Highly Efficient Production of UDP-Glucose from Sucrose via the Semirational Engineering of Sucrose Synthase and a Cascade Route Design.

Nucleotide sugars are essential precursors for carbohydrate synthesis but are in scarce supply. Uridine diphosphate (UDP)-glucose is a core building block in nucleotide sugar preparation, making its efficient synthesis critical. Here, a process for producing valuable UDP-glucose and functional mannose from sucrose was established and improved via a semirational sucrose synthase (SuSy) design and the accurate D-mannose isomerase (MIase) cascade. Engineered SuSy exhibited enzyme activity 2.2-fold greater than that of the WT. The structural analysis identified a latch-hinge combination as the hotspot for enhancing enzyme activity. Coupling MIase, process optimization, and reaction kinetic analysis revealed that MIase addition during the high-speed UDP-glucose synthesis phase distinctly accelerated the entire process. The simultaneous triggering of enzyme modules halved the reaction time and significantly increased the UDP-glucose yield. A maximum UDP-glucose yield of 83%, space-time yield of 70 g/L/h, and mannose yield of 32% were achieved. This novel and efficient strategy for sucrose value-added exploitation has industrial promise.

Patterned dense Janus membranes with simultaneously robust fouling, wetting and scaling resistance for membrane distillation.

Membrane fouling, wetting and scaling are three prominent challenges that severely hinder the practical applications of membrane distillation (MD). Herein, polyamide/polyvinylidene fluoride (PA/PVDF) Janus membrane comprising a hydrophobic PVDF substrate and a patterned dense PA layer by reverse interfacial polymerization (R-IP) was developed. Direct contact MD experiments demonstrated that PA/PVDF Janus membrane could exhibit simultaneously superior resistance towards surfactant-induced wetting, oil-induced fouling and gypsum-induced scaling without compromising flux. Importantly, the size-sieving effect, rather than the breakthrough pressure of the membrane, was revealed as the critical factor that probably endowed its resistance to wetting. Furthermore, a unique possible anti-scaling mechanism was unveiled. The superhydrophilic patterned dense PA layer with strong salt rejection capability not only prevented scale-precursor ions from intruding the substrate but also resulted in the high surface interfacial energy that inhibited the adhesion and growth of gypsum on the membrane surface, while its relatively low surface -COOH density benefited from R-IP process further ensured the membrane with a low scaling propensity. This study shall provide new insights and novel strategies in designing high-performance MD membranes and enable robust applications of MD facing the challenges of membrane fouling, wetting and scaling.

Engineering the Thermostability of Sucrose Synthase by Reshaping the Subunit Interaction Contributes to Efficient UDP-Glucose Production.

The restricted availability of UDP-glucose, an essential precursor that targets oligo/polysaccharide and glycoside synthesis, makes its practical application difficult. Sucrose synthase (Susy), which catalyzes one-step UDP-glucose synthesis, is a promising candidate. However, due to poor thermostability of Susy, mesophilic conditions are required for synthesis, which slow down the process, limit productivity, and prevent scaled and efficient UDP-glucose preparation. Here, we obtained an engineered thermostable Susy (mutant M4) from Nitrosospira multiformis through automated prediction and greedy accumulation of beneficial mutations. The mutant improved the T1/2 value at 55 °C by 27-fold, resulting in UDP-glucose synthesis at 37 g/L/h of space-time yield that met industrial biotransformation standards. Furthermore, global interaction between mutant M4 subunits was reconstructed by newly formed interfaces according to molecular dynamics simulations, with residue Trp162 playing an important role in strengthening the interface interaction. This work enabled effective, time-saving UDP-glucose production and paved the way for rational thermostability engineering of oligomeric enzymes.

Dual-layer Janus charged nanofiltration membranes constructed by sequential electrospray polymerization for efficient water softening.

The separation of hardness ions such as calcium and magnesium from hard water can improve water quality, which is important but technically challenging. Nanofiltration (NF) has attracted much attention because of its efficiency, environmental friendliness and low cost. However, common NF membranes with a singly (either positively or negatively) charged layer have insufficient water softening capacity. In this work, two types of dual-layer Janus charged polyamide NF membranes composed of oppositely charged inner and outer layers were developed for the first time by sequential electrospray polymerization strategy for efficient water softening. The effect of the microstructure of the dually charged barrier layer on the separation performance of divalent salt ions was explored. Detailed mechanistic studies revealed that the microstructure of the outer layer of the barrier layer played a crucial role in the ion separation of the Janus membrane due to its control of the reverse transport of ions. Janus charged polyamide NF membrane with a loose outer layer exhibited better water softening performance (93.6% of hardness removed) compared to the singly charged NF membranes due to the simultaneous dual electrostatic effect and no ion reverse transport confinement. This Janus charged NF membrane also possessed good antifouling performance, mainly due to its negatively charged outer layers. The mechanistic insights gained in this study reveal the huge potential of microstructural design toward high-performance Janus charged NF membranes, and provide important guidance on the future development of high-efficiency water softening NF membranes.

Insights into the role of dual reaction sites for single Ni atom Fenton-like catalyst towards degradation of various organic contaminants.

The trade-off of Fenton-like catalysts in activity and stability remains a challenge in practical remediation applications. In this work, we successfully synthesized an efficient and stable catalyst comprised of single nickel (Ni) atoms dispersed on N-doped porous carbon (named Ni-SAs@CN) through a simple micropore confinement strategy. The catalyst exhibited outstanding catalytic performance with 25.8 min-1 turnover frequency for peroxymonosulfate (PMS) activation toward degradation of various organic pollutants (e.g., antibiotics, dyes, and plasticizers) in a wide pH range (4.5-10.8). Electron paramagnetic resonance and in situ Raman analyses demonstrated that both radical (including SO4•- and •OH) and Ni-PMS* dominated nonradical (via electron transfer) pathways played pivotal role in the decomposition of organics. The X-ray adsorption fine structure analysis and computational pieces of evidence demonstrate that the atomically dispersed NiN4 coordination is the intrinsic catalytic site for PMS activation. Meanwhile, pyrrolic N acts as a functional site to anchor target contaminants to the surface region for oxidation. In this process which is benefited from the dual active sites, the target contaminants were degraded via combined radical and nonradical pathways, which significantly boost the overall oxidation and mineralization kinetics.

Efficient degradation of Bisphenol A by dielectric barrier discharge non-thermal plasma: Performance, degradation pathways and mechanistic consideration.

The discharge of recalcitrant and persistent organic pollutants into the environment and subsequent adverse impacts on the ecosystem has aroused a great concern all over the world. In this study, dielectric barrier discharge (DBD) non-thermal plasma was employed to eliminate bisphenol A (BPA). The influences of several vital experimental parameters, including discharge voltage, initial pH of solution, and rate of water flow on degradation of BPA, were explored in detail. In addition, the real wastewater from pharmaceutical factory was utilized to test the oxidation performance of DBD system. 96.8% chemical oxygen demand removal was achieved using DBD system. Radical quenching experiment as well as electron paramagnetic resonance test demonstrated that •OH was the main reactive oxygen species for the degradation of BPA. Moreover, eight major BPA degradation intermediates were identified by UPLC-MS. Ultimately, based on the UPLC-MS test results, a possible degradation pathway of BPA was proposed.

Key aroma compounds and metabolic profiling of Debaryomyces hansenii L1-1-fermented Flos Sophorae.

The extract from Debaryomyces hansenii L1-1-fermented Flos Sophorae has a unique aroma and could be used as a natural spice. The influence of yeast growth and culture medium pH on organoleptic properties of fermented substrates, as well as on the content of volatile aromatic compounds, total sugars, polysaccharides, reducing sugars, total proteins, and amino acids, were analyzed. Metabolic pathways were annotated to compare and contrast key aromatic compounds and metabolic profiles of water and ethanol extracts of D. hansenii L1-1-fermented Flos Sophorae. We found that cells grew most rapidly, pH values changed significantly, and the largest consumption of sugars and amino acids occurred within 48 hr, producing bouquet-like, fruity, and sweet odors, as well as the highest content of volatile aromatic compounds in the extracts. The main aroma metabolites were 2-phenylethanol, linalool, and α-terpineol. The sensory quality of the ethanol extracts was superior to that of the water extracts. Five aromatic compounds, isoamyl alcohol, 2-methylbutan-1-ol, isobutyric acid, 2,3-hexanedione, and 1-hexanol, were positively correlated with the water extract group and negatively correlated with the ethanol extract group, whereas 13 aromatic compounds, styrene, acetophenone, 2-octen-1-ol, linalool, naphthalene, α-terpineol, dihydrocarveol, (-)-myrtenol, methyl anthranilate, eugenol, γ-nonanolactone, jasmone, and ß-ionone, showed the converse trend. Although 2-phenylethanol displayed the highest concentration in the extracts, it did not significantly contribute to the separation of ethanol and water extracts. In Flos Sophorae medium, D. hansenii mainly produces 2-phenylethanol from phenylalanine by the Ehrlich reaction, whereas it produces linalool and α-terpineol by the terpenoid backbone and monoterpenoid biosynthetic pathways; the variable contents of proline, arginine, and glutamate could respond to the arginine and proline metabolic pathways. PRACTICAL APPLICATIONS: Flos Sophorae, a collection of buds of Sophora japonica L., is a traditional Chinese medicinal and edible plant for its good aroma, taste, and nutritional value. Debaryomyces hansenii is a common, aroma-producing yeast. D. hansenii L1-1-fermented Flos Sophorae had a unique, bouquet-like aroma, slightly softer than the typical Flos Sophorae-like aroma. This study enriches our understanding of predominant aroma components and determines their contribution to the profiles of Flos Sophorae ferments obtained using D. hansenii L1-1. Researchers and manufacturers specializing in spices making can use these data to improve the aromatic profiles of natural spices produced by microorganisms, thereby obtaining unique aromas.

Biological strategies for oligo/polysaccharide synthesis: biocatalyst and microbial cell factory.

Oligosaccharides and polysaccharides constitute the principal components of carbohydrates, which are important biomacromolecules that demonstrate considerable bioactivities. However, the variety and structural complexity of oligo/polysaccharides represent a major challenge for biological and structural explorations. To access structurally defined oligo/polysaccharides, biological strategies using glycoenzyme biocatalysts have shown remarkable synthetic potential attributed to their regioselectivity and stereoselectivity that allow mild, structurally controlled reaction without addition of protecting groups necessary in chemical strategies. This review summarizes recent biotechnological approaches of oligo/polysaccharide synthesis, which mainly includes in vitro enzymatic synthesis and cell factory synthesis. We have discussed the important factors involved in the production of nucleotide sugars. Furthermore, the strategies established in the cell factory and enzymatic syntheses are summarized, and we have highlighted concepts like metabolic flux rebuilding and regulation, enzyme engineering, and route design as important strategies. The research challenges and prospects are also outlined and discussed.

Development of an Efficient Strategy to Improve Extracellular Polysaccharide Production of Ganoderma lucidum Using L-Phenylalanine as an Enhancer.

Ganoderma lucidum has been a well-known species of basidiomycetes for a long time, and has been widely applied in the fields of food and medicine. Based on the simulation results of model iZBM1060 in our previous research, the effect of L-phenylalanine on G. lucidum extracellular polysaccharides (EPSs) was investigated in this study. EPS production reached 0.91 g/L at 0.4 g/L L-phenylalanine after a 24 h culture, which was 62.5% higher than that of control (0.56 g/L). Transcriptome and genome analysis showed that L-phenylalanine deaminase and benzoate 4-hydroxylase (related to L-phenylalanine metabolism) were significantly up-regulated, while the cell wall mannoprotein gene was down-regulated. Transmission electronic microscopy (TEM) and atomic force microscopy results showed that the cell wall thickness decreased by 58.58%, and cell wall porosity increased in cells treated with L-phenylalanine, which probably contribute to the increasing EPS production. This study provides an efficient strategy for fungal polysaccharide production with high output and low cost.

An Epidermis-like Hierarchical Smart Coating with a Hardness of Tooth Enamel.

We overcome the fundamental dilemma in achieving hard materials with self-healing capability by integrating an epidermis-like hierarchical stratified structure with attractive mechanical and barrier properties of graphene oxide and show that such biomimetic design enables a smart hierarchical coating system with a synergetic healing effect and a record-high stiffness (31.4 ± 1.8 GPa)/hardness (2.27 ± 0.09 GPa) among all self-healable polymeric films even comparable to that of tooth enamel. A quasi-linear layer-by-layer (LBL) film with constituent graphene oxide is deposited on top of an exponential LBL counterpart as a protective hard layer, forming a hierarchical stratified assembly mimicking the structure of epidermis. The hybrid multilayers can achieve a complete restoration after scratching thanks to the mutual benefit: The soft underneath cushion can provide additional polymers to assist the recovery of the outer hard layer, which in turn can be a sealing barrier promoting the self-healing of the soft layer during stimulated polymer diffusion. The presenting hybridization mode of LBL assembly represents a promising tool for integrating seemingly contradictory properties in artificial materials with potential performances surpassing those in nature.

Reconstruction and Analysis of a Genome-Scale Metabolic Model of Ganoderma lucidum for Improved Extracellular Polysaccharide Production.

In this study, we reconstructed for the first time a genome-scale metabolic model (GSMM) of Ganoderma lucidum strain CGMCC5.26, termed model iZBM1060, containing 1060 genes, 1202 metabolites, and 1404 reactions. Important findings based on model iZBM1060 and its predictions are as follows: (i) The extracellular polysaccharide (EPS) biosynthetic pathway was elucidated completely. (ii) A new fermentation strategy is proposed: addition of phenylalanine increased EPS production by 32.80% in simulations and by 38.00% in experiments. (iii) Eight genes for key enzymes were proposed for EPS overproduction. Model iZBM1060 provides a useful platform for regulating EPS production in terms of system metabolic engineering for G. lucidum, as well as a guide for future metabolic pathway construction of other high value-added edible/ medicinal mushroom species.