An In Vitro Hybrid Biocatalytic System Enabled by a Combination of Surface-Displayed, Purified, and Cell-Free Expressed Enzymes.

Enzymatic cascades have become a green and sustainable approach for the synthesis of valuable chemicals and pharmaceuticals. Using sequential enzymes to construct a multienzyme complex is an effective way to enhance the overall performance of biosynthetic routes. Here we report the design of an efficient in vitro hybrid biocatalytic system by assembling three enzymes that can convert styrene to (S)-1-phenyl-1,2-ethanediol. Specifically, we prepared the three enzymes in different ways, which were cell surface-displayed, purified, and cell-free expressed. To assemble them, we fused two orthogonal peptide-protein pairs (i.e., SpyTag/SpyCatcher and SnoopTag/SnoopCatcher) to the three enzymes, allowing their spatial organization by covalent assembly. By doing this, we constructed a multienzyme complex, which could enhance the production of (S)-1-phenyl-1,2-ethanediol by 3 times compared to the free-floating enzyme system without assembly. After optimization of the reaction system, the final product yield reached 234.6 µM with a substrate conversion rate of 46.9% (based on 0.5 mM styrene). Taken together, our strategy integrates the merits of advanced biochemical engineering techniques, including cellular surface display, spatial enzyme organization, and cell-free expression, which offers a new solution for chemical biosynthesis by enzymatic cascade biotransformation. We, therefore, anticipate that our approach will hold great potential for designing and constructing highly efficient systems to synthesize chemicals of agricultural, industrial, and pharmaceutical significance.

Transfer learning improves predictions in lignin content of Chinese fir based on Raman spectra.

Lignin in biomass plays significant role in substitution of synthetic polymer and reduction of energy expenditure, and the lignin content was usually determined by wet chemical methods. However, the methods' heavy workload, low efficiency, huge consumption of chemicals and use of toxic reagents render them unsuitable for sustainable development and environmental protection. Chinese fir, a prevalent angiosperm tree, holds immense importance for various industries. Since our previous work found that Raman spectroscopy could accurately predict the lignin content in poplar, we propose that the lignin content of Chinese fir can be estimated by similar strategy. The results suggested that the peak at 2895 cm-1 is the optimal choice of internal standard peak and algorithm of XGBoost demonstrates the highest accuracy among all algorithms. Furthermore, transfer learning was successfully introduced to enhance the accuracy and robustness of the model. Ultimately, we report that a machine learning algorithm, combining transfer learning with XGBoost or LightGBM, offers an accurate, high-efficiency and environmental friendly method for predicting the lignin content of Chinese fir using Raman spectra.

A Large-Scale Fabrication of Flexible, Ultrathin, and Robust Solid Electrolyte for Solid-State Lithium-Sulfur Batteries.

All-solid-state lithium metal batteries (ASSLMBs) are considered as the most promising candidates for the next-generation high-safety batteries. To achieve high energy density in ASSLMBs, it is essential that the solid-state electrolytes (SSEs) are lightweight, thin, and possess superior electrochemical stability. In this study, a feasible and scalable fabrication approach to construct 3D supporting skeleton using an electro-blown spinning technique is proposed. This skeleton not only enhances the mechanical strength but also hinders the migration of Li-salt anions, improving the lithium-ion transference number of the SSE. This provides a homogeneous distribution of Li-ion flux and local current density, promoting uniform Li deposition. As a result, based on the mechanically robust and thin SSEs, the Li symmetric cells show outstanding Li plating/stripping reversibility. Besides, a stable interface contact between SSE and Li anode has been established with the formation of an F-enriched solid electrolyte interface layer. The solid-state Li|sulfurized polyacrylonitrile (Li|SPAN) cell achieves a capacity retention ratio of 94.0% after 350 cycles at 0.5 C. Also, the high-voltage Li|LCO cell shows a capacity retention of 92.4% at 0.5 C after 500 cycles. This fabrication approach for SSEs is applicable for commercially large-scale production and application in high-energy-density and high-safety ASSLMBs.

Cell-free biosynthesis and engineering of ribosomally synthesized lanthipeptides.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural products with diverse chemical structures and potent biological activities. A vast majority of RiPP gene clusters remain unexplored in microbial genomes, which is partially due to the lack of rapid and efficient heterologous expression systems for RiPP characterization and biosynthesis. Here, we report a unified biocatalysis (UniBioCat) system based on cell-free gene expression for rapid biosynthesis and engineering of RiPPs. We demonstrate UniBioCat by reconstituting a full biosynthetic pathway for de novo biosynthesis of salivaricin B, a lanthipeptide RiPP. Next, we delete several protease/peptidase genes from the source strain to enhance the performance of UniBioCat, which then can synthesize and screen salivaricin B variants with enhanced antimicrobial activity. Finally, we show that UniBioCat is generalizable by synthesizing and evaluating the bioactivity of ten uncharacterized lanthipeptides. We expect UniBioCat to accelerate the discovery, characterization, and synthesis of RiPPs.

Expanding the toolbox of probiotic Escherichia coli Nissle 1917 for synthetic biology.

Escherichia coli Nissle 1917 (EcN) is a probiotic microbe that has the potential to be developed as a promising chassis for synthetic biology applications. However, the molecular tools and techniques for utilizing EcN remain to be further explored. To address this opportunity, the EcN-based toolbox was systematically expanded, enabling EcN as a powerful platform for more applications. First, two EcN cryptic plasmids and other compatible plasmids were genetically engineered to enrich the manipulable plasmid toolbox for multiple gene coexpression. Next, two EcN-based technologies were developed, including the conjugation strategy for DNA transfer, and quantification of protein expression capability. Finally, the EcN-based applications were further expanded by developing EcN native integrase-mediated genetic engineering and establishing an in vitro cell-free protein synthesis (CFPS) system. Overall, this study expanded the toolbox for manipulating and making full use of EcN as a commonly used probiotic chassis, providing several simplified, dependable, and predictable strategies for researchers working in synthetic biology fields.

De Novo Design and Synthesis of Polypeptide Immunomodulators for Resetting Macrophage Polarization.

Modulating the extracellular matrix microenvironment is critical for achieving the desired macrophage phenotype in immune investigations or tumor therapy. Combining de novo protein design and biosynthesis techniques, herein, we designed a biomimetic polypeptide self-assembled nano-immunomodulator to trigger the activation of a specific macrophage phenotype. It was intended to be made up of (​GGS​GGP​GGG​PAS​AAA​NSA​SRA​TSN​SP)n, the RGD motif from collagen, and the IKVAV motif from laminin. The combination of these domains allows the biomimetic polypeptide to assemble into extracellular matrix-like nanofibrils, creating an extracellular matrix-like milieu for macrophages. Furthermore, changing the concentration further provides a facile route to fine-tune macrophage polarization, which enhances antitumor immune responses by precisely resetting tumor-associated macrophage immune responses into an M1-like phenotype, which is generally considered to be tumor-killing macrophages, primarily antitumor, and immune-promoting. Unlike metal or synthetic polymer-based nanoparticles, this polypeptide-based nanomaterial exhibits excellent biocompatibility, high efficacy, and precise tunability in immunomodulatory effectiveness. These encouraging findings motivate us to continue our research into cancer immunotherapy applications in the future.

Mesostructured Fibrils Exfoliated in Deep Eutectic Solvent as Building Blocks of Collagen Membranes.

The mesoscale components of collagen (nanofibrils, fibrils, and fiber bundles) are well organized in native tissues, resulting in superior properties and diverse functions. In this paper, we present a simple and controlled liquid exfoliation method to directly extract medium-sized collagen fibers ranging from 102 to 159 nm in diameter from bovine Achilles tendon using urea/hydrochloric acid and a deep eutectic solvent (DES). In situ observations under polarized light microscopy (POM) and molecular dynamics simulations revealed the effects of urea and GuHCl on tendon collagen. FTIR study results confirmed that these fibrils retained the typical structural characteristics of type I collagen. These shed collagen fibrils were then used as building blocks to create independent collagen membranes with good and stable mechanical properties, excellent barrier properties, and cell compatibility. A new method for collagen processing is provided in this work by using DES-assisted liquid exfoliation for constructing robust collagen membranes with mesoscale collagen fibrils as building blocks.

Humanoid Intelligent Display Platform for Audiovisual Interaction and Sound Identification.

This study proposes a rational strategy for the design, fabrication and system integration of the humanoid intelligent display platform (HIDP) to meet the requirements of highly humanized mechanical properties and intelligence for human-machine interfaces. The platform's sandwich structure comprises a middle light-emitting layer and surface electrodes, which consists of silicon elastomer embedded with phosphor and silk fibroin ionoelastomer, respectively. Both materials are highly stretchable and resilient, endowing the HIDP with skin-like mechanical properties and applicability in various extreme environments and complex mechanical stimulations. Furthermore, by establishing the numerical correlation between the amplitude change of animal sounds and the brightness variation, the HIDP realizes audiovisual interaction and successful identification of animal species with the aid of Internet of Things (IoT) and machine learning techniques. The accuracy of species identification reaches about 100% for 200 rounds of random testing. Additionally, the HIDP can recognize animal species and their corresponding frequencies by analyzing sound characteristics, displaying real-time results with an accuracy of approximately 99% and 93%, respectively. In sum, this study offers a rational route to designing intelligent display devices for audiovisual interaction, which can expedite the application of smart display devices in human-machine interaction, soft robotics, wearable sound-vision system and medical devices for hearing-impaired patients.

Fluorescence-coded logarithmic-dilution digital droplet PCR for ultrawide-dynamic-range nucleic acid quantification.

Digital PCR (dPCR) is considered the next generation of nucleic acid detection for its ability of absolute quantification and high sensitivity. However, when compared to the current gold standard, quantitative PCR (qPCR), dPCR is falling behind by several orders of magnitude in dynamic range, which limits its clinical applicability. Here we present fluorescence-coded logarithmic-dilution digital droplet PCR (Flodd-PCR) that features a dynamic range across 7 orders of magnitude, over 2 orders higher than conventional dPCR (4-5 log range) and approaching that of qPCR (7-8 log range). Flodd-PCR realizes such a wide dynamic range by dividing ∼20,000 droplets into 4 groups, each featuring a unique dilution factor of the loaded DNA template and thus a shifted dynamic range. This is achieved by a microfluidic chip that performs multi-step serial dilution (20-925 folds) and droplet generation. The post-PCR droplets can be clustered in silico based on their dilution indicator fluorescence and analyzed independently. Experimentally, Flodd-PCR can detect 4-20,000,000 copies/µL (cp./µL) of the synthetic human papillomavirus (HPV) DNA and outperforms standard dPCR when analyzing clinical HPV samples. Furthermore, Flodd-PCR can be implemented with existing dPCR system set-up with minimal adjustment, and therefore will also have wide practicality in different applications which conventional dPCR has already demonstrated.

Tendon Repair and Regeneration Using Bioinspired Fibrillation Engineering That Mimicked the Structure and Mechanics of Natural Tissue.

Replicating the controlled nanofibrillar architecture of collagenous tissue represents a promising approach in the design of tendon replacements that have tissue-mimicking biomechanics─outstanding mechanical strength and toughness, defect tolerance, and fatigue and fracture resistance. Guided by this principle, a fibrous artificial tendon (FAT) was constructed in the present study using an engineering strategy inspired by the fibrillation of a naturally spun silk protein. This bioinspired FAT featured a highly ordered molecular and nanofibrillar architecture similar to that of soft collagenous tissue, which exhibited the mechanical and fracture characteristics of tendons. Such similarities provided the motivation to investigate FAT for applications in Achilles tendon defect repair. In vitro cellular morphology and expression of tendon-related genes in cell culture and in vivo modeling of tendon injury clearly revealed that the highly oriented nanofibrils in the FAT substantially promoted the expression of tendon-related genes combined with the Achilles tendon structure and function. These results provide confidence about the potential clinical applications of the FAT.

Silk nanofibrils-MOF composite membranes for pollutant removal from water.

Membrane separation technology is considered an effective strategy to remove pollutants in sewage. However, it remains a significant challenge to fabricate inexpensive membranes with high purification efficiency. Therefore, the present study proposes the integration of silk nanofibrils (SNFs) and polydopamine⊂metal-organic framework (PDA⊂MOF) nanoparticles to prepare self-supporting membranes, which can effectively intercept nanoparticle pollutants through the size exclusion effect and can strongly adsorb organic dyes and metal ions by SNF. In addition, PDA⊂MOF enables these membranes to adsorb small molecules and heavy metal ions during the filtration process, thereby effectively removing various pollutants from sewage. The integration of size-exclusion and adsorption capabilities enables the SNF/PDA⊂MOF membrane to remove nanoparticles, small-molecule dyes, heavy metal ions, and radioactive elements. This work provides a rational approach for the design and development of the next generation of water treatment membranes and is expected to be used in environmental, food-related, and biomedical fields.

Biomimetic Silk Architectures Outperform Animal Horns in Strength and Toughness.

Structural biomimicry is an intelligent approach for developing lightweight, strong, and tough materials (LSTMs). Current fabrication technologies, such as 3D printing and two-photon lithography often face challenges in constructing complex interlaced structures, such as the sinusoidal crossed herringbone structure that contributes to the ultrahigh strength and fracture toughness of the dactyl club of peacock mantis shrimps. Herein, bioinspired LSTMs with laminated or herringbone structures is reported, by combining textile processing and silk fiber "welding" techniques. The resulting biomimetic silk LSTMs (BS-LSTMs) exhibit a remarkable combination of lightweight with a density of 0.6-0.9 g cm-3 , while also being 1.5 times stronger and 16 times more durable than animal horns. These findings demonstrate that BS-LSTMs are among the toughest natural materials made from silk proteins. Finite element simulations further reveal that the fortification and hardening of BS-LSTMs arise primarily from the hierarchical organization of silk fibers and mechanically transferable meso-interfaces. This study highlights the rational, cost-effective, controllable mesostructure, and transferable strategy of integrating textile processing and fiber "welding" techniques for the fabrication of BS-LSTMs with advantageous structural and mechanical properties. These findings have significant implications for a wide range of applications in biomedicine, mechanical engineering, intelligent textiles, aerospace industries, and beyond.

Embedding Living Cells with a Mechanically Reinforced and Functionally Programmable Hydrogel Fiber Platform.

Living materials represent a new frontier in functional material design, integrating synthetic biology tools to endow materials with programmable, dynamic, and life-like characteristics. However, a major challenge in creating living materials is balancing the tradeoff between structural stability, mechanical performance, and functional programmability. To address this challenge, a sheath-core living hydrogel fiber platform that synergistically integrates living bacteria with hydrogel fibers to achieve both functional diversity and structural and mechanical robustness is proposed. In the design, microfluidic spinning is used to produce hydrogel fiber, which offers advantages in both structural and functional designability due to their hierarchical porous architectures that can be tailored and their mechanical performance that can be enhanced through a variety of post-processing approaches. By introducing living bacteria, the platform is endowed with programmable functionality and life-like capabilities. This work reconstructs the genetic circuits of living bacteria to express chromoproteins and fluorescent proteins as two prototypes that enable the coloration of living fibers and sensing water pollutants by monitoring the amount of fluorescent protein expressed. Altogether, this study establishes a structure-property-function optimized living hydrogel fiber platform, providing a new tool for accelerating the practical applications of the emerging living material systems.

IntelliSense Bio-Ionotronics Battery for Early Warning of Geological Seepage.

Water seepage-induced geological hazards (SIGHs), including landslides, collapse, debris flow, and ground fissures, often cause substantial human mortality, economic losses, and environmental damage. However, an early warning of geological water seepage remains a significant challenge. A self-powered, cost-effective, reliable, and susceptible SIGH early warning system (SIGH-EWS) is reported herein. This system designed the all-solid, sustainable, fire retardant, and safe-to-use bio-ionotronic batteries to provide a stable power supply for Internet of Things chipsets. Furthermore, the batteries' outstanding humidity and water sensitivity allow sensing of the water seepage emergence. Integrating energy management and wireless communication systems, the SIGH-EWS realizes timely alerts for early water seepage in different water and soil environments with a time resolution in seconds. Based on these merits, the SIGH-EWS demonstrates promising application prospects for early warning of geological disasters and corresponding design strategies that can potentially guide the designs of next-generation geological hazard alarm systems.

Combining Cell-Free Expression and Multifactor Optimization for Enhanced Biosynthesis of Cinnamyl Alcohol.

Cell-free expression systems have emerged as a potent and promising platform for the biosynthesis of chemicals by reconstituting in vitro expressed enzymes. Here, we report cell-free biosynthesis of cinnamyl alcohol (cinOH) with enhanced productivity by using the Plackett-Burman experimental design for multifactor optimization. Initially, four enzymes were individually expressed in vitro and directly mixed to reconstitute a biosynthetic route for the synthesis of cinOH. Then, the Plackett-Burman experimental design was used to screen multiple reaction factors and found three crucial parameters (i.e., reaction temperature, reaction volume, and carboxylic acid reductase) for the cinOH production. With the optimum reaction conditions, approximately 300 µM of cinOH was synthesized after 10 h of cell-free biosynthesis. Extending the production time to 24 h also increased the production to a maximum yield of 807 µM, which is nearly 10 times higher than the initial yield without optimization. This study demonstrates that cell-free biosynthesis can be combined with other powerful optimization methodologies such as the Plackett-Burman experimental design for enhanced production of valuable chemicals.

The Fractal Network Structure of Silk Fibroin Molecules and Its Effect on Spinning of Silkworm Silk.

Animal silk is usually considered to exist as a solid fiber with a highly ordered structure, formed by the hierarchical assembly starting from a single silk fibroin (SF) chain. However, this study showed that silk protein molecules existed in the form of a fractal network structure in aqueous solution, rather than as a single chain. This type of network was relatively rigid with low fractal dimension. Finite element analysis revealed that this network structure significantly helped in the stable storage of SF prior to the spinning process and in the rapid formation of a ß-sheeted nanocrystalline and nematic texture during spinning. Further, the strong but brittle mechanical properties of Bombyx mori silk could also be well-explained through the fractal network model of silk fibroin. The strength was mainly derived from the dual network structure, consisting of nodes and ß-sheet cross-links, whereas the brittleness could be attributed to the rigidity of the SF chains between these nodes and cross-links. In summary, this study presents insights from network topology for understanding the spinning process of natural silk and the structure-property relationship in silk materials.

Cell-Free Biosynthesis of Lysine-Derived Unnatural Amino Acids with Chloro, Alkene, and Alkyne Groups.

Crude extract-based cell-free expression systems have been used to produce natural products by reconstitution of their biosynthetic pathways in vitro. However, the chemical scope of cell-free synthesized natural compounds is still limited, which is partially due to the length of biosynthetic gene clusters. To expand the product scope, here, we report cell-free biosynthesis of several lysine-derived unnatural amino acids with functional moieties such as chloro, alkene, and alkyne groups. Specifically, five related enzymes (i.e., halogenase, oxidase, lyase, ligase, and hydroxylase) involved in ß-ethynylserine biosynthesis are selected for cell-free expression. These enzymes can be expressed in single, in pairs, or in trios to synthesize different compounds, including, for instance, 4-Cl-l-lysine, 4-Cl-allyl-l-glycine, and l-propargylglycine. The final product of γ-l-glutamyl-l-ß-ethynylserine (a dipeptide with an alkyne group) can also be synthesized by cell-free expression of the full biosynthetic pathway (i.e., five enzymes). Our results demonstrate the flexibility of cell-free systems, enabling easy regulation and rational optimization for target compound formation. Overall, this work expands not only the type of enzymes (e.g., halogenase) but also the scope of natural products (e.g., terminal-alkyne amino acid) that can be rapidly produced in cell-free systems. With the development of cell-free biotechnology, we envision that cell-free strategies will create a new frontier for natural product biosynthesis.

In-situ growth of low-dimensional perovskite-based insular nanocrystals for highly efficient light emitting diodes.

Regulation of perovskite growth plays a critical role in the development of high-performance optoelectronic devices. However, judicious control of the grain growth for perovskite light emitting diodes is elusive due to its multiple requirements in terms of morphology, composition, and defect. Herein, we demonstrate a supramolecular dynamic coordination strategy to regulate perovskite crystallization. The combined use of crown ether and sodium trifluoroacetate can coordinate with A site and B site cations in ABX3 perovskite, respectively. The formation of supramolecular structure retard perovskite nucleation, while the transformation of supramolecular intermediate structure enables the release of components for slow perovskite growth. This judicious control enables a segmented growth, inducing the growth of insular nanocrystal consist of low-dimensional structure. Light emitting diode based on this perovskite film eventually brings a peak external quantum efficiency up to 23.9%, ranking among the highest efficiency achieved. The homogeneous nano-island structure also enables high-efficiency large area (1 cm2) device up to 21.6%, and a record high value of 13.6% for highly semi-transparent ones.

Engineering Escherichia coli to Utilize Erythritol as Sole Carbon Source.

Erythritol, one of the natural sugar alcohols, is widely used as a sugar substitute sweetener in food industries. Humans themselves are not able to catabolize erythritol and their gut microbes lack related catabolic pathways either to metabolize erythritol. Here, Escherichia coli (E. coli) is engineered to utilize erythritol as sole carbon source aiming for defined applications. First, the erythritol metabolic gene cluster is isolated and the erythritol-binding transcriptional repressor and its DNA-binding site are experimentally characterized. Transcriptome analysis suggests that carbohydrate metabolism-related genes in the engineered E. coli are overall upregulated. In particular, the enzymes of transaldolase (talA and talB) and transketolase (tktA and tktB) are notably overexpressed (e.g., the expression of tktB is improved by nearly sixfold). By overexpression of the four genes, cell growth can be increased as high as three times compared to the cell cultivation without overexpression. Finally, engineered E. coli strains can be used as a living detector to distinguish erythritol-containing soda soft drinks and can grow in the simulated intestinal fluid supplemented with erythritol. This work is expected to inspire the engineering of more hosts to respond and utilize erythritol for broad applications in metabolic engineering, synthetic biology, and biomedical engineering.