MFC (28 mA m-2, 4.20 mW m-2) the current density and maximum power density of the anode MFC modified by COFBTA-DPPD-rGO (134.5 mA m-2, 21.78 mW m-2) were increased by 380.3% and 423.6%, respectively. The transferred electron number n and charge transfer coefficient α of the modified COFBTA-DPPD-rGO anode (4 and 0.43) compared to the unmodified electrode (2.4 and 0.38) were increased by 67% and 13%, respectively. The decolorization ratio of MO could reach 90.3% at 10 h. Compared with the unmodified electrode MFC (53.0%), the decolorization ratio and kinetic constant of decolorization process were enhanced by 26% and 372%, respectively. Therefore, COFBTA-DPPD-rGO could be a new choice for applying to the MFCs.

High-Entropy Alloy Activating Laves-Phase Network for Multi-Component Metallic Coatings with High Hardness.

The low hardness and poor wear resistance of laser-cladding 316L stainless steel impose significant constraints on its practical applications. In this study, a strategy for strengthening laser-cladding 316L stainless steel with WMoTaNb refractory high-entropy alloy as a reinforcement material is proposed. The results confirm that the coating primarily comprises a body-centered cubic (BCC) Fe-based solid solution, a network-distributed hexagonal Fe2X (X = W, Mo, Ta, and Nb) Laves phase, and a diffusely distributed face-centered cubic (FCC) (Ta, Nb)C phase. The Fe-based solid solution distributes along columnar and fine dendrites, while the Laves phase and (Ta, Nb)C phase are in the inter-dendrites. The presence of a significant number of network Laves phases exhibiting high strength and hardness is the primary factor contributing to the enhancement of coating microhardness. The hardness of the composite coating is increased by nearly twice compared to that of the 316L coating, resulting in an improved wear resistance. The present work can shed light on designing and fabricating 316L stainless steel coating with enhanced hardness and wear resistance.

ATP-free in vitro biotransformation of starch-derived maltodextrin into poly-3-hydroxybutyrate via acetyl-CoA.

In vitro biotransformation (ivBT) facilitated by in vitro synthetic enzymatic biosystems (ivSEBs) has emerged as a highly promising biosynthetic platform. Several ivSEBs have been constructed to produce poly-3-hydroxybutyrate (PHB) via acetyl-coenzyme A (acetyl-CoA). However, some systems are hindered by their reliance on costly ATP, limiting their practicality. This study presents the design of an ATP-free ivSEB for one-pot PHB biosynthesis via acetyl-CoA utilizing starch-derived maltodextrin as the sole substrate. Stoichiometric analysis indicates this ivSEB can self-maintain NADP+/NADPH balance and achieve a theoretical molar yield of 133.3%. Leveraging simple one-pot reactions, our ivSEBs achieved a near-theoretical molar yield of 125.5%, the highest PHB titer (208.3 mM, approximately 17.9 g/L) and the fastest PHB production rate (9.4 mM/h, approximately 0.8 g/L/h) among all the reported ivSEBs to date, and demonstrated easy scalability. This study unveils the promising potential of ivBT for the industrial-scale production of PHB and other acetyl-CoA-derived chemicals from starch.

Enzymatic biofuel cell-powered iontophoretic facial mask for enhanced transdermal drug delivery.

Recent advances in enzymatic biofuel cells (EBFCs) have resulted in great progress in health monitoring and supplying power to medical applications, such as drug delivery. On the other hand, to enhance the electric field-assisted transdermal permeation for facial mask application, an external power source is usually required. Herein, we attempted to combine an EBFC with a facial mask so that the microcurrent generated can boost the transdermal permeability of target molecules in the facial mask essence. When screen-printed onto a polypropylene-based non-woven fabric, the three-layered flexible EBFC could produce a voltage of ∼0.4 V and a maximum power density of 23.3 µW cm-2, leading to an approximately 2-3-fold increase in permeated nicotinamide, arbutin, and aspirin levels within 15 min compared to non-iontophoretic transdermal drug delivery. Both cell viability and animal experiments further demonstrated that the EBFC-powered iontophoresis worked well in living animals with good biocompatibility. These results suggest that the EBFC-powered iontophoretic facial mask can effectively improve the permeation of drugs and holds a promise for the possible cosmetic application.

Construction of Cupriavidus necator displayed with superoxide dismutases for enhanced growth in bioelectrochemical systems.

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-IgAß 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.

Embracing a low-carbon future by the production and marketing of C1 gas protein.

Food scarcity and environmental deterioration are two major problems that human populations currently face. Fortunately, the disruptive innovation of raw food materials has been stimulated by the rapid evolution of biomanufacturing. Therefore, it is expected that the new trends in technology will not only alter the natural resource-dependent food production systems and the traditional way of life but also reduce and assimilate the greenhouse gases released into the atmosphere. This review article summarizes the metabolic pathways associated with C1 gas conversion and the production of single-cell protein for animal feed. Moreover, the protein function, worldwide authorization, market access, and methods to overcome challenges in C1 gas assimilation microbial cell factory construction are also provided. With widespread attention and increasing policy support, the production of C1 gas protein will bring more opportunities and make tremendous contributions to our sustainable future.

Photoswitchable Enzymatic Biofuel Cell Based on Fusion Protein with Natural Photoreceptor Vivid.

Enzymatic biofuel cells (EBFCs) have increasingly been the subject of research, but the control of the EBFC output remains difficult. In this study, we fuse glucose 6-phosphate dehydrogenase (G6PDH) and diaphorase (DI) with the natural photoreceptor Vivid named "Mag". The output current and power density of EBFCs with the fusion protein exhibit a sensitive and efficient response to blue light. Following optimizations, the power density increases nearly 4-fold from 1.32 to 6.26 µW cm-2, whereas the current rises from 5.9 to 10.8 µA after 20 min of illumination, dropping back within 30 min under dark conditions.

Protein engineering for electrochemical biosensors.

The development of electrochemical biosensors has gained tremendous attention. Protein engineering has been applied for enhancing properties of native redox enzymes, such as selectivity, sensitivity, and stability required for applicable biosensors. This review highlights recent advances of protein engineering to improve enzymatic catalysis of biosensors, facilitate electron transfer and enzyme immobilization, and construct allosteric protein biosensors. The pros and cons of different protein engineering strategies are briefly discussed, and perspectives are further provided.

An Enzymatic Biosensor for the Detection of D-2-Hydroxyglutaric Acid in Serum and Urine.

D-2-hydroxyglutaric acid (D2HG) is overproduced as a result of the D-2-hydroxyglutaric aciduria and relevant cancers, caused by gene mutation. Accurate analysis of D2HG could help rapid diagnosis of these diseases and allow for timely treatment. In this work, a D-2-hydroxyglutarate dehydrogenase from Ralstonia solanacearum (RsD2HGDH) is cloned and recombinantly expressed. This enzyme features the direct electron transfer to chemical electron mediators (such as methylene blue (MB)) in the absence of additional coenzymes. Therefore, NAD+, a natural electron acceptor for the commercial D2HGDH and usually known for being unstable and difficult for immobilization can be avoided in the preparation of biosensors. The RsD2HGDH and MB are co-immobilized on a two-dimensional material, Ti3C2 MXene, followed by drop-coating on the gold screen-printed electrode (AuSPE) to construct a compact and portable biosensor. The D2HG in samples can be catalyzed by RsD2HGDH, where the current change is measured by chronoamperometry at -0.23 V. The biosensor shows a D2HG detection range of 0.5 to 120 µM (R2 = 0.9974) with a sensitivity of 22.26 µA mM-1 cm-2 and a detection limit of 0.1 µM (S/N = 3). The biosensor retains 72.52% performance of its incipient state after 30 days of storage. The samples of D2HG-containing fetal bovine serum and artificial urine were analyzed with the recovery of 99.56% to 106.83% and 97.30% to 102.47% further indicating the great application potential of our portable D2HG biosensor.