Valinomycin is a nonribosomal cyclodepsipeptide with diverse biological functions such as antitumor, antiviral, antifungal, and insecticidal activity, besides, it is also used as a potassium ionophore. It is intracellularly synthesized by several Streptomyces isolates with a low titer (<85 mg/L). The new strain Streptomyces sp. ZJUT-IFE354 was previously isolated with a good capability for valinomycin synthesis (>400 mg/L). To explore the fedbatch fermentation strategies, the parallel fermentation experiments were used to evaluate the effect of fermentation conditions. The results showed that the time point for activating valinomycin synthesis was in the mid-log cell growth phase. The optimal pH condition is to keep pH >= 6.5 by adding NaOH instead of ammonium hydroxide because ammonium hydroxide will inhibit valinomycin synthesis. KNO3 was proved to be an effective nitrogen source to remarkably enhance the titer of valinomycin up to 1183 mg/L. High residual glucose concentrations show almost no inhibitory effect on valinomycin production, even though feeding is required for high yields. These results indicated that valinomycin synthesis might be regulated by nitrogen source metabolism. A very high valinomycin titer (>1100 mg/L) is successfully achieved in this study, which suggests that strain ZJUTIFE-354 is suitable for the scale-up production.

Ergothioneine (EGT) is a unique natural chiral compound endowed with potent antioxidative, anti-inflammatory, and cytoprotective properties. Currently, EGT is primarily produced via bioextraction from mushrooms and chemical synthesis; however, the low efficiency and high costs associated with these methods hinder their ability to meet the growing market demand. Consequently, heterologous EGT production in non-native host strains (e.g., Escherichia coli and Corynebacterium glutamicum) has garnered increasing attention. With the rapid advancement of synthetic biology and metabolomics, remarkable progress has been achieved in EGT production in recent years, with the high titers have reached 7.2 g/L in E. coli and 9.3 g/L in Yarrowia lipolytica. Meanwhile, the development of a "chemoenzymatic catalytic cascade″ route has achieved the highest titer: 47.3 g/L. This review focuses on the latest advances in the discovery and identification of key enzymes involved in EGT biosynthetic and catabolic pathways and metabolic engineering strategies for EGT production. Additionally, the multifunctional roles and practical applications of EGT in the food, cosmetics, and pharmaceutical industries are summarized.

The sewage sludge anaerobic digestion process produces a large amount of biogas residue. This study proposes a new path for the resource utilization of sludge biogas residue. Biogas residue biochar (BRBC) showed efficient periodate (PI) activation capability after hydrothermal loading of Mn. Under neutral conditions, the optimal removal rate of sulfadiazine (SDZ) could reach 100% within 5 min, and the pseudo-first-order kinetic reaction constant of the Mn-modified biogas residue biochar (Mn-BRBC) activating the PI system was 0.6942 min-1, which was 8.97 times that of the BRBC/PI system. It was confirmed that 1O2 plays a dominant role in the degradation process of SDZ. Characterization results showed that the hydrothermal process changes the vacancy structure of the BRBC surface, and the rearrangement of surface biomass during the process produces abundant oxygen-containing functional groups. Electrochemical experiments demonstrated that the ability of BRBC-mediated electron transfer was significantly enhanced after Mn loading. The biotoxicity of the sulfadiazine degradation products was greatly reduced compared with that of the parent pollutant. Moreover, the system achieved sulfadiazine degradation rates of 88.4% and 83.35% in tap water and lake water, respectively. This study offers a highly promising method for the resource utilization of sludge digestate.

Apigenin, a naturally occurring flavonoid with notable biological activities, is difficult to produce efficiently due to the low yield of plant extraction and the constraints of limited natural resources. To overcome this challenge, three type I flavonoid synthases (FNSI) from Petroselinum crispum (PcFNSI), Apium graveolens (AgFNSI), and Daucus carota (DcFNSI) were heterologously expressed in Escherichia coli, and their catalytic efficiencies in converting naringenin to apigenin were systematically evaluated. Among these, PcFNSI exhibited superior solubility in E. coli and demonstrated higher apigenin-synthesizing activity compared to AgFNSI and DcFNSI. Subsequent application studies revealed that the optimal reaction conditions for PcFNSI-mediated apigenin production included 600 mu M naringenin, 5 mM alpha-ketoglutarate, 0.5 mM Fe2+, and 1% DMSO. Under these conditions, the conversion rate of naringenin reached up to 80.4%, yielding 130.34 mg/L apigenin. Notably, variations in apigenin/succinic acid concentrations, substrate configurations, feeding strategies, or enzyme addition methods had no significant impact on the substrate conversion rate. This study establishes an optimized FNSI-based reaction system for synthesizing apigenin from naringenin and systematically examines the factors affecting apigenin production. The findings provide a foundation for the large-scale industrial production of apigenin and related flavonoid compounds.

Traditional wood adhesives such as phenolic and urea-formaldehyde, which continuously release carcinogenic formaldehyde during production and use, seriously endanger human health and pollute the environment. Therefore, the development of high-performance bio-based adhesives without formaldehyde has become a research hotspot in the field of wood processing. This study developed a green and efficient strategy for preparing bio-based formaldehyde-free adhesives via deep eutectic solvent (DES)-assisted hydrothermal extraction of wheat straw lignin and its modification. The extraction process featured mild conditions (120 °C, 3 h) with high energy efficiency, achieving a lignin yield of 80.2 ± 1.06% without harsh reagents. Notably, oxidative modification of lignin used only hydrogen peroxide (without additional chemicals), achieving a non-toxic and pollution-free treatment while effectively introducing carboxyl groups to enhance reactivity. The oxidized recovered lignin (ORL) reacted with polyvinyl alcohol (PVA) and glycerol triglycidyl ether (GTE) to form a stable cross-linked network. The oxidized lignin adhesive (OLA) exhibited a wet shear strength of 1.07 ± 0.22 MPa, meeting the Chinese national standard GB/T 9846-2015 (≥0.7 MPa). This work emphasizes the mild and green extraction and modification process, providing a sustainable method for the value-added utilization of straw and promoting the industrialization process of environmentally friendly wood adhesives.

Sucrose phosphorylase (SPase) is widely used for the glycosylation of polyhydroxy compounds, while low regioselectivity will limit its application. In this study, the regioselectivity of wild‐type SPase from Limosilactobacillus reuteri for the synthesis of 2‐ O ‐α‐d‐glucosylglycerol (2‐αGG) (84.7% ± 0.5%) was enhanced by combinational semi‐rational strategies. A key residue R137 was identified by sequence alignment analysis of non‐conserved residues in the catalytic pocket, molecular docking analysis, and alanine scanning at the key flexible loops. The regioselectivity of R137M increased to 95.4% ± 0.4%. Iteration mutation, loop engineering, and triple code saturation mutagenesis were used to further increase the 2‐OH selectivity (>97%) and double mutant R137M/L337T (98.0% ± 0.5%) was the best one. Whole‐cell transformation with R137M/L337T produced a higher yield of 2‐αGG with the increased final 2‐OH selectivity (>99%); besides, the amount of byproduct glucose greatly declined, meaning a decreased hydrolysis activity. Hence, the combinational semi‐rational mutagenesis methods are effective for improving the regioselectivity of SPase.

This study presents an approach combining molecular dynamics (MD) simulations and near-attack conformation (NAC) analysis to mine and engineer UDP-glucosyltransferases. Using the MD-NAC strategy, we identified that BsYjiC from Bacillus subtilis possesses menthol glycosylating activity for the first time, and it exhibited a 28% higher kcat/KM value compared to the known l-menthol glycosylating enzyme, BlYjiC from Bacillus licheniformis. The strategy was further employed to modify BsYjiC by preventing nonproductive substrate motion and stabilizing NACs through engineering key residues identified by MD. Based on this strategy, 14 positive mutants were identified out of 16 candidates, and the double mutant A76D/V108L (M2) demonstrated a 10.64-fold increase in kcat/KM value compared to the starting point. Whole-cell cascade reactions with sucrose synthase yield 160 mM glucoside after catalyzing for 96 h, demonstrating its application merit. This work underscores MD-driven enzyme engineering as a promising strategy to overcome limitations of static structure-based approaches.

Paenibacillus has attracted considerable scientific and practical attention in recent years owing to its diverse biological characteristics and extensive range of applications. Its applicability spans multiple fields, including agriculture, medicine, and industrial biotechnology. However, the widespread utilization of Paenibacillus is hindered by several challenges, such as environmental variability, biosafety concerns, and technical barriers. Current research efforts are increasingly directed toward elucidating the diversity and mechanisms of action of its bioactive metabolites. This review offers a comprehensive synthesis of the current state of knowledge on Paenibacillus, covering its metabolic capabilities, practical applications across various sectors, existing limitations, and prospective avenues for future research.

Bacillus subtilis is widely used for industrial enzyme production due to its food safety and good capability of protein synthesis and secretion. However, the production of heterologous proteins is often inefficient, partly due to poor compatibility and versatility of genetic elements in B. subtilis. Recent study shows that transcription and translation is uncoupled in B. subtilis, which is quite different from general knowledge about the transcription-translation coupling mechanism in bacteria. The uncoupling mechanism in B. subtilis shows that the transcription rate is much faster than translation rate. Therefore, the translation regulation will play an important role in highly-effective synthesis of heterologous protein. To better understanding the different regulation strategies at the translation level in B. subtilis, this review will summarize the translation process in B. subtilis cell and its regulatory mechanisms as well as the differences in comparison to other bacteria. Besides, the genetic engineering strategies for engineering the translation regulatory elements are also summarized.

Paenibacillus, a plant-growth-promoting rhizobacterium, exhibits broad-spectrum biocontrol activity through the production of diverse antibacterial metabolites, competitive niche colonization, induction of systemic resistance, and enhancement of nutrient uptake. The review summarizes recent advances in elucidating the synergistic interactions among its biocontrol mechanisms and their responses to environmental factors. Subsequently, it outlines gene editing and regulatory technologies applicable to Paenibacillus. Next, key synthetic biology strategies employed to enhance biosynthetic capabilities are examined. Finally, future prospects and challenges associated with advancing Paenibacillus toward precision engineering and high-efficiency applications are discussed. Notably, its role in industrial biotechnology—particularly in the scalable production of industrial enzymes and high-value chemicals—is increasingly recognized as a focal point of growing scientific and commercial interest.

Background: Hydroxytyrosol (HT), a bioactive polyphenol derived from olive oil, exhibits antioxidant, antibacterial, anti-inflammatory, and anticancer properties. The global market size of HT reached 751.2 million USD in 2024 and is growing at a compound annual growth rate (CAGR) of 6.6 % reported by Future Market Insights (FMI). It is projected to reach 1425.5 million USD by 2034. However, the extraction of HT from plants is regionally dependent, involves high pollution, and requires significant energy consumption. Therefore, developing sustainable alternatives to plant extraction for HT production is critical. Scope and approach: This study conducted a systematic review of HT biosynthesis, applications, and optimization strategies of production reported in recent years using Web of Science, PubMed, and Scopus, offering a reference for achieving higher levels of HT biosynthesis in the future. Key findings and conclusions: The level of biological synthesis of HT has been increasing year by year. Currently, the highest titer of biocatalytic synthesis of HT is 31.2 g/L, and the titer of de novo synthesis of HT has also reached 9.87 g/L. However, there is still room for improvement in the selection of key enzymes, cofactor regeneration, metabolic pathway modification, reduction of oxidation of HT in the fermentation process, and alleviation of cytotoxicity in the biosynthesis of HT. These strategies will bring new insights into the biosynthesis of HT and broaden its applications in fields such as food, pharmaceuticals, and cosmetics.

Sorgoleone, a lipophilic benzoquinone allelochemical exuded by sorghum [Sorghum bicolor (L) Moench] root, represents a promising multitarget botanical herbicide with significant potential for sustainable weed management. Sorgoleone inhibits weed growth through concurrent disruption of mitochondrial respiration, photosystem II electron transfer, carotenoid biosynthesis, and root H+-ATPase activity. It exhibits broad-spectrum activity against terrestrial weeds and aquatic plants, with heightened efficacy against small-seeded species and dicots. Environmentally, sorgoleone is also a biological nitrification inhibitor (BNI) that could suppress soil nitrification (enhancing nitrogen use efficiency by 15-20%) and enhances arbuscular mycorrhizal symbiosis. Due to its important bioactivities and potential application value, plant extraction, chemical synthesis, and biosynthetic synthesis have been explored to overcome production constraints of sorgoleone. This review provides a summary and discussion of the biological activities, herbicidal mechanisms, total synthesis, and biosynthesis of sorgoleone, serving as a basis for further research and applications.

Brivaracetam is a third-generation antiepileptic drug containing two chiral centers (2S, 4 R). To achieve effective asymmetric synthesis of 4 R chiral center, a novel one-pot chemo-enzymatic cascade strategy was developed to synthesize the 4 R chiral precursor, (R)-4-propyldihydrofuran-2(3H)-one (1b). The butenolide 5-hydroxy-4propylfuran-2(5H)-one was selected as the starting substrate due to the presence of an active electron-withdrawing group, which enabled rapid enzymatic reduction by an ene-reductase with > 99 % conversion. However, this reaction generated a racemization-prone intermediate. Subsequent reduction using a newly identified alcohol dehydrogenase (YahK) demonstrated high activity but exhibited S-configuration stereo-selectivity. Through semi-rational engineering, the stereoselectivity of a YahK triple mutant (G132T/T182A/ M313R) was successfully reversed to favor the R-configuration. The chemo-enzymatic cascade combining ADH reduction and chemical cyclization yielded the chiral product 4R-1b with an enantiomeric excess (ee) of > 91 % (R). Notably, the one-pot mode further enhanced the ee value of the final product to > 98 %. This strategy effectively bypassed the limitations of low-reactivity alkene substrates, enabling efficient construction of the 4 R chiral center in brivaracetam.

Sucrose phosphorylase (SPase) is widely used for the preparation of functional glycosides like 2-O-α-d-glucopyranosyl glycerol (2-αGG). Its heterologous expression in Bacillus subtilis has attracted wide interest. Here, the expression of LreSP-MT derived from Limosilactobacillus reuteri was tested through a combinatorial strategy. The promoter adjacent to gene was found to be a key factor in achieving high-level expression, and the best promoter PyvyD increased extracellular enzyme activity by 310 % compared to the strong promoter P43. The dual promoter PydjO-PyvyD further enhanced enzymatic activity by 22 %. Optimal RBS increased enzymatic activity by 14 % after RBS screening and engineering. The optimal recombinant strain B. subtilis WB800 (pPydjO-PyvyD-RBSB15-LreSP-MT) produced a high extracellular enzyme activity of 9.1 U/mL and extracellular recombinant protein yield of up to 8.0 g/L in a fed-batch fermentation. The 10-fold diluted fermentation broth yielded up to 322.6 g/L of 2-αGG through a sucrose-feeding biotransformation strategy, suggesting it is a high-efficiency bioprocess to produce 2-αGG. The results also highlight the critical role of genetic element compatibility for heterologous gene expression in B. subtilis.

Hyaluronidase is used extensively across various domains, with food industry, healthcare, and surgical applications. Hyaluronidase produces low molecular weight hyaluronate through three mechanisms, substrate cleavage and two different hydrolysis pathways. Heterologous expression enhances the production of hyaluronidase in order to meet application demands. Several directed evolution strategies have been implemented to modify the properties of hyaluronidase. Hyaluronic acid of various molecular weights has been generated using metabolic engineering and enzyme engineering techniques. Both hyaluronidase and different Mw forms of hyaluronate have found widespread use in numerous fields. Although research on hyaluronidase and hyaluronic acid oligosaccharides faces opportunities and challenges, there are significant application prospects. This work underscores the potential for using hyaluronidase and hyaluronic acids of different Mw, with broad applicability and innovative prospects in bioresource exploration.

Asiaticoside (AS) and madecassoside (MS) are two key triterpenoid saponin compounds found in Centella asiatica. Despite their structural similarities, they exhibit distinct biological activities. AS is known for its anti-inflammatory properties and its effectiveness in treating rheumatoid arthritis, while MS is recognized for its ability to promote wound healing and significantly improve burn scars. Additionally, AS is widely utilized in the cosmetics industry due to its antioxidant and moisturizing properties. Currently, separating and purifying these two compounds simultaneously using existing methods is challenging. This study focuses on synthesizing molecularly imprinted polymers (MIPs) through precipitation polymerization with AS as the template. By optimizing various preparation parameters, such as the molar ratios of functional monomers, porogens, and cross-linking agents, high selectivity was achieved. The maximum adsorption capacity reached 19.16 mg/g, with an imprinting factor of 2.86. The MIPs were characterized using techniques like scanning electron microscopy (SEM), TA, Brunauer-Emmett-Teller (BET) adsorption, and Fourier-transform infrared (FT-IR). Their effectiveness was assessed through static and dynamic adsorption, desorption, and reusability tests. When applied in molecularly imprinted solid-phase extraction (MISPE) of crude extracts from C. asiatica, the recovery of AS was 39.90%, and its purity increased by 3.01 times, whereas the purity of MS improved by 6.51 times. These findings indicate that the preparation of MIPs is cost-effective, highly selective, reproducible, and user-friendly, making it a promising method for enriching and isolating AS from complex mixtures.

Ectoine is a compatible solute that functions as a cell protector from various stresses, protecting cells and stabilizing biomolecules, and is widely used in medicine, cosmetics, and biotechnology. Microbial fermentation has been widely used for the large-scale production of ectoine, and a number of fermentation strategies have been developed to increase the ectoine yield, reduce production costs, and simplify the production process. Here, Corynebacterium glutamicum was engineered for ectoine production by heterologous expression of the ectoine biosynthesis operon ectBAC gene from Halomonas elongata, and a series of genetic modifications were implemented. This included introducing the de3 gene from Escherichia coli BL21 (DE3) to express the T7 promoter, eliminating the lysine transporter protein lysE to limit lysine production, and performing a targeted mutation lysCS301Y on aspartate kinase to alleviate feedback inhibition of lysine. The new engineered strain Ect10 obtained an ectoine titer of 115.87 g/L in an optimized fed-batch fermentation, representing the highest ectoine production level in C. glutamicum and achieving the efficient production of ectoine in a low-salt environment.

A retrospective study reported that empagliflozin reduced the risk of urinary stone events in patients with diabetes mellitus. To further investigate empagliflozin's potential, we conducted an animal experiment to determine whether empagliflozin can prevent renal stone formation in hyperoxaluria rats. Hyperoxaluria rat models were constructed by administrating 0.75 % ethylene glycol and 1 % ammonium chloride in water. The empagliflozin-treated rats were gauged with empagliflozin at different concentrations, and their body weight and blood sugar data were recorded. After 30 days of treatment, we obtained 24-h urine, kidney, and blood samples. The urine samples were subjected to component detection. Blood samples were prepared for component detection and cytokines detection. Renal samples were subjected to von Kossa staining, transmission electron microscopy, immunohistochemistry, and transcriptome sequencing analysis. Results showed that in empagliflozin-treated hyperoxaluria rats, renal crystal deposition and mitochondria injury, urinary concentration, and excretion of oxalate were significantly decreased. Additionally, plasma levels of VEGF, IL-2, IL-1β, and MCP-1 were decreased. Immunohistochemistry showed that renal expression of KIM-1, MCP-1 was significantly decreased in empagliflozin-treated hyperoxaluria rats. Transcriptome sequencing of renal tissue represented that 25 genes were down-regulated while 12 were up-regulated in empagliflozin-treated hyperoxaluria rats. These regulated genes were mainly enriched in fatty acid metabolism, insulin resistance, muscle contraction, bile secretion, and parathyroid metabolism. Our animal experiments found that empagliflozin could reduce urinary concentration and excretion of oxalate and inhibit renal inflammation, then abating renal calcium oxalate deposition in hyperoxaluria rats in a non-diabetic state.

Cyclodextrin glycosyltransferase (CGTase) catalyzes intermolecular transglycosylation through either disproportionation or cyclization-coupling pathway. Kinetics analysis reveals that the hesperidin glycosylation process catalyzed by a CGTase variant (M1) is primarily accomplished through the disproportionation pathway. The cyclization-coupling pathway exhibits a lower reaction rate and competitively consumes glycosyl donor and yield byproducts that impair disproportionation. Under the guidance of reaction kinetics, mutagenesis was targeted at residues in the -3, +1, and +2 subsites, known to control the selectivity between disproportionation and cyclization. A quadruple variant was identified with 2.9 times hesperidin glycosylation activity compared to M1, and 20.3 times compared to the wild-type. Kinetic analysis reveals a fourfold improvement of kcat/KmA for disproportionation and an 85.5% reduction in kcat/Km for cyclization after mutagenesis. Binding free energy analysis further confirms that the mutagenesis favors the binding of hesperidin, and destabilizes the binding of cyclodextrin.

Ene-reductase (ER) has been widely applied for asymmetrical synthesis of chiral intermediates due to its substrate promiscuity, photoexcited reactivity, and excellent property with producing two chiral centers at a time. Natural ERs often exhibit the same stereoselectivity, and they need to be engineered for opposite configuration of chiral compounds. The hydrogenation process toward activated alkenes by ERs is composed of reductive half reaction and oxidative half reaction, which are dependent upon two cofactors NAD(P)H and flavin mononucleotide. The catalytic activity of ERs will be affected by the size of the substrate, the activating strength of the electron-withdrawing groups, redox potential of cofactors, and the loop flexibility around catalytic cavity. Currently, protein engineering to ERs has been successfully employed to enhance various catalytic properties, including photoexcited asymmetric synthesis. This review summarizes the approaches to reverse the stereoselectivity and enhance catalytic activity of ERs and new applications of the engineered ERs in photobiocatalytic asymmetric synthesis, besides the discussion with the existing molecular mechanisms of mutants regarding the improved catalytic performance.