ATP regeneration by ATPases for in vitro biotransformation.

Adenosine triphosphate (ATP) regeneration is a significant step in both living cells and in vitro biotransformation (ivBT). Rotary motor ATP synthases (ATPases), which regenerate ATP in living cells, have been widely assembled in biomimetic structures for in vitro ATP synthesis. In this review, we present a comprehensive overview of ATPases, including the working principle, orientation and distribution density properties of ATPases, as well as the assembly strategies and applications of ATPase-based ATP regeneration modules. The original sources of ATPases for in vitro ATP regeneration include chromatophores, chloroplasts, mitochondria, and inverted Escherichia coli (E. coli) vesicles, which are readily accessible but unstable. Although significant advances have been made in the assembly methods for ATPase-artificial membranes in recent decades, it remains challenging to replicate the high density and orientation of ATPases observed in vivo using in vitro assembly methods. The use of bioproton pumps or chemicals for constructing proton motive forces (PMF) enables the versatility and potential of ATPase-based ATP regeneration modules. Additionally, overall robustness can be achieved via membrane component selection, such as polymers offering great mechanical stability, or by constructing a solid supporting matrix through layer-by-layer assembly techniques. Finally, the prospects of ATPase-based ATP regeneration modules can be expected with the technological development of ATPases and artificial membranes.

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.

Consecutive Reduction of Five Carbon Dioxide Molecules by Gas-Phase Niobium Carbide Cluster Anions Nb3C4-: Unusual Mechanism for Enhanced Reactivity by the Carbon Ligands.

Studying the cleavage of the C═O bond during CO2 activation at room temperature is highly significant for comprehending the CO2 conversion processes. Herein, mass spectrometry experiments and density functional theory calculations indicate that the niobium carbide anions Nb3C4- can continuously convert five CO2 molecules to CO under thermal collision conditions, while the other clusters with less carbon ligands Nb3C1-3- reduce fewer CO2 molecules. Size-dependent reactivity of Nb3C1-4- cluster anions toward CO2 is observed. Interestingly, the carbon atoms in Nb3C4- not only act as highly active adsorption sites for CO2 but also serve as electron donors to reduce CO2. The stored electrons are released through a carbon-carbon coupling process. Our findings on the role of carbon ligands in enhancing transition metal carbide reactivity can offer new insights for designing active sites on catalysts with both high activity and selectivity.

Logical rotation of non-separable states via uniformly self-assembled chiral superstructures.

The next generation of high-capacity, multi-task optical informatics requires sophisticated manipulation of multiple degrees of freedom (DoFs) of light, especially when they are coupled in a non-separable way. Vector beam, as a typical non-separable state between the spin and orbital angular momentum DoFs, mathematically akin to entangled qubits, has inspired multifarious theories and applications in both quantum and classical regimes. Although qubit rotation is a vital and ubiquitous operation in quantum informatics, its classical analogue is rarely studied. Here, we demonstrate the logical rotation of vectorial non-separable states via the uniform self-assembled chiral superstructures, with favorable controllability, high compactness and exemption from formidable alignment. Photonic band engineering of such 1D chiral photonic crystal renders the incident-angle-dependent evolution of the spatially-variant polarizations. The logical rotation angle of a non-separable state can be tuned in a wide range over 4π by this single homogeneous device, flexibly providing a set of distinguished logic gates. Potential applications, including angular motion tracking and proof-of-principle logic network, are demonstrated by specific configuration. This work brings important insight into soft matter photonics and present an elegant strategy to harness high-dimensional photonic states.

Nitrogen adsorption on Nb2C6H4+ cations: the important role of benzyne (ortho-C6H4).

N2 adsorption is a prerequisite for activation and transformation. Time-of-flight mass spectrometry experiments show that the Nb2C6H4+ cation, resulting from the gas-phase reaction of Nb2+ with C6H6, is more favorable for N2 adsorption than Nb+ and Nb2+ cations. Density functional theory calculations reveal the effect of the ortho-C6H4 ligand on N2 adsorption. In Nb2C6H4+, interactions between the Nb-4d and C-2p orbitals enable the Nb2+ cation to form coordination bonds with the ortho-C6H4 ligand. Although the ortho-C6H4 ligand in Nb2C6H4+ is not directly involved in the reaction, its presence increases the polarity of the cluster and brings the highest occupied molecular orbital (HOMO) closer to the lowest occupied molecular orbital (LUMO) of N2, thereby increasing the N2 adsorption energy, which effectively facilitates N2 adsorption and activation. This study provides fundamental insights into the mechanisms of N2 adsorption in "transition metal-organic ligand" systems.

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.

Polychromatic Dual-Mode Imaging with Structured Chiral Photonic Crystals.

Optical spatial differentiation is a typical operation of optical analog computing and can single out the edge to accelerate the subsequent image processing, but in some cases, overall information about the object needs to be presented synchronously. Here, we propose a multifunctional optical device based on structured chiral photonic crystals for the simultaneous realization of real-time dual-mode imaging. This optical differentiator is realized by self-organized large-birefringence cholesteric liquid crystals, which are photopatterned to encode with a special integrated geometric phase. Two highly spin-selective modes of second-order spatial differentiation and bright-field imaging are exhibited in the reflected and transmitted directions, respectively. Two-dimensional edges of both amplitude and phase objects have been efficiently enhanced in high contrast and the broadband spectrum. This work extends the ingenious building of hierarchical chiral nanostructures, enriches their applications in the emerging frontiers of optical computing, and boasts considerable potential in machine vision and microscopy.

Construction of an artificial phosphoketolase pathway that efficiently catabolizes multiple carbon sources to acetyl-CoA.

The canonical glycolysis pathway is responsible for converting glucose into 2 molecules of acetyl-coenzyme A (acetyl-CoA) through a cascade of 11 biochemical reactions. Here, we have designed and constructed an artificial phosphoketolase (APK) pathway, which consists of only 3 types of biochemical reactions. The core enzyme in this pathway is phosphoketolase, while phosphatase and isomerase act as auxiliary enzymes. The APK pathway has the potential to achieve a 100% carbon yield to acetyl-CoA from any monosaccharide by integrating a one-carbon condensation reaction. We tested the APK pathway in vitro, demonstrating that it could efficiently catabolize typical C1-C6 carbohydrates to acetyl-CoA with yields ranging from 83% to 95%. Furthermore, we engineered Escherichia coli stain capable of growth utilizing APK pathway when glycerol act as a carbon source. This novel catabolic pathway holds promising route for future biomanufacturing and offering a stoichiometric production platform using multiple carbon sources.

Bi-Chiral Nanostructures Featuring Dynamic Optical Rotatory Dispersion for Polychromatic Light Multiplexing.

Chiral nanostructures featuring the unique optical activity have attracted broad interests from scientists. The typical polarization rotation of transmitted light is usually wavelength dependent, namely the optical rotatory dispersion. However, its dynamic tunability and intriguing collaboration with other optical degrees of freedom, especially the highly desired spatial phase, remain elusive. Herein, a bi-chiral liquid crystalline nanostructure is proposed to induce an effect called reflective optical rotatory dispersion. Thanks to the independent manipulation of opposite-handed self-assembled helices, spin-decoupled geometric phases are induced simultaneously. These naturally unite multi-dimensions of light and versatile stimuli-responsiveness of soft matter. Dynamic holography driven by heat and electric field is demonstrated with a fast response. For polychromatic light, the hybrid multiplexed holographic painting is exhibited with fruitful tunable colors. This study extends the ingenious construction of soft chiral superstructures, presents an open-ended strategy for on-demand light control, and enlightens advanced applications of display, optical computing, and communication.

Industrial-scale extraction of high value-added kaolin from excavation waste: Demonstration from Xiamen, China.

Excavation waste from the construction of subways and other underground infrastructures is mainly composed of gravel, sand and clay of minimal economic value, which commonly ends up in landfills. Although the coarse sand and gravel of the excavation waste are typically recycled on site, a large amount of the fine-grained residue must be disposed of due to the prohibition of marine land reclamation in Xiamen, China, leading to an increasingly severe shortage of landfills. In this contribution, a new strategy was successfully developed for industrial-scale extraction of high value-added kaolin from the excavation waste of Xiamen. This strategy can overcome the challenges of complex and variable chemical compositions, high iron contents, low industrial grade, and organic contaminants in the raw materials. Characterization using chemical analysis, powder X-ray diffraction, scanning electron microscopy, and infrared spectroscopy showed that the Xiamen excavation waste originated from granite weathering is mainly composed of kaolinite and quartz, along with high Fe contents and other impurities. The excavation waste was subjected to an intensive process of blunging, grinding, sieving, and classifying, as well as successive iron removal by magnetic separation. Subsequently, the extracted products meet commercial requirements, including those for high-quality kaolin with whiteness and plasticity larger than 90° and 17%, respectively. Moreover, an industrial-scale green production line with an annual treatment capacity of one million tonnes of excavation waste at the utilization rate of 100% was implemented. Hence, this work presents an effective approach for exploiting similar excavation waste around the world to promote sustainable development.

Design, Synthesis and Antitumor Activity of FAK/PLK1 Dual Inhibitors with Quinazolinone as the Skeleton.

Febrifugine is a kind of quinazolinone compound with high biological activity from a Chinese herb called Chang Shan (Dichroa febrifuga). Febrifugine and its derivatives possess extensive biological activities, some of which exhibited anti-tumor activities as FAK inhibitors. However, they are not very effective at inhibiting tumor metastasis, perhaps because tumors gain energy through compensatory activation of other signaling pathways that promote cell migration and invasion. Therefore, seventeen novel febrifugine derivatives with quinazolinone skeleton were designed, synthesized and acted as potential FAK/PLK1 dual inhibitors. These compounds were determined by 1 H-NMR, 13 C-NMR and MS. Most of the compounds exhibited good inhibitory activity against cancer cell lines by computer-assisted screening, antitumor activity test and FAK/PLK1 inhibitory activity test, wherein compound 3b was screened as a high-efficiency lead compound.

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.

Self-Assembled Core-Shell Nanoscale Coordination Polymer Nanoparticles Carrying a Sialyltransferase Inhibitor for Cancer Metastasis Inhibition.

Despite hypersialylation of cancer cells together with a significant upregulation of sialyltransferase (ST) activity contributes to the metastatic cascade at multiple levels, there are few dedicated tools to interfere with their expression. Although transition state-based ST inhibitors are well-established, they are not membrane permeable. To tackle this problem, herein, we design and construct long-circulating, self-assembled core-shell nanoscale coordination polymer (NCP) nanoparticles carrying a transition state-based ST inhibitor, which make the inhibitor transmembrane and potently strip diverse sialoglycans from various cancer cells. In the experimental lung metastasis and metastasis prevention models, the nanoparticle device (NCP/STI) significantly inhibits metastases formation without systemic toxicity. This strategy enables ST inhibitors to be applied to cells and animals by providing them with a well-designed nanodelivery system. Our work opens a new avenue to the development of transition state-based ST inhibitors and demonstrates that NCP/STI holds great promise in achieving metastases inhibition for multiple cancers.

Alignment rule and geometric confinement lead to stability of a vortex in active flow.

Vortices are hallmarks of a wide range of nonequilibrium phenomena in fluids at multiple length scales. In this work, we numerically study the whirling motion of self-propelled soft point particles confined in circular domain, and aim at addressing the stability issue of the coherent vortex structure. By the combination of dynamical and statistical analysis at the individual particle level, we reveal the persistence of the whirling motion resulting from the subtle competition of activity and geometric confinement. In the stable whirling motion, the scenario of the coexistence of the irregular microscopic motions of individual particles and the regular global whirling motion is fundamentally different from the motion of a vortex in passive fluid. Possible orientational order coexisting with the whirling are further explored. This work shows the stability mechanism of vortical dynamics in active media under the alignment rule in confined space and may have implications in creating and harnessing macroscale coherent dynamical states by tuning the confining geometry.

Biosynthesis of artificial starch and microbial protein from agricultural residue.

Growing populations and climate change pose great challenges to food security. Humankind is confronting a serious question: how will we feed the world in the near future? This study presents an out-of-the-box solution involving the highly efficient biosynthesis of artificial starch and microbial proteins from available and abundant agricultural residue as new feed and food sources. A one-pot biotransformation using an in vitro coenzyme-free synthetic enzymatic pathway and baker's yeast can simultaneously convert dilute sulfuric acid-pretreated corn stover to artificial starch and microbial protein under aerobic conditions. The ß-glucosidase-free commercial cellulase mixture plus an ex vivo two-enzyme complex containing cellobiose phosphorylase and potato α-glucan phosphorylase displayed on the surface of Saccharomyces cerevisiae, showed better cellulose hydrolysis rates than a commercial ß-glucosidase-rich cellulase mixture. This is because the channeling of the hydrolytic product from the solid cellulosic feedstock to the yeast mitigated the inhibition of the cellulase cocktail. Animal tests have shown that the digestion of artificial amylose results in slow and relatively small changes in blood sugar levels, suggesting that it could be a new health food component that prevents obesity and diabetes. A combination of the utilization of available agricultural residue and the biosynthesis of starch and microbial protein from non-food biomass could address the looming food crisis in the food-energy-water nexus.

Carrier-Free Immobilization of Multi-Enzyme Complex Facilitates In†Vitro Synthetic Enzymatic Biosystem for Biomanufacturing.

A method is developed for carrier-free immobilization of multi-enzyme complexes with more than four enzymes by utilization of polypeptide interactions (SpyCatcher-SpyTag and dockerin-cohesin) and enzyme component self-oligomerization. Two pairs of scaffoldins with different arrangements of SpyCatcher-SpyTag and cohesins are prepared to recruit the four dockerin-containing cascade enzymes (i. e., alpha-glucan phosphorylase, phosphoglucomutase, inositol 1-phosphate synthase, and inositol 1-phosphatase) that can convert starch into inositol, forming multi-enzyme complexes. These self-assembled enzyme complexes show higher initial reaction rates than the four-enzyme cocktail. Moreover, water-insoluble self-assembled multi-enzyme complexes are observed, being the carrier-free immobilized multi-enzyme complex aggregates. These immobilized enzyme complexes can be recycled easily by simple centrifuging followed by resuspension for another round of reaction. Not only can these immobilized enzyme complexes be obtained by mixing the purified enzyme components, but also by the mixing of crude cell extracts. Therefore, the strategy for the carrier-free immobilization of enzyme complex sheds light on improving the catalytic capability of in vitro synthetic enzymatic biosystems.

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.

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.

Spin-Decoupled Transflective Spatial Light Modulations Enabled by a Piecewise-Twisted Anisotropic Monolayer.

Wavefront control lies at the heart of modern optics. Metasurfaces with specifically tailored resonators can encode different phases to two orthogonal polarization components, but suffer from wavelength-dependent efficiency, sophisticated fabrication, and limited size. Liquid crystals, another excellent candidate for planar optics, are restricted to spin-coupled conjugated phase modulations. Planar optics with spin-decoupled functions is expected to release the multifunctionality of modern optics. Here, a spin-decoupled transflective spatial light modulator is presented with a piecewise-twisted anisotropic monolayer. The phases of reflected and transmitted light can be independently customized by preprogramming the initial orientations of the periodic helix and mirror-symmetric dual-twist configuration, respectively. A transflective orbital angular momentum encoder and decoder is demonstrated, which is simultaneously compatible with different multiplexing techniques. This work releases the multifunctionality of advanced planar optics and may upgrade existing devices in optical informatics.

Engineering of a thermophilic dihydroxy-acid dehydratase toward glycerate dehydration for in vitro biosystems.

Dihydroxy-acid dehydratase (DHAD) plays an important role in the utilization of glycerol or glucose for the production of value-added chemicals in the in vitro synthetic enzymatic biosystem. The low activity of DHAD in the dehydration of glycerate to pyruvate hampers its applications in biosystems. Protein engineering of a thermophilic DHAD from Sulfolobus solfataricus (SsDHAD) was performed to increase its dehydration activity. A triple mutant (I161M/Y145S/G205K) with a 10-fold higher activity on glycerate dehydration was obtained after three rounds of iterative saturation mutagenesis (ISM) based on computational analysis. The shrunken substrate-binding pocket and newly formed hydrogen bonds were the reason for the activity improvement of the mutant. For the in vitro synthetic enzymatic biosystems of converting glucose or glycerol to L-lactate, the biosystems with the mutant SsDHAD showed 3.32- and 2.34-fold higher reaction rates than the wild type, respectively. This study demonstrates the potential of protein engineering to improve the efficiency of in vitro synthetic enzymatic biosystems by enhancing the enzyme activity of rate-limited enzymes. KEY POINTS: • A screening method was established for the protein engineering of SsDHAD. • A R3 mutant of SsDHAD with 10-fold higher activity was obtained. • The R3 mutant exhibits higher productivity in the in vitro biosystems.