Leloir Glycosyltransferases as Biocatalysts for Chemical Production

Glycosylation is a chemical transformation that is centrally important in all glycoscience and related technologies. Catalysts offering good control over reactivity and selectivity in synthetic glycosylations are much sought. The enzymes responsible for glycosylations in natural biosynthesis are sugar-nucleotide-dependent (Leloir) glycosyltransferases. Discovery-oriented synthesis and pilot batch production of oligosaccharides and glycosylated natural products have previously relied on Leloir glycosyltransferases. However, despite their perceived synthetic utility, Leloir glycosyltransferases are yet to see widespread application in industrial biocatalysis. Here we show progress and limitations in the development of Leloir glycosyltransferases into robust biocatalytic systems for use in glycosylations for chemical production. Obtaining highly active and stable (whole-cell) catalysts that can promote the desired glycosylation(s) coupled to an in situ sugar nucleotide supply remains a difficult problem. Optimizing glycosyltransferase cascade reactions for high process efficiency is another. Glycosylations of some natural products (e.g., flavonoids, terpenoids) involve acceptor substrate solubility as a special challenge for biocatalytic process design. Strategies to overcome these problems are illustrated from examples of integrated biocatalytic process development with this class of enzymes

Thermal Cycling Cascade Biocatalysis of <i>myo</i>-Inositol Synthesis from Sucrose

<i>myo</i>-Inositol belongs to the vitamin B group (vitamin B8) and is widely used in the drug, cosmetic, and food and feed industries. It is produced by acid hydrolysis of phytate, but this method suffers from costly feedstock and serious phosphorus pollution. Here a four-enzyme pathway containing thermophilic sucrose phosphorylase, phosphoglucomutase, inositol 1-phosphate synthase, and inositol monophosphatase was designed to convert sucrose to inositol and fructose. To enable the use of enzymes with different optimal temperatures and thermostabilities, we developed a thermal cycling cascade biocatalysis that can selectively add less-thermostable sucrose phosphorylase immobilized on cellulose-containing magnetic nanoparticles into the cold enzyme cocktail or remove it from the hot enzyme cocktail by using a magnetic field (ON/OFF) switch. A series of exergonic reactions push the overall reaction forward, resulting in a high product molar yield (0.98 mol of inositol/mol of sucrose). This cascade biocatalysis platform could open a door to the large-scale production of less-costly inositol and upgrade sucrose to a value-added nutraceutical and functional sweetener

Programming Integrative Extracellular and Intracellular Biocatalysis for Rapid, Robust, and Recyclable Synthesis of Trehalose

We herein introduce a strategy that leverages and integrates the attributes of whole-cell catalysis with enhanced stability of extracellular immobilized enzymes for rapid, robust, recyclable enzyme cascade reactions in a scalable fashion. We demonstrated the utility of the integrative strategy for catalytic synthesis of trehalose from soluble starch with two-step sequential bioconversion enzymatic reactions, implemented by coupling the enzymatic immobilization of β-amylase (BA), based upon E. coli biofilm curli display technique, with intracellular expression of trehalose synthase (TreS) within the same cells. This integrative strategy, compared with a strategy based on cells coupled with isolated BA, enabled a 103.5 ± 18.7% increase in the maximum trehalose formation rate by efficiently reducing the average distance of BA to intracellluar TreS enzyme. In addition, the maximum yield of starch into trehalose reached as high as 59.0 ± 1.3% at a relatively high starch concentration (10% w/v) with 15 g/L of engineered cells. We further showed that the productivity of trehalose and the percentages of cell viability remained 89.1 ± 4.4% and 85.2 ± 3.6%, respectively, even after 8 continuous rounds of biocatalysis. In addition, this strategy exhibited superb operational stability even under harsh conditions, for example, solutions rich in high amount of organic solvents. The strategy demonstrated here opens up research opportunities of combining extracellular catalysis with intracellular reactions for rapid and robust production of various value-based products

Self-Assembled Nanofibers for Strong Underwater Adhesion: The Trick of Barnacles

Developing adhesives that can function underwater remains a major challenge for bioengineering, yet many marine creatures, exemplified as mussels and barnacles, have evolved their unique proteinaceous adhesives for strong wet adhesion. The mechanisms underlying the strong adhesion of these natural adhesive proteins provide rich information for biomimetic efforts. Here, combining atomic force microscopy (AFM) imaging and force spectroscopy, we examine the effects of pH on the self-assembly and adhesive properties of cp19k, a key barnacle underwater adhesive protein. For the first time, we confirm that the bacterial recombinant <i>Balanus albicostatus</i> cp19k (rBalcp19k), which contains no 3,4-dihydroxyphenylalanine (DOPA) or any other amino acids with post-translational modifications, can self-assemble into aggregated nanofibers at acidic pHs. Under moderately acidic conditions, the adhesion strength of unassembled monomeric rBalcp19k on mica is only slightly lower than that of a commercially available mussel adhesive protein mixture, yet the adhesion ability of rBalcp19k monomers decreases significantly at increased pH. In contrast, upon preassembly at acidic and low-salinity conditions, rBalcp19k nanofibers keep stable in basic and high-salinity seawater and display much stronger adhesion and thus show resistance to its adverse impacts. Besides, we find that the adhesion ability of Balcp19k is not impaired when it is combined with an N-terminal Thioredoxin (Trx) tag, yet whether the self-assembly property will be disrupted is not determined. Collectively, the self-assembly-enhanced adhesion presents a previously unexplored mechanism for the strong wet adhesion of barnacle cement proteins and may lead to the design of barnacle-inspired adhesive materials

<b>Supplemental Material - Rizatriptan benzoate-loaded dissolving microneedle patch for management of acute migraine therapy</b>

Supplemental Material for Rizatriptan benzoate-loaded dissolving microneedle patch for management of acute migraine therapy by Chao Zhong, Xiufeng Zhang, Yanfang Sun, Zhong Shen, Yanan Mao, Tianqiu Liu, Rui Wang, Lei Nie, Amin Shavandi, Khaydar E Yunusov and Guohua Jiang in Journal of Biomaterials Applications.</p

DataSheet_1_Genome-wide identification of TPS and TPP genes in cultivated peanut (Arachis hypogaea) and functional characterization of AhTPS9 in response to cold stress.zip

IntroductionTrehalose is vital for plant metabolism, growth, and stress resilience, relying on Trehalose-6-phosphate synthase (TPS) and Trehalose-6-phosphate phosphatase (TPP) genes. Research on these genes in cultivated peanuts (Arachis hypogaea) is limited.MethodsThis study employed bioinformatics to identify and analyze AhTPS and AhTPP genes in cultivated peanuts, with subsequent experimental validation of AhTPS9’s role in cold tolerance.ResultsIn the cultivated peanut genome, a total of 16 AhTPS and 17 AhTPP genes were identified. AhTPS and AhTPP genes were observed in phylogenetic analysis, closely related to wild diploid peanuts, respectively. The evolutionary patterns of AhTPS and AhTPP genes were predominantly characterized by gene segmental duplication events and robust purifying selection. A variety of hormone-responsive and stress-related cis-elements were unveiled in our analysis of cis-regulatory elements. Distinct expression patterns of AhTPS and AhTPP genes across different peanut tissues, developmental stages, and treatments were revealed, suggesting potential roles in growth, development, and stress responses. Under low-temperature stress, qPCR results showcased upregulation in AhTPS genes (AhTPS2-5, AhTPS9-12, AhTPS14, AhTPS15) and AhTPP genes (AhTPP1, AhTPP6, AhTPP11, AhTPP13). Furthermore, AhTPS9, exhibiting the most significant expression difference under cold stress, was obviously induced by cold stress in cultivated peanut, and AhTPS9-overexpression improved the cold tolerance of Arabidopsis by protect the photosynthetic system of plants, and regulates sugar-related metabolites and genes.DiscussionThis comprehensive study lays the groundwork for understanding the roles of AhTPS and AhTPP gene families in trehalose regulation within cultivated peanuts and provides valuable insights into the mechanisms related to cold stress tolerance.</p

Diverse Supramolecular Nanofiber Networks Assembled by Functional Low-Complexity Domains

Self-assembling supramolecular nanofibers, common in the natural world, are of fundamental interest and technical importance to both nanotechnology and materials science. Despite important advances, synthetic nanofibers still lack the structural and functional diversity of biological molecules, and the controlled assembly of one type of molecule into a variety of fibrous structures with wide-ranging functional attributes remains challenging. Here, we harness the low-complexity (LC) sequence domain of fused in sarcoma (FUS) protein, an essential cellular nuclear protein with slow kinetics of amyloid fiber assembly, to construct random copolymer-like, multiblock, and self-sorted supramolecular fibrous networks with distinct structural features and fluorescent functionalities. We demonstrate the utilities of these networks in the templated, spatially controlled assembly of ligand-decorated gold nanoparticles, quantum dots, nanorods, DNA origami, and hybrid structures. Owing to the distinguishable nanoarchitectures of these nanofibers, this assembly is structure-dependent. By coupling a modular genetic strategy with kinetically controlled complex supramolecular self-assembly, we demonstrate that a single type of protein molecule can be used to engineer diverse one-dimensional supramolecular nanostructures with distinct functionalities

Novel β‑Hairpin Antimicrobial Peptide Containing the β‑Turn Sequence of -NG- and the Tryptophan Zippers Facilitate Self-Assembly into Nanofibers, Exhibiting Excellent Antimicrobial Performance

Antimicrobial peptides (AMPs) have emerged as promising agents to combat the antibiotic resistance crisis due to their rapid bactericidal activity and low propensity for drug resistance. However, AMPs face challenges in terms of balancing enhanced antimicrobial efficacy with increased toxicity during modification processes. In this study, de novo d-type β-hairpin AMPs are designed. The conformational transformation of self-assembling peptide W-4 in the environment of the bacterial membrane and the erythrocyte membrane affected its antibacterial activity and hemolytic activity and finally showed a high antibacterial effect and low toxicity. Furthermore, W-4 displays remarkable stability, minimal occurrence of drug resistance, and synergistic effects when combined with antibiotics. The in vivo studies confirm its high safety and potent wound-healing properties at the sites infected by bacteria. This study substantiates that nanostructured AMPs possess enhanced biocompatibility. These advances reveal the superiority of self-assembled AMPs and contribute to the development of nanoantibacterial materials

Novel β‑Hairpin Antimicrobial Peptide Containing the β‑Turn Sequence of -NG- and the Tryptophan Zippers Facilitate Self-Assembly into Nanofibers, Exhibiting Excellent Antimicrobial Performance

Antimicrobial peptides (AMPs) have emerged as promising agents to combat the antibiotic resistance crisis due to their rapid bactericidal activity and low propensity for drug resistance. However, AMPs face challenges in terms of balancing enhanced antimicrobial efficacy with increased toxicity during modification processes. In this study, de novo d-type β-hairpin AMPs are designed. The conformational transformation of self-assembling peptide W-4 in the environment of the bacterial membrane and the erythrocyte membrane affected its antibacterial activity and hemolytic activity and finally showed a high antibacterial effect and low toxicity. Furthermore, W-4 displays remarkable stability, minimal occurrence of drug resistance, and synergistic effects when combined with antibiotics. The in vivo studies confirm its high safety and potent wound-healing properties at the sites infected by bacteria. This study substantiates that nanostructured AMPs possess enhanced biocompatibility. These advances reveal the superiority of self-assembled AMPs and contribute to the development of nanoantibacterial materials