Advancements in heparosan production through metabolic engineering and improved fermentation.

Heparin is one of the most widely used natural drugs, and has been the preferred anticoagulant and antithrombotic agent in the clinical setting for nearly a century. Heparin also shows increasing therapeutic potential for treating inflammation, cancer, and microbial and viral diseases, including COVID-19. With advancements in synthetic biology, heparin production through microbial engineering of heparosan offers a cost-effective and scalable alternative to traditional extraction from animal tissues. Heparosan serves as the starting carbon backbone for the chemoenzymatic synthesis of bioengineered heparin, possessing a chain length that is critically important for the production of heparin-based therapeutics with specific molecular weight (MW) distributions. Recent advancements in metabolic engineering of microbial cell factories have resulted in high-yield heparosan production. This review systematically analyzes the key modules involved in microbial heparosan biosynthesis and the latest metabolic engineering strategies for enhancing production, regulating MW, and optimizing the fermentation scale-up of heparosan. It also discusses future studies, remaining challenges, and prospects in the field.

Detection and analysis of hyaluronic acid raw materials from different sources by NIR and aquaphotomics.

Hyaluronic acid (HA), a polysaccharide, is widely used for its essential physiological functions. Although the structures of low molecular weight HA produced by both acid and enzyme degradation methods are extremely similar, there are still differences due to the different degradation principles. There is currently no clear way to distinguish between HA prepared by acidolysis and enzymatic hydrolysis. Based on near-infrared (NIR) spectroscopy and aquaphotomics technology, a method for distinguishing HA raw materials and their mixtures from different sources was proposed, and HA with different mixed ratios was accurately quantified. First, NIR spectra of the HA samples were collected. The spectra were then preprocessed to improve the spectral resolution. Spectral information was extracted based on wavelet transform and principal component analysis, resulting in a final selection of 12 characteristic wavelengths containing classification information. The discriminative and quantitative models were then constructed using the 12 wavelengths. The discriminative model achieved a 100% identification rate for HA from different sources. The correlation coefficient of calibration (Rc), validation (Rp), external test (Rt), root mean square error of cross validation (RMSECV), calibration (RMSEC), validation (RMSEP), and external test (RMSET) of the mixed proportion quantitative model were 0.9876, 0.9876, 0.9898, 0.0546, 0.0433, 0.0440, and 0.0347, respectively. In this study, the problem of structural similarity and non-identifiability of HA produced by acidolysis and enzymatic hydrolysis was addressed, and quality monitoring of HA feedstock in HA circulating links was achieved. This is the first time to achieve accurate quantification of solid mixtures using the aquaphotomics method.

Structural Analysis and Classification of Low-Molecular-Weight Hyaluronic Acid by Near-Infrared Spectroscopy: A Comparison between Traditional Machine Learning and Deep Learning.

Confusing low-molecular-weight hyaluronic acid (LMWHA) from acid degradation and enzymatic hydrolysis (named LMWHA-A and LMWHA-E, respectively) will lead to health hazards and commercial risks. The purpose of this work is to analyze the structural differences between LMWHA-A and LMWHA-E, and then achieve a fast and accurate classification based on near-infrared (NIR) spectroscopy and machine learning. First, we combined nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectroscopy, two-dimensional correlated NIR spectroscopy (2DCOS), and aquaphotomics to analyze the structural differences between LMWHA-A and LMWHA-E. Second, we compared the dimensionality reduction methods including principal component analysis (PCA), kernel PCA (KPCA), and t-distributed stochastic neighbor embedding (t-SNE). Finally, the differences in classification effect of traditional machine learning methods including partial least squares-discriminant analysis (PLS-DA), support vector classification (SVC), and random forest (RF) as well as deep learning methods including one-dimensional convolutional neural network (1D-CNN) and long short-term memory (LSTM) were compared. The results showed that genetic algorithm (GA)-SVC and RF were the best performers in traditional machine learning, but their highest accuracy in the test dataset was 90%, while the accuracy of 1D-CNN and LSTM models in the training dataset and test dataset classification was 100%. The results of this study show that compared with traditional machine learning, the deep learning models were better for the classification of LMWHA-A and LMWHA-E. Our research provides a new methodological reference for the rapid and accurate classification of biological macromolecules.

Enhanced Stability and Function of Probiotic Streptococcus thermophilus with Self-Encapsulation by Increasing the Biosynthesis of Hyaluronan.

The harsh conditions of the gastrointestinal tract limit the potential health benefits of oral probiotics. It is promising that oral bioavailability is improved by strengthening the self-protection of probiotics. Here, we report the encapsulation of a probiotic strain by endogenous production of hyaluronan to enhance the effects of oral administration of the strain. The traditional probiotic Streptococcus thermophilus was engineered to produce hyaluronan shells by using traceless genetic modifications and clustered regularly interspaced short palindromic repeat interference. After oral delivery to mice in the form of fermented milk, hyaluronan-coated S. thermophilus (204.45 mg/L hyaluronan in the milk) exhibited greater survival and longer colonization time in the gut than the wild-type strain. In particular, the engineered probiotic strain could also produce hyaluronan after intestinal colonization. Importantly, S. thermophilus self-encapsulated with hyaluronan increased the number of goblet cells, mucus production, and abundance of the microorganisms related to the biosynthesis of short-chain fatty acids, resulting in the enhancement of the intestinal barrier. The coating formed by endogenous hyaluronan provides an ideal reference for the effective oral administration of probiotics.

Cell-based high-throughput screening of polysaccharide biosynthesis hosts.

Valuable polysaccharides are usually produced using wild-type or metabolically-engineered host microbial strains through fermentation. These hosts act as cell factories that convert carbohydrates, such as monosaccharides or starch, into bioactive polysaccharides. It is desirable to develop effective in vivo high-throughput approaches to screen cells that display high-level synthesis of the desired polysaccharides. Uses of single or dual fluorophore labeling, fluorescence quenching, or biosensors are effective strategies for cell sorting of a library that can be applied during the domestication of industrial engineered strains and metabolic pathway optimization of polysaccharide synthesis in engineered cells. Meanwhile, high-throughput screening strategies using each individual whole cell as a sorting section are playing growing roles in the discovery and directed evolution of enzymes involved in polysaccharide biosynthesis, such as glycosyltransferases. These enzymes and their mutants are in high demand as tool catalysts for synthesis of saccharides in vitro and in vivo. This review provides an introduction to the methodologies of using cell-based high-throughput screening for desired polysaccharide-biosynthesizing cells, followed by a brief discussion of potential applications of these approaches in glycoengineering.

Cell-based and cell-free biocatalysis for the production of D-glucaric acid.

D-Glucaric acid (GA) is a value-added chemical produced from biomass, and has potential applications as a versatile platform chemical, food additive, metal sequestering agent, and therapeutic agent. Marketed GA is currently produced chemically, but increasing demand is driving the search for eco-friendlier and more efficient production approaches. Cell-based production of GA represents an alternative strategy for GA production. A series of synthetic pathways for GA have been ported into Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris, respectively, and these engineered cells show the ability to synthesize GA de novo. Optimization of the GA metabolic pathways in host cells has leapt forward, and the titer and yield have increased rapidly. Meanwhile, cell-free multi-enzyme catalysis, in which the desired pathway is constructed in vitro from enzymes and cofactors involved in GA biosynthesis, has also realized efficient GA bioconversion. This review presents an overview of studies of the development of cell-based GA production, followed by a brief discussion of potential applications of biosensors that respond to GA in these biosynthesis routes.

Self-healing on mismatched fractured composite surfaces of SiC with a diameter of 180 nm.

Self-healing on fractured surfaces of silicon carbide (SiC) is highly desirable, to avoid the catastrophic failure of high-performance devices working at extreme environments. Nevertheless, self-healing on a fractured surface of an amorphous and crystalline (AAC) composite structure of a brittle nanowire (NW) has not been demonstrated. In this study, self-healing is demonstrated on mismatched fractured surfaces of the AAC composite structure of a brittle solid for a SiC NW with a diameter of 187 nm. Fracture strength is 10.18 GPa for the AAC structure, recovering 11.7% after self-healing on its mismatched fractured surfaces. To the best of our knowledge, we firstly report the self-healing on mismatched fractured surfaces of the AAC structure for a brittle NW. This is a breakthrough of the previous prediction that self-healing could not be realized on a brittle NW with a diameter over 150 nm. A growth of 3 nm was found after self-healing on the gap induced by mismatched fractured surfaces, which is different from previous reports for pure amorphous and monocrystalline brittle NWs. To reduce the potential energy, coherent rebonding and debonding were performed to realize the atomic migration to fill the gap, resulting in the growth of gap of 3 nm to perform self-healing. Our findings shed light on the potential of self-healing for design and fabrication of next-generation high-performance SiC devices used in the vacuum and aerospace industries.

Macroscale Superlubricity Enabled by Graphene-Coated Surfaces.

Friction and wear remain the primary modes for energy dissipation in moving mechanical components. Superlubricity is highly desirable for energy saving and environmental benefits. Macroscale superlubricity was previously performed under special environments or on curved nanoscale surfaces. Nevertheless, macroscale superlubricity has not yet been demonstrated under ambient conditions on macroscale surfaces, except in humid air produced by purging water vapor into a tribometer chamber. In this study, a tribological system is fabricated using a graphene-coated plate (GCP), graphene-coated microsphere (GCS), and graphene-coated ball (GCB). The friction coefficient of 0.006 is achieved in air under 35 mN at a sliding speed of 0.2 mm s-1 for 1200 s in the developed GCB/GCS/GCP system. To the best of the knowledge, for the first time, macroscale superlubricity on macroscale surfaces under ambient conditions is reported. The mechanism of macroscale superlubricity is due to the combination of exfoliated graphene flakes and the swinging and sliding of the GCS, which is demonstrated by the experimental measurements, ab initio, and molecular dynamics simulations. These findings help to bridge macroscale superlubricity to real world applications, potentially dramatically contributing to energy savings and reducing the emission of carbon dioxide to the environment.

Understanding hyaluronic acid induced variation of water structure by near-infrared spectroscopy.

In order to understand the hydration effect of hyaluronic acid (HA) in aqueous solution, near-infrared (NIR) spectroscopy was used to investigate the HA aqueous solutions at different concentrations and temperature. As HA concentration was raised, there was a nonlinear change in absorption value in the first overtone region of OH, indicating the changes of hydration water. A reconstructed spectrum based on principal component analysis (PCA) was established and analyzed with the concept of aquaphotomics. The results showed that HA acted as a structure maker to make water molecules arranged in order. Water species with two hydrogen bonds (S2) and three hydrogen bonds (S3) showed the decrease at low concentration range of 0-40 mg/mL, but increased at higher concentration, indicating the difference in water species at different HA concentration. Meanwhile, HA had the ability to improve the thermal stability of water structure, suggesting a potential bio-protective function. This study provides a unique perspective on the molecular interactions between HA and water molecules, which is helpful for understanding the role of HA in life process and may serve as the basis for HA applications.

Formation mechanism of fivefold deformation twins in a face-centered cubic alloy.

The formation mechanism considers fivefold deformation twins originating from the grain boundaries in a nanocrystalline material, resulting in that fivefold deformation twins derived from a single crystal have not been reported by molecular dynamics simulations. In this study, fivefold deformation twins are observed in a single crystal of face-centered cubic (fcc) alloy. A new formation mechanism is proposed for fivefold deformation twins in a single crystal. A partial dislocation is emitted from the incoherent twin boundaries (ITBs) with high energy, generating a stacking fault along {111} plane, and resulting in the nucleating and growing of a twin by the successive emission of partials. A node is fixed at the intersecting center of the four different slip {111} planes. With increasing stress under the indentation, ITBs come into being close to the node, leading to the emission of a partial from the node. This generates a stacking fault along a {111} plane, nucleating and growing a twin by the continuous emission of the partials. This process repeats until the formation of fivefold deformation twins.

Comparison of the cytotoxicities and wound healing effects of hyaluronan, carbomer, and alginate on skin cells in vitro.

OBJECTIVE: To compare the cytotoxicities and efficacy of hyaluronan (HA), carbomer, and sodium alginate on repairing thermal-injured cells and promoting cell migration. DESIGN: The 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetra-zoliumromide method was used to evaluate the cytotoxicities of HA, carbomer, and sodium alginate on L929 mouse fibroblasts and their repairing ability to thermal-injured HaCaT keratinocytes. A scratch test was used to observe the effects of the 3 materials on cell migration. RESULTS: Hyaluronan with different molecular weights were nontoxic, even at the concentration of 0.5%, whereas carbomer and sodium alginate showed mild or moderate cytotoxicities when their concentrations were higher than 0.1%. Cell viability and cell density of the thermal-injured keratinocytes treated with HA (600, 1070, and 1500 kDa) were increased significantly compared with that of model control (P < .05), whereas carbomer aggravated cell injury, and sodium alginate had no obvious repairing ability. Hyaluronan promoted cell migration significantly with higher cell density in the scratch area, compared with the control after culture for 48 hours; both carbomer and sodium alginate inhibited the cell migration, and carbomer altered the cell morphology completely. CONCLUSIONS: Hyaluronan can repair cell injury and promote cell migration and proliferation. It also has good biocompatibility. As a new type of hydrogel matrix, HA is superior to carbomer and sodium alginate if it is used in wound caring preparations.

Current research on hyaluronic acid-drug bioconjugates.

Hyaluronic acid (HA) is a mucopolysaccharide acid composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine. Based on numerous characteristics such as viscoelastic properties, water-binding ability, biocompatibility and non-immunogenicity, HA has been approved by FDA for biological and medical applications. In addition, multifarious receptors of HA like CD44, RHAMM and TSG6 are over-expressed on the surface of malignant cells, which play important roles in targeting ability. Bioconjugates linking drugs to HA could improve solubility, prolong half-life, provide active targeting capability and then increase the bioavailability of these coupled drugs by pro-drug strategy. Therefore, a large number of HA-drug bioconjugates have been studied. The purpose of this review was to summarize these HA-drug bioconjugates and further discuss synthetic methods and the relevant application in pharmaceuticals.

Oral absorption of hyaluronic acid and phospholipids complexes in rats.

AIM: To prepare a complex of hyaluronic acid (HA) and phospholipids (PL), and study the improvement effect of PL on the oral absorption of HA. METHODS: The complex of HA-PL (named Haplex) was prepared by film dispersion and sonication method, its physico-chemical properties were identified by infrared spectra and differential scanning calorimetry (DSC). The oral absorption of Haplex was studied. Thirty-two healthy rats were divided into 4 groups randomly: (1) a normal saline (NS) control group; (2) an HA group; (3) a mixture group and (4) a Haplex group. After intragastric administration, the concentration of HA in serum was determined. RESULTS: The physico-chemical properties of Haplex were different from HA or PL or their mixture. After Haplex was administered to rats orally, the serum concentration of HA was increased when compared with the mixture or HA control groups from 4 h to 10 h (P<0.05). The DeltaAUC0-12 h of Haplex was also greater than that of the other three groups (P<0.05). CONCLUSION: The method of film dispersion and sonication can prepare HA and PL complex, and PL can enhance the oral absorption of exogenous HA.