An engineered monolignol 4-o-methyltransferase depresses lignin biosynthesis and confers novel metabolic capability in Arabidopsis.

Although the practice of protein engineering is industrially fruitful in creating biocatalysts and therapeutic proteins, applications of analogous techniques in the field of plant metabolic engineering are still in their infancy. Lignins are aromatic natural polymers derived from the oxidative polymerization of primarily three different hydroxycinnamyl alcohols, the monolignols. Polymerization of lignin starts with the oxidation of monolignols, followed by endwise cross-coupling of (radicals of) a monolignol and the growing oligomer/polymer. The para-hydroxyl of each monolignol is crucial for radical generation and subsequent coupling. Here, we describe the structure-function analysis and catalytic improvement of an artificial monolignol 4-O-methyltransferase created by iterative saturation mutagenesis and its use in modulating lignin and phenylpropanoid biosynthesis. We show that expressing the created enzyme in planta, thus etherifying the para-hydroxyls of lignin monomeric precursors, denies the derived monolignols any participation in the subsequent coupling process, substantially reducing lignification and, ultimately, lignin content. Concomitantly, the transgenic plants accumulated de novo synthesized 4-O-methylated soluble phenolics and wall-bound esters. The lower lignin levels of transgenic plants resulted in higher saccharification yields. Our study, through a structure-based protein engineering approach, offers a novel strategy for modulating phenylpropanoid/lignin biosynthesis to improve cell wall digestibility and diversify the repertories of biologically active compounds.

Formulating Spray-Dried Albumin-Modified Lipid Nanoparticles Encapsulating Acyclovir for Enhanced Pulmonary Drug Delivery.

BACKGROUND: Viral pneumonia, a pressing global health issue, necessitates innovative therapeutic approaches. Acyclovir, a potent ring-opening antiviral agent with broad-spectrum activity, faces water solubility, oral bioavailability, and drug resistance challenges. The aim of this study was to increase the efficacy of acyclovir through respiratory delivery by encapsulating it within albumin-modified lipid nanoparticles and formulate it as a spray. METHODS: Nanoparticles was synthesized via the reverse evaporation method; its physicochemical characteristics were rigorously evaluated, including particle size, zeta potential, morphology, encapsulation efficiency, drug loading, and release profile. Furthermore, the cytotoxicity of nanoparticles and its therapeutic potential against viral pneumonia were assessed through cellular and animal model experiments. Result s: Nanoparticles exhibited a spherical morphology, with a mean particle size of 97.48 ± 5.36 nm and a zeta potential of 30.28 ± 4.72 mv; they demonstrated high encapsulation efficiency (93.26 ± 3.27%), drug loading (11.36 ± 0.48%), and a sustained release profile of up to 92% under neutral conditions. Notably, nanoparticles showed low cytotoxicity and efficient intracellular delivery of acyclovir. In vitro studies revealed that nanoparticles significantly reduced interleukin-6 levels induced by influenza virus stimulation. In vivo, nanoparticles treatment markedly decreased mortality, attenuated the inflammatory markers interleukin-6 and tumor necrosis factor-α levels, and mitigated inflammatory lung injury in mice with viral pneumonia. CONCLUSIONS: In this study, albumin was modified with polyethylene glycol (PEG) containing cationic lipid nanoparticles (LN) to prepare albumin-modified lipid nanoparticles encapsulating acyclovir (ALN-Acy), which can effectively deliver Acy into tissues and cells, prolong the survival of mice, and reduce lung injury and inflammatory factors. White albumin LN can be used as efficient drug delivery carriers, and the delivery of Acy via albumin LN is expected to be a therapeutic strategy for treating inflammatory diseases.

Hydrated alginate polysaccharide fabrics grafted with sliver nanocrystals for wearable thermal and health management.

The creation of functional components with precise chemistries on carbohydrate polymers is of great significance for future wearable biomedicine and health management. Among various carbohydrate polymers, marine polysaccharide featured with antimicrobial, biodegradable and biocompatible properties is an ideal platform while the water-swelling nature makes it difficult to form stable interface. Here, well-dispersed silver nanoparticles have been in-situ assembled on hydrated alginate fabric (AF), involving chemical absorption of Ag ions and in-situ reduction of conductive Ag layer. Owing to the stable complex formed between Ag ions and carboxyl groups, the Ag-grafted AF exhibits superior Joule heating capability, including low operating voltage (1-3 V), high saturation temperature (63 °C), rapid response time (25 s) and outstanding durability against harsh conditions. Furthermore, the Ag-grafted AF demonstrates noticeable inhibition against E. coli and S. aureus as compared with the pristine AF. This work provides a rational strategy for the functionalization of hydrated polysaccharide and enables wearable thermotherapy devices for human health management.

CRISPR/Cas9 mutated p-coumaroyl shikimate 3'-hydroxylase 3 gene in Populus tomentosa reveals lignin functioning on supporting tree upright.

The lignin plays one of the most important roles in plant secondary metabolism. However, it is still unclear how lignin can contribute to the impressive height of wood growth. In this study, C3'H, a rate-limiting enzyme of the lignin pathway, was used as the target gene. C3'H3 was knocked out by CRISPR/Cas9 in Populus tomentosa. Compared with wild-type popular trees, c3'h3 mutants exhibited dwarf phenotypes, collapsed xylem vessels, weakened phloem thickening, decreased hydraulic conductivity and photosynthetic efficiency, and reduced auxin content, except for reduced total lignin content and significantly increased H-subunit lignin. In the c3'h3 mutant, the flavonoid biosynthesis genes CHS, CHI, F3H, DFR, ANR, and LAR were upregulated, and flavonoid metabolite accumulations were detected, indicating that decreasing the lignin biosynthesis pathway enhanced flavonoid metabolic flux. Furthermore, flavonoid metabolites, such as naringenin and hesperetin, were largely increased, while higher hesperetin content suppressed plant cell division. Thus, studying the c3'h3 mutant allows us to deduce that lignin deficiency suppresses tree growth and leads to the dwarf phenotype due to collapsed xylem and thickened phloem, limiting material exchanges and transport.

Sequestration and transport of lignin monomeric precursors.

Lignin is the second most abundant terrestrial biopolymer after cellulose. It is essential for the viability of vascular plants. Lignin precursors, the monolignols, are synthesized within the cytosol of the cell. Thereafter, these monomeric precursors are exported into the cell wall, where they are polymerized and integrated into the wall matrix. Accordingly, transport of monolignols across cell membranes is a critical step affecting deposition of lignin in the secondarily thickened cell wall. While the biosynthesis of monolignols is relatively well understood, our knowledge of sequestration and transport of these monomers is sketchy. In this article, we review different hypotheses on monolignol transport and summarize the recent progresses toward the understanding of the molecular mechanisms underlying monolignol sequestration and transport across membranes. Deciphering molecular mechanisms for lignin precursor transport will support a better biotechnological solution to manipulate plant lignification for more efficient agricultural and industrial applications of cell wall biomass.

Monolignol acyltransferase for lignin p-hydroxybenzoylation in Populus.

Plant lignification exhibits notable plasticity. Lignin in many species, including Populus spp., has long been known to be decorated with p-hydroxybenzoates. However, the molecular basis for such structural modification remains undetermined. Here, we report the identification and characterization of a Populus BAHD family acyltransferase that catalyses monolignol p-hydroxybenzoylation, thus controlling the formation of p-hydroxybenzoylated lignin structures. We reveal that Populus acyltransferase PHBMT1 kinetically preferentially uses p-hydroxybenzoyl-CoA to acylate syringyl lignin monomer sinapyl alcohol in vitro. Consistently, disrupting PHBMT1 in Populus via CRISPR-Cas9 gene editing nearly completely depletes p-hydroxybenzoates of stem lignin; conversely, overexpression of PHBMT1 enhances stem lignin p-hydroxybenzoylation, suggesting PHBMT1 functions as a prime monolignol p-hydroxybenzoyltransferase in planta. Altering lignin p-hydroxybenzoylation substantially changes the lignin solvent dissolution rate, indicative of its structural significance on lignin physiochemical properties. Identification of monolignol p-hydroxybenzoyltransferase offers a valuable tool for tailoring lignin structure and physiochemical properties and for engineering the industrially important platform chemical in woody biomass.

Carbonized polymer dots activated hierarchical tungsten oxide for efficient and stable triethylamine sensor.

Hierarchical metal oxide semiconductors present great potential in detecting toxic and hazardous gases with special emphasis on the regulation of their structures and compositions to advance sensor performance. Herein, marine polysaccharide derived carbonized polymer dots (CPDs) are presented to activate hierarchical tungsten oxide (WO3) as efficient and stable triethylamine sensor. Owing to the promoted receptor and transducer function of the oxide/polymer/carbon heterostructure, the CPDs/WO3 sensor exhibits extraordinary sensing characteristics for triethylamine detection, including higher response (4.3 times), faster response/recovery (4.3 times/2.1 times), lower operating temperature (30 °C) and lower detection limit (2.4 times) as compared with hierarchical WO3 sensor, which are also superior to most of the previous reports related to triethylamine detection. Importantly, the adsorption-desorption kinetic of WO3 is found to be enhanced by 67 times after introducing CPDs, mainly derived from abundant slit-like channels for gas diffuse, desirable defect feature as reactive sites, and favorable 0D-2D interface for charge transfer and transport. This work not only establishes an alternative strategy for promoting metal oxide semiconductor gas sensors but also provides a fundamental understanding of CPDs in gas-sensing field.

Effects of annealing on the structure and magnetic properties of Fe80B20 magnetostrictive fibers.

BACKGROUND: Fe80B20 amorphous alloys exhibit excellent soft magnetic properties, high abrasive resistance and outstanding corrosion resistance. In this work, Fe80B20 amorphous micro-fibers with HC of 3.33 Oe were firstly fabricated and the effects of annealing temperature on the structure and magnetic properties of the fibers were investigated. METHODS: In this study, Fe80B20 amorphous fibers were prepared by the single roller melt-spinning method. The structures of as-spun and annealed fibers were investigated by X-ray diffractometer (XRD) (PANalytical X,Pert Power) using Cu Kα radiation. The morphology of the fibers was observed by scanning electron microscopy (SEM) (HITACHI-S4800). Differential scanning calorimetry (DSC) measurements of the fibers were performed on Mettler Toledo TGA/DSC1 device under N2 protection. Vibrating sample magnetometer (VSM, Versalab) was used to examine the magnetic properties of the fibers. The resonance behavior of the fibers was characterized by an impedance analyzer (Agilent 4294A) with a home-made copper coil. RESULTS: The X-ray diffusion (XRD) patterns show that the fibers remain amorphous structure until the annealing temperature reaches 500°C. The differential scanning calorimetry (DSC) results show that the crystallization temperature of the fibers is 449°C. The crystallization activation energy is calculated to be 221 kJ/mol using Kissinger formula. The scanning electron microscopy (SEM) images show that a few dendrites appear at the fiber surface after annealing. The result indicates that the coercivity HC (//) and HC (⊥) slightly increases with increasing annealing temperature until 400°C, and then dramatically increases with further increasing annealing temperature which is due to significant increase in magneto-crystalline anisotropy and magneto-elastic anisotropy. The Q value firstly increases slightly when the annealing temperature rises from room temperature (RT) to 300°C, then decreases until 400°C. Eventually, the value of Q increases to ~2000 at annealing temperature of 500°C. CONCLUSIONS: In this study, Fe80B20 amorphous fibers with the diameter of 60 µm were prepared by the single roller melt-spinning method and annealed at 200°C, 300°C, 400°C, and 500°C, respectively. XRD results indicate that the fiber structure remains amorphous when the annealing temperature is below 400°C. α-Fe phase and Fe3B phase appear when the annealing temperature rises to 500°C, which is above the crystallization temperature of 449°C. The recrystallization activation energy is calculated to be 221 kJ/mol. The coercivity increases with increasing annealing temperature, which attributes to the increase of total anisotropy. All the as-spun and annealed fibers exhibit good resonance behavior for magnetostrictive sensors.

Enhancing digestibility and ethanol yield of Populus wood via expression of an engineered monolignol 4-O-methyltransferase.

Producing cellulosic biofuels and bio-based chemicals from woody biomass is impeded by the presence of lignin polymer in the plant cell wall. Manipulating the monolignol biosynthetic pathway offers a promising approach to improved processability, but often impairs plant growth and development. Here, we show that expressing an engineered 4-O-methyltransferase that chemically modifies the phenolic moiety of lignin monomeric precursors, thus preventing their incorporation into the lignin polymer, substantially alters hybrid aspens' lignin content and structure. Woody biomass derived from the transgenic aspens shows a 62% increase in the release of simple sugars and up to a 49% increase in the yield of ethanol when the woody biomass is subjected to enzymatic digestion and yeast-mediated fermentation. Moreover, the cell wall structural changes do not affect growth and biomass production of the trees. Our study provides a useful strategy for tailoring woody biomass for bio-based applications.

Hybridized electromagnetic-triboelectric nanogenerator for scavenging biomechanical energy for sustainably powering wearable electronics.

We report a hybridized electromagnetic-triboelectric nanogenerator for highly efficient scavenging of biomechanical energy to sustainably power wearable electronics by human walking. Based on the effective conjunction of triboelectrification and electromagnetic induction, the hybridized nanogenerator, with dimensions of 5 cm × 5 cm × 2.5 cm and a light weight of 60 g, integrates a triboelectric nanogenerator (TENG) that can deliver a peak output power of 4.9 mW under a loading resistance of 6 MΩ and an electromagnetic generator (EMG) that can deliver a peak output power of 3.5 mW under a loading resistance of 2 kΩ. The hybridized nanogenerator exhibits a good stability for the output performance and a much better charging performance than that of an individual energy-harvesting unit (TENG or EMG). Furthermore, the hybridized nanogenerator integrated in a commercial shoe has been utilized to harvest biomechanical energy induced by human walking to directly light up tens of light-emitting diodes in the shoe and sustainably power a smart pedometer for reading the data of a walking step, distance, and energy consumption. A wireless pedometer driven by the hybrid nanogenerator can work well to send the walking data to an iPhone under the distance of 25 m. This work pushes forward a significant step toward energy harvesting from human walking and its potential applications in sustainably powering wearable electronics.

GOLD HULL AND INTERNODE2 encodes a primarily multifunctional cinnamyl-alcohol dehydrogenase in rice.

Lignin content and composition are two important agronomic traits for the utilization of agricultural residues. Rice (Oryza sativa) gold hull and internode phenotype is a classical morphological marker trait that has long been applied to breeding and genetics study. In this study, we have cloned the GOLD HULL AND INTERNODE2 (GH2) gene in rice using a map-based cloning approach. The result shows that the gh2 mutant is a lignin-deficient mutant, and GH2 encodes a cinnamyl-alcohol dehydrogenase (CAD). Consistent with this finding, extracts from roots, internodes, hulls, and panicles of the gh2 plants exhibited drastically reduced CAD activity and undetectable sinapyl alcohol dehydrogenase activity. When expressed in Escherichia coli, purified recombinant GH2 was found to exhibit strong catalytic ability toward coniferaldehyde and sinapaldehyde, while the mutant protein gh2 completely lost the corresponding CAD and sinapyl alcohol dehydrogenase activities. Further phenotypic analysis of the gh2 mutant plants revealed that the p-hydroxyphenyl, guaiacyl, and sinapyl monomers were reduced in almost the same ratio compared to the wild type. Our results suggest GH2 acts as a primarily multifunctional CAD to synthesize coniferyl and sinapyl alcohol precursors in rice lignin biosynthesis.