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.

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.

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.

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.

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.

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.

A miniaturized bionic ocean-battery mimicking the structure of marine microbial ecosystems.

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.

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.

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.

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.

[Advances in a new energy system based on electricity-hydrogen-carbohydrate cycle].

The development of green and low-carbon renewable energy systems has become an important international consensus. It is also an essential path for China to implement the dual-carbon strategy, ensure national energy security, and achieve sustainable development. This review introduces the theory of a new energy system based on electricity-hydrogen-carbohydrate (EHC) cycle, and highlights the biotransformations of carbohydrate/water-to-hydrogen, carbohydrate-to-electricity, and CO2-to-carbohydrate powered by hydrogen- or electric-energy based on the in vitro synthetic enzymatic biosystems (ivSEB) developed by Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences in the past decade. We elaborate the design principle and the molecular basis of ivSEB, and further expand from the EHC cycle to in vitro biomanufacturing with starch as the feedstock. Combined with the latest research advances, we analyze and discuss advantages and disadvantages of ivSEB, prospect future directions, so as to promote the green, low-carbon and sustainable development of economy and society.

[Artificial bioconversion of carbon dioxide].

Utilization of carbon dioxide (CO2) is a huge challenge for global sustainable development. Biological carbon fixation occurs in nature, but the low energy efficiency and slow speed hamper its commercialization. Physical-chemical carbon fixation is efficient, but relies on high energy consumption and often generates unwanted by-products. Combining the advantages of biological, physical and chemical technologies for efficient utilization of CO2 remains to be an urgent scientific and technological challenge to be addressed. Here, based on the development of Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences in the past decade, we summarize the important progress in the design and construction of functional parts, pathways and systems for artificial bioconversion of carbon dioxide, including the breakthrough on the artificial synthesis of starch from CO2. Moreover, we prospect how to further develop the technologies for artificial bioconversion of carbon dioxide. These progress and perspectives provide new insight for achieving the goal of "carbon peaking and carbon neutrality".

Stoichiometric Conversion of Maltose for Biomanufacturing by In Vitro Synthetic Enzymatic Biosystems.

Maltose is a natural α-(1,4)-linked disaccharide with wide applications in food industries and microbial fermentation. However, maltose has scarcely been used for in vitro biosynthesis, possibly because its phosphorylation by maltose phosphorylase (MP) yields ß-glucose 1-phosphate (ß-G1P) that cannot be utilized by α-phosphoglucomutase (α-PGM) commonly found in in vitro synthetic enzymatic biosystems previously constructed by our group. Herein, we designed an in vitro synthetic enzymatic reaction module comprised of MP, ß-phosphoglucomutase (ß-PGM), and polyphosphate glucokinase (PPGK) for the stoichiometric conversion of each maltose molecule to two glucose 6-phosphate (G6P) molecules. Based on this synthetic module, we further constructed two in vitro synthetic biosystems to produce bioelectricity and fructose 1,6-diphosphate (FDP), respectively. The 14-enzyme biobattery achieved a Faraday efficiency of 96.4% and a maximal power density of 0.6 mW/cm2, whereas the 5-enzyme in vitro FDP-producing biosystem yielded 187.0 mM FDP from 50 g/L (139 mM) maltose by adopting a fed-batch substrate feeding strategy. Our study not only suggests new application scenarios for maltose but also provides novel strategies for the high-efficient production of bioelectricity and value-added biochemicals.

Biomass sugar-powered enzymatic fuel cells based on a synthetic enzymatic pathway.

Biomass stores a tremendous amount of chemical energy and is considered as an abundant and sustainable alternative to fossil fuels. However, the use of biomass to produce mW-level electricity for portable devices suffers from its structural complexity and therefore low energy conversion efficiency. In this study, we design an enzymatic pathway that could co-utilize and completely oxidize glucose and xylose from biomass hydrolysate to achieve high energy density in EFC. Faraday efficiency of 92% and maximum power density of 0.14 mW cm-2 are achieved in this EFC. After the systematically optimization of enzyme loading and temperature as well as the removal of enzyme inhibitor from biomass hydrolysate by activated charcoal, the biomass sugar-powered EFC could reach a maximum power density of 0.5 mW cm-2 and remain 60% of the initial value after 10 days. These results offer a feasible way to extract the energy stored in biomass as much as possible without the side effects of biomass hydrolysate on EFC.

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.

Wastewater-powered high-value chemical synthesis in a hybrid bioelectrochemical system.

A microbial electrochemical system could potentially be applied as a biosynthesis platform by extracting wastewater energy while converting it to value-added chemicals. However, the unfavorable thermodynamics and sluggish kinetics of in vivo whole-cell cathodic catalysis largely limit product diversity and value. Herein, we convert the in vivo cathodic reaction to in vitro enzymatic catalysis and develop a microbe-enzyme hybrid bioelectrochemical system (BES), where microbes release the electricity from wastewater (anode) to power enzymatic catalysis (cathode). Three representative examples for the synthesis of pharmaceutically relevant compounds, including halofunctionalized oleic acid based on a cascade reaction, (4-chlorophenyl)-(pyridin-2-yl)-methanol based on electrochemical cofactor regeneration, and l-3,4-dihydroxyphenylalanine based on electrochemical reduction, were demonstrated. According to the techno-economic analysis, this system could deliver high system profit, opening an avenue to a potentially viable wastewater-to-profit process while shedding scientific light on hybrid BES mechanisms toward a sustainable reuse of wastewater.

An integrated NAD+-dependent dehydrogenase-based biosensor for xylose fermentation sample analysis.

NAD+-dependent dehydrogenase-based biosensors usually suffer from the low accuracy due to the interference of cofactors in the complex environment, such as fermentation samples. Herein, we demonstrate the example of an integrated biosensor device that can be applied for analyzing xylose fermentation samples. The device is composed of one chamber for the elimination of NAD+ and NADH in the fermentation broth and another chamber for the sample analysis. In the first chamber, a cyclic voltammetry method coupled with Ni foam as a working electrode was proven to be effective in removing NAD+ and NADH in the fermentation broth. In the other chamber, xylose dehydrogenase, as the recognition element, and diaphorase, used for the regeneration of bioactive NAD+ mediated by vitamin K3, were co-immobilized on the surface of the magnetic nanoparticles, which was further coated onto a magnetic glassy carbon electrode. The detection range of the constructed biosensor was from 0.5 to 10 g L-1 with a detection limit of 0.01 g L-1 at a signal-to-noise ratio of 3. Moreover, the biosensor achieved high selectivity, recovery, reproducibility, and good long-time stability when analyzing real xylose fermentation samples, suggesting its promising application potential.

High-efficiency transformation of archaea by direct PCR products with its application to directed evolution of a thermostable enzyme.

Hyperthermophilic archaea with unique biochemical and physiological characteristics are important organisms for fundamental research of life science and have great potential for biotechnological applications. However, low transformation efficiency of foreign DNA molecules impedes developments in genetic modification tools and industrial applications. In this study, we applied prolonged overlap extension PCR (POE-PCR) to generate multimeric DNA molecules and then transformed them into two hyperthermophilic archaea, Thermococcus kodakarensis KOD1 and Pyrococcus yayanosii A1. This study was the first example to demonstrate the enhanced transformation efficiencies of POE-PCR products by a factor of approximately 100 for T. kodakarensis KOD1 and 8 for P. yayanosii A1, respectively, relative to circular shuttle plasmids. Furthermore, directed evolution of a modestly thermophilic enzyme, Methanothermococcus okinawensis 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), was conducted to obtain more stable ones due to high transformation efficiency of T. kodakarensis (i.e. ~3 × 104  CFU per µg DNA). T. kodakarensis harbouring the most thermostable MoHMGR mutant can grow in the presence of a thermostable antibiotic simvastatin at 85°C and even higher temperatures. This high transformation efficiency technique could not only help develop more hyperthermophilic enzyme mutants via directed evolution but also simplify genetical modification of archaea, which could be novel hosts for industrial biotechnology.

Platinum nanoparticle-deposited multi-walled carbon nanotubes as a NADH oxidase mimic: characterization and applications.

The effective regeneration of bioactive NAD+ plays an important role in numerous dehydrogenase-dependent applications including biocatalysis and biosensing. However, this process usually suffers from high thermodynamic barrier, instability and high cost associated with natural enzymes. The emergence of nanomaterials with enzyme mimic characteristics has offered a potential alternative to many enzyme-catalyzed processes. Platinum nanoparticles (PtNPs), for example, have been extensively studied for their peroxidase- and oxidase-like activities. However, their behavior as a NADH oxidase mimic has barely been characterized in detail. Herein, we report a facile approach for preparing PtNP-deposited multi-walled carbon nanotubes (PtNPs@MWCNTs) as the nanozyme for NADH oxidation. Its enzymatic activity was investigated in depth, revealing that it is a NADH oxidase instead of a peroxidase and the catalytic process generates O2˙-, rather than OH˙ or 1O2, from dissolved O2. The recovery yield of bioactive NAD+ regeneration by the nanozyme could reach ∼100% with a total turnover number of ∼6000. Besides, it exhibited terrific electrochemical performance for NADH oxidation and sensing by greatly boosting the response and lowering the oxidation overpotential. It could also work on biomimetic cofactors with even higher activity. Finally, xylose dehydrogenase was immobilized with the nanozyme to constitute a hybrid bioelectrode for xylose sensing. The biosensor had a xylose detecting range of 5-400 µM with the limit of detection as low as 1 µM and can retain its performance after being reused several times. Our results suggest that the PtNPs@MWCNTs characterized as a NADH oxidase nanozyme hold great promise in the applications of biocatalysis and biosensing, which intensively deal with dehydrogenases and natural or biomimetic cofactors.