Epigallocatechin gallate (EGCG), the most abundant and bioactive catechin in green tea, has limited applications due to its chemical instability, low solubility, and poor bioavailability. Enzymatic glycosylation offers a sustainable method to improve these properties. However, the regioselectivity and catalytic efficiency of enzymatic glycosylation remain challenging. In the present study, a loop-exchanged variant of sucrose phosphorylase from Streptococcus mutans (SmSP), designated SmSP_LoopB, shifted the major product from EGCG-4'-O-α-d-glucopyranoside to EGCG-4″-O-α-d-glucopyranoside. The SmSP_LoopB I235S/V297A mutant, generated through a tailored, tunnel-focused mutagenesis strategy, exhibited a 140.5-fold increase in catalytic efficiency compared to SmSP_LoopB. Under optimized reaction conditions (15 g/L EGCG, 336 g/L sucrose, 2 U/mL enzyme), the yield of EGCG-4″-O-α-d-glucopyranoside reached 83.3% (16.9 g/L) within 4 h. The mutant also demonstrated excellent performance and stability in the whole-cell biocatalytic system. This work establishes a synergistic engineering approach that enhances specificity and efficiency, enabling the scalable production of well-defined EGCG glycosides.

The major green tea polyphenol, epigallocatechin gallate (EGCG), has beneficial antioxidant and anti-inflammatory activities but suffers from poor solubility and stability. Its monoglycosylated derivative, (-)-epigallocatechin gallate 4'-O-α-d-glucopyranoside (EGCG-G1), partially overcomes these limitations. In this study, we engineered Leuconostoc mesenteroides sucrose phosphorylase (LmSPase) for efficient EGCG-G1 production. The triple mutant M3 (T219L/E393I/N335G), created via loop engineering, consensus design, and Rosetta Dock design, exhibited 4.09-fold higher transglycosylase activity at 30 °C, a 1.69-fold longer half-life at 45 °C, and significantly improved regioselectivity compared to the wild type. In a fed-batch reaction at 30 °C and pH 6.0, 25 g/L EGCG was converted within 24 h, producing 31.11 g/L (91.92% yield) of EGCG-G1 with 87.27% purity. This semirational design strategy enhanced the key properties of LmSPase and provides an effective biocatalyst for EGCG-G1 production.

The consumption of a high-sugar diet has been widely associated with various health complications. Due to its low caloric content, favorable safety profile, and high sweetness, rubusoside has been recognized as a promising alternative to sucrose and synthetic sweeteners. In this study, we report a β-glucosidase from Bacteroides thetaiotaomicron (BtBGL), which specifically hydrolyzes the β-1,2-glucosidic bond in the sophorosyl disaccharide at the C-13 hydroxyl group of stevioside to produce rubusoside. The M98V/Y718F mutant of BtBGL was obtained through consensus design and site-directed mutagenesis. Compared to the wild-type BtBGL, the optimal temperature of the M98V/Y718F mutant increased from 45 °C to 50 °C, demonstrating improved thermostability at this temperature. Additionally, the optimal pH decreased from 6.0 to 5.5, while enzyme activity at pH 5.5 increased from 61.17 U/mg (at pH 6.0) to 83.34 U/mg. This shift in optimal pH remains consistent when stevioside is used as the substrate. The increased surface charge, hydrophobicity, and hydrogen bonding in this double-point mutant may contribute to its enhanced activity at the altered pH and improvement in thermal stability. Stevioside (200 g/L) can be completely converted by the M98V/Y718F mutant within 1 h, and the highest rubusoside yields of 80

With the growing health concern on sugar consumption, the high sweetness and low calorie natural sweeteners, such as steviol glycosides, mogrosides, glycyrrhizin, and trilobatin, offer a viable alternative. Glycosylation is an effective means to enhance the functional properties of natural sweeteners. UDP-glycosyltransferases (UGTs), key enzymes that catalyze transglycosylation reactions, play a crucial role in the glycosylation of natural sweeteners. However, natural UGTs often face limitations, such as low activity, narrow substrate specificity, and undesirable regioselectivity, which hinder their applications in industrial-scale production of glycosylated natural sweeteners. This review summarizes the advancements in omics-based approaches, as well as machine learning tools for mining and discovering novel UGTs. Subsequent protein engineering methods have successfully optimized UGTs performance, resulting in enhanced activity, higher stability, improved regioselectivity, and broader substrate promiscuity. Finally, the applications of engineered UGTs in the synthesis of high-value natural sweeteners are also discussed. From our perspective, the discovery and engineering of UGTs present a promising strategy for developing new natural sweeteners.

Sucrose synthase (SuSy) is a unique glycosyltransferase that can be utilized in the production of nucleoside monosaccharides, such as diphosphate (UDP)-glucose, which serve as essential sugar donors for the glycosylation reactions catalyzed by UDP-dependent glycosyltransferases (UGTs). The selection of an appropriate SuSy coupled with a UGT is crucial for achieving the efficient synthesis of glycoside products. In this study, three candidate SuSy genes were identified from the transcriptome sequencing of Stevia rebaudiana, among which SrSUS1 was found to be expressed and active in Escherichia coli. The optimal temperature and pH for SrSUS1 were determined to be 55°C and pH 7.0, respectively. A variant SrSUS1T49A/L90P/V104E was generated based on a consensus sequence strategy, exhibiting a 3.6-fold increase in activity and the enhanced affinity for sucrose (Km = 52.32 mM), as well as the improved thermal stability and catalytic efficiency. By coupling SrSUS1T49A/L90P/V104E with glycosyltransferase UGTAn85Q23E/N65D or UGT76G4, respectively, the production of 163.32 mM (43.63 g/L) of 2-phenylethyl-β-D-pyranoside and 72.29 mM (93.35 g/L) of rebaudioside M was achieved within 24 h in one-pot, two-enzyme fed-batch reactions. This study provides new insights into plant-derived SuSys and presents a promising biocatalyst for industrial glycosylation applications.

Pyrrosia calvata is a traditional Chinese medicinal fern endemic to Guangxi, China. However, its phytochemical profile remains largely unexplored, and the comprehensive characterization of its chemical constituents is still lacking. In this study, the ethanolic crude extract of P. calvata was analyzed using ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) in both negative and positive ionization modes, employing a data-dependent acquisition (DDA) strategy. The acquired data were processed using MS-DIAL and further analyzed through the Feature-Based Molecular Networking (FBMN) workflow on the Global Natural Products Social (GNPS) platform to facilitate the discovery and annotation of both known and putatively novel natural products. As a result, fourteen compounds were identified or tentatively characterized, with flavonoids representing the predominant chemical class. Notably, eleven of these compounds were reported for the first time from P. calvata. The results demonstrate the effectiveness of FBMN-assisted metabolomics in expanding the chemical coverage of trace constituents and provide a foundation for future targeted isolation and pharmacological evaluation of bioactive compounds from this medicinal fern.

Ginsenoside Rh2 (3-β-O-Glc-protopanaxadiol) and Rg3 (20-β-O-Glc-3-β-O-Glc-protopanaxadiol) are rare tetracyclic triterpenoids known for their significant medicinal properties, including antitumor and anti-inflammatory activities. The glycosylation of the C3-OH and C20-OH groups of protopanaxadiol (PPD) by UDP-glycosyltransferase (UGT) is a crucial biological modification that contributes to the extensive structural and functional diversity of PPD-type ginsenosides. In this study, we identified a 3-O-glycosyltransferase (GuUGT73F15) from Glycyrrhiza uralensis, which specifically glycosylates the C3-OH of PPD to produce ginsenoside Rh2. Through site-directed mutagenesis and combinatorial active-site engineering, an optimized variant, GuUGT73F15H47P/R84K/N211T, exhibiting a 2.65-fold increase in catalytic efficiency (kcat/Km) for PPD conversion and a remarkable 26.7-fold extension in half-life (t1/2 = 292.47 min at 37 °C) compared to the wild-type enzyme. Molecular docking and dynamics simulations provided mechanistic insights into the enhanced activity and thermostability of the enzyme. Furthermore, this variant was combined with a glycosyltransferase variant (UGT29R91M/D184M/A287V/A342L) and sucrose synthases, resulting in a high-level production of Rg3 (19.88 mM, 15.61 g/L) in a 24-h fed-batch reaction using PPD as the substrate. This study presents an efficient and thermostable O-glycosyltransferase for the targeted biosynthesis of PPD-type ginsenosides.

Uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) play important roles in plant growth and development. As an important branch of plant UGTs, the UGT73 family participates in secondary metabolism, hormone regulation, and stress responses. Studies have shown that this family is involved in the synthesis of flavonoids, terpenoids, and other substances as well as the regulation of hormone homeostasis through precise glycosylation modifications. This review has collated the relevant properties of the plant UGT73 family in recent years and aimed to (1) analyze the structural characteristics of UGT73 family glycosyltransferase genes in different plant species; (2) outline the substrate specificity, catalytic sites, and mechanisms of UGT73 family glycosyltransferases; and (3) elaborate on their notable roles in growth and development, hormone regulation, and stress resistance. In-depth investigations are required to analyze the catalytic structure of the UGT73 family, complex regulatory networks, and interspecific functional differences. Future studies should combine multi-omic and synthetic biology technologies to explore new functions of the UGT73 family, thereby providing theoretical support and practical guidance for the development of plant metabolic engineering and green biotechnology.

D-allulose is a rare sugar with promising applications in food and health industries, owing to its low caloric value and multiple health benefits. In this study, we systematically investigated a thermostable D-allulose 3-epimerase (TcDAEase) from Thermogemmatispora carboxidivorans for food-compatible continuous production. The enzyme exhibited remarkable thermostability, with over 70% activity retained at 80 °C, and showed broad pH tolerance across the range of 8.0 to 13.0. Notably, TcDAEase exhibited high catalytic activity toward D-allulose and D-fructose even without the addition of metal ions. Moreover, food-grade Mg2+ was identified as enhancing enzyme activity by 14.3%, thus ensuring compliance with Generally Recognized as Safe (GRAS) standards for food applications. To improve industrial applicability, the enzyme was immobilized using a chitosan-diatomaceous earth (DE) matrix via three-step adsorption–crosslinking–embedding strategy. The immobilized TcDAEase achieved 48.7% ± 2.4% activity recovery and retained 90.3% ± 1.5% activity over seven reaction cycles. Furthermore, continuous production of D-allulose was realized in a packed-bed reactor, operating stably at 60 °C, pH 8.0 and 5 mM Mg2+ for 150 days, producing 756 kg of D-allulose with a conversion yield exceeding 89.7% of the theoretical maximum. Overall, this study provides a feasible strategy for the continuous and efficient production of high-value-added D-allulose in the food industry.

Plant pathogenic fungi are seriously threatening agricultural production. There is an urgent need to develop novel fungicides with low toxicity and high efficiency. In this study, we synthesized 26 oxime ester derivatives of matrine and systematically evaluated their antifungicidal activities toward seven common phytopathogenic fungi. The in vitro assay against Sclerotinia sclerotiorum (S. sclerotiorum) showed that the EC50 value of compound 5-11 (EC50: 7.2 μg/mL) is almost 4 times lower than that of azoxystrobin (EC50: 28.3 μg/mL). Preliminary fungicidal research indicated that compound 5-11 has a certain destructive effect on the hyphae of S. sclerotiorum. Furthermore, molecular docking and molecular dynamics simulation analysis demonstrated that the potent insecticidal activity of 5-11 is likely mediated by its specific interactions with SDH primarily through the π-cation interaction and the hydrophobic interactions with key residues. This work lays a foundation for the application of matrix derivatives in plant protection.

Phenylethanoid glycosides (PhGs) are glycoconjugates composed of phenylethyl alcohols and glycoside moieties, exhibiting diverse bioactivities. 2-Phenylethyl-β-d-glucopyranoside (2PE-Glc) is the simplest form of PhGs. We successfully designed and implemented a novel multienzyme cascade system with cofactor self-recycling capabilities for the efficient synthesis of 2PE-Glc from l-phenylalanine (L-Phe). First, different phenylalanine dehydrogenases involved in the cascade system were compared, and LsPheDHV33A/A206D/L283F was selected for the deamination reaction. Subsequently, the rate-limiting step was determined to be phenylacetaldehyde reduction, and PmADH was screened out and evolved by consensus design, leading to a mutant PmADHI32V, which exhibited 1.74 times the activity of the wild type. Finally, five enzymes were coexpressed in E. coli BL21 (DE3) to construct a multienzyme cascade system, and the reaction conditions of conversion of l-Phe to 2PE-Glc catalyzed by the multienzyme cascade system were optimized. Under the optimal reaction conditions, the yield of 2PE-Glc reached 27.62 mM (7.85 g/L) in a fed-batch reaction. This work not only provides an effective strategy for the biosynthesis of 2PE-Glc but also demonstrates the potential of the multienzyme cascade system in the synthesis of biologically active complex glycoconjugates.

Traditional metal-organic frameworks (MOFs) preparation is generally time-consuming, polluting, and lacking specificity for enzyme immobilization. This paper introduced a facile, rapid, and green method to produce three MOFs subsequently employed to purify and coimmobilize recombinant glycosyltransferase (UGT) and recombinant sucrose synthetase (SUSy) using histidine tag (His-tag) for the specific adsorption of Ni2+ and Co2+ from MOFs. This method simplified enzyme purification from crude extracts and enabled enzymes to be reused. The results demonstrated that NiCo-MOF exhibited a higher enzyme load (115.9 mg/g) than monometallic MOFs. Additionally, the NiCo-MOF@UGT&SUSy demonstrated excellent stability and efficiently produced the rare ginsenoside Rh2 by catalyzing a coupling reaction (95.6 μg/mL), solving the problem of the substrate cost of uridine diphosphate glucose (UDPG). The NiCo-MOF@UGT&SUSy retained 68.97% of the initial activity after 10 cycles. Finally, molecular docking studies elucidated the conversion mechanism of the target product Rh2. This technique is important in the industrialization of ginsenoside production and enzyme purification.

Phenylethanoid glycosides (PhGs) are naturally occurring glycosides derived from plants with various biological activities. Glycosyltransferases catalyze the production of PhGs from phenylethanols via a transglycosylation reaction. The low activity and stability of glycosyltransferase limit its industrial application. An ancestral glycosyltransferase, UGTAn85, with heat resistance, alkali resistance, and high stability was resurrected using ancestral sequence reconstruction technology. This enzyme can efficiently convert phenylethanols to PhGs. The optimal reaction temperature and pH for UGTAn85 were found to be 70 °C and pH 10.0, respectively. This study employed a combination of structure-guided rational design and co-evolution analysis to enhance its catalytic activity. Potential mutation sites were identified through computer-aided design, including homology modeling, molecular docking, Rosetta dock design, molecular dynamics simulation, and co-evolution analysis. By targeted mutagenesis, the UGTAn85 mutant Q23E/N65D exhibited a 2.2-fold increase in enzyme activity (11.85 U/mg) and elevated affinity (Km = 0.11 mM) for 2-phenylethanol compared to UGTAn85. Following a fed-batch reaction, 36.16 g/L 2-phenylethyl-β-d-glucopyranoside and 51.49 g/L salidroside could be produced within 24 h, respectively. The findings in this study provide a new perspective on enhancing the stability and activity of glycosyltransferases, as well as a potential biocatalyst for the industrial production of PhGs.

The oxidation of long-chain fatty alcohols with Jones reagent to prepare long-chain fatty acids is an efficient and economical method. The by-product of chromium sulfate is electrolyzed and oxidized to hexavalent chromium, which is recycled to avoid the emission of toxic by-products. In this article, the process conditions for the preparation of carboxylic acid by chromium reagent oxidizing alcohol are optimized, and the effects of various reaction conditions on the experiment are explored. Innovatively, the reaction was optimized by reverse addition. The two synthesis methods are compared by orthogonal experiments, and the optimal reaction conditions for oxidation are determined: the organic solvent of alcohol is added to the acidified Jones reagent by reverse addition; the amount of chromium reagent is 1.5 times the required stoichiometry; the chromium concentration in chromium reagent is 2.5 mol.L-1; the reaction temperature was lower than 30 degrees C and the reaction time was 4 h. Under the optimized reaction conditions, the formation of by-products was effectively inhibited, and the conversion rate of alcohol oxidation to carboxylic acid was increased from 80% to more than 90%.

A distinct protocol for secondary amide formation through a catalyst- and additive-free cross-coupling of isocyanates with triarylboranes has been described.

An organo-photoredox catalyzed gem-difluoroallylation of glycine with alpha-trifluoromethyl alkenes via direct C(sp(3))-H functionalization of glycine and C-F bond activation of alpha-trifluoromethyl alkenes has been described. As a consequence, a broad range of gem-difluoroalkene-containing unnatural amino acids are afforded in moderate to excellent yields. This reaction exhibits multiple merits such as readily available starting materials, broad substrate scope, and mild reaction conditions. The feasibility of this reaction has been highlighted by the late-stage modification of several peptides as well as the improved in vitro antifungal activity of compound 3v toward Valsa mali compared to that with commercial azoxystrobin.

Alcohols are the most commercially abundant, synthetically versatile and operationally convenient functional groups in organic chemistry. Therefore, a strategy that utilizes hydroxy-containing compounds to develop novel bond disconnection and formation process would achieve molecular diversity. Herein, a deconstructive strategy for the generation of quinoxalin-2(1H)-one derivatives has been developed from alcohol precursors via oxy-radical-induced β-fragmentation. Additionally, 1,5-HAT and deoxygenation by P(III) along with oxy-radical were demonstrated as alternative pathways for this transformation. Furthermore, with the deep-seated reorganization of a few terpenes carbon framework, a unique activity with inhibition against the growth of pathogenic fungi was observed.

A straightforward method for the phosphorylation of electron-deficient alkenes and aryl alkynes has been developed, leading to C(sp3)-P and C(sp2)-P bond formation. This process involves the generation of phosphorus radical cation intermediates through the photocatalyzed oxidation of ethyl diarylphosphinites. The coupling with electron-deficient alkenes encompasses a variety of heteroaromatics, including pyridine, (benzo)thiazole, and benzoxazole, as well as α,β-unsaturated esters and amides. Impressively, the coupling of radical cations with aryl alkynes demonstrated remarkable regioselectivity, thereby facilitating the synthesis of rare α-aryl vinyl phosphine oxides.

Rare ginsenosides Rg3 and Rh2, which exhibit diverse pharmacological effects, are derivatives of protopanaxadiol (PPD). UDP-glycosyltransferases, such as the M315F variant of Bs-YjiC (Bs-YjiCm) from Bacillus subtilis and UGTPg29 from Panax ginseng, can efficiently convert PPD into Rh2 and Rh2 into Rg3, respectively. In the present study, the N178I mutation of Bs-YjiCm was introduced, resulting in an increase in Rh2 production. UDP-glycosyltransferase UGTPg29 was then engineered to improve its robustness through semi-rational design. The variant R91M/D184M/A287V/A342L, which indicated desirable stability and activity, was utilized in coupling with the N178I variant of Bs-YjiCm and sucrose synthase AtSuSy from Arabidopsis thaliana to set up a "one-pot" three-enzyme reaction for the biosynthesis of Rg3. The influential factors, including the ratio and concentration of UDP-glycosyltransferases, pH, and the concentrations of UDP, sucrose, and DMSO, were optimized. On this basis, a fed-batch strategy was adopted to achieve a Rg3 yield as high as 12.38 mM (9.72 g/L) with a final yield of 68.78% within 24 h. This work may provide promising UDP-glycosyltransferase candidates for ginsenoside biosynthesis.