Journal: Synthetic and Systems Biotechnology

Article Title: Functional characterization of four glycosyltransferases for biosynthesis of steroidal saponins in medicinal plant Paris polyphylla

doi: 10.1016/j.synbio.2026.04.002

Figure Lengend Snippet: Reaction schemes and LC-MS analysis of Pp UGTs reactions with diosgenin and pennogenin . ( a ) Biosynthesis of trillin mediated by UGT91BP2 and UGT703R1–3 through transferring glucose from UDP-glucose to the C-3 OH position of diosgenin. ( b ) Extracted ion chromatograms (EICs) of the in vitro enzyme activity assays of recombinant UGT91BP2 and UGT703R1–3 with UDP-glucose and diosgenin showing the formation of trillin, as compared to the authentic trillin standard. Control is the empty expression vector. ( c ) MS spectra of the enzymatic reaction products in hydrogen and sodium ion adducts compared to the fragmentation pattern of the trillin standard. The product molecular ions [M + H] + and [M +Na] + and the feature fragment ion [M + H − Glc] + with m / z 415.32 are marked. Of note, additional peaks observed in the diosgenin EIC ( m / z 415.32) for UGT703R1–3 might correspond to substrate isomeric impurities, in-source adducts, or minor non-enzymatic by-products; these peaks, which arose from the extracted ion flow of the substrate m / z , did not co-elute with the product, trillin , and therefore did not affect the interpretation of the product formation. ( d ) Biosynthesis of pennogenin 3- O -glucoside mediated by the UGT703R1–3 through transferring glucose from UDP-glucose to the C-3 OH position of pennogenin. ( e ) EICs of the in vitro enzyme activity assays of recombinant UGT91BP2 and UGT703R1–3 with UDP-glucose and pennogenin showing the formation of pennogenin 3- O -glucoside, compared to the control empty expression vector. ( f ) MS/MS spectra ([M + H – Glc] + and [M + H – Glc − H 2 O] + ) of the enzymatic reaction products.

Article Snippet: Diosgenin, pennogenin, and trillin standard compounds were purchased from Chengdu Push Biotechnology Co., Ltd., and Shanghai Yuanye Biotechnology Co., Ltd. (China).

Techniques: Liquid Chromatography with Mass Spectroscopy, Transferring, In Vitro, Activity Assay, Recombinant, Control, Expressing, Plasmid Preparation, Tandem Mass Spectroscopy

Journal: Synthetic and Systems Biotechnology

Article Title: Functional characterization of four glycosyltransferases for biosynthesis of steroidal saponins in medicinal plant Paris polyphylla

doi: 10.1016/j.synbio.2026.04.002

Figure Lengend Snippet: Transient expression and characterization of Pp UGTs in Nicotiana benthamiana . ( a ) UHPLC-MS traces of the trillin formation in N. benthamiana leaf extracts transiently expressing UGT91BP2 and UGT703R1–3 with the infiltration of diosgenin substrate, compared to trillin authentic standard and empty vector control. Q1: parent ion; Q3: daughter ion. ( b ) Confocal microscopic images of the localization of UGT91BP2-GFP, UGT703R1-GFP, UGT703R2-GFP and UGT703R3-GFP in N. benthamiana leaves. DAPI (a nuclear-specific fluorescence dye) acts as a nucleus indicator. Scale bars, 20 μm. The experiments were repeated two times with similar results.

Article Snippet: Diosgenin, pennogenin, and trillin standard compounds were purchased from Chengdu Push Biotechnology Co., Ltd., and Shanghai Yuanye Biotechnology Co., Ltd. (China).

Techniques: Expressing, Plasmid Preparation, Control, Fluorescence

Journal: Synthetic and Systems Biotechnology

Article Title: Reconstructing the hydrogenobyrinic acid synthetic toolkit by combining cell-free systems and metabolic engineering

doi: 10.1016/j.synbio.2026.01.012

Figure Lengend Snippet: Screening of bottleneck reaction steps in the 1w–SmHBA operon by multienzyme catalysis. a, Schematic illustration of the reaction components. The in vitro reaction system consisted of crude cell extracts (CCE) from strains harboring the 1w–SmHBA plasmid as the basic catalytic source, supplemented with multienzyme-expressing strains carrying plasmids encoding 4–5, 2–3, or single heterologously expressed Cob enzymes to alter the enzyme composition in each reaction. b, Comparison of in vitro HBA production using CCE from strains H1, H2, and LvH0. Two-sided unpaired t -test is carried out between H1, H2, and LvH0. Unpaired t -test of data: H1 to H2, ∗∗∗∗, P < 0.0001 (t = 35.12); LvH0 to H2, ∗∗∗∗, P < 0.0001 (t = 30.84). c, In vitro HBA production using CCE from H21–H28 strains, each harboring an additional RcCob enzyme. Red bars indicate values higher than those of the H2 reactant, whereas blue bars indicate values lower than those of H2 reactant. Two-sided unpaired t -test is carried out between H1 to H21-28. Unpaired t -test of data:H2 to H21, ∗, P = 0.0227 (t = 3.605); H24 to H2, ∗∗∗∗, P < 0.0001 (t = 24.60); H27 to H2, ∗, P = 0.5363 (t = 0.6757). d, In vitro HBA production using CCE from H37–H44 strains, in which SmCob enzymes were replaced with the corresponding RcCob enzymes. Two-sided unpaired t -test is carried out between H1 to H37-44. Unpaired t -test of data:H37 to H2, ∗, P = 0.0145 (t = 4.129); H41 to H2, ∗, P = 0.0315 (t = 3.246); H42 to H2, ∗∗∗, P = 0.0003 (t = 11.66); H43 to H2, ∗, P = 0.0229 (t = 3.592); H44 to H2, ∗∗∗∗, P < 0.0001 (t = 15.76). e, Screening of reactions supplemented with crude cell extracts from strains heterologously expressing 4–5 Cob enzymes. HBA-A: CCE with pET28a–CobAIGJM; HBA-B: CCE with pACYCDuet-1–CobFKLH. Unpaired t -test of data: HBA-A 45 OD 600 to Control in HBA titer, ns, P = 0.0729 (t = 2.418); HBA-B 45 OD 600 to Control in HBA titer, ∗∗, P = 0.0029 (t = 6.502); HBA-A 45 OD 600 to Control in Urogen III titer, ∗∗, P = 0.0022 (t = 7.002); HBA-B 45 OD 600 to Control in Urogen III titer, ∗∗, P = 0.0011 (t = 8.309); f, Screening of reactions supplemented with crude cell extracts from strains heterologously expressing 2–3 Cob enzymes. AIG: CCE with pet28a-CobAIG; JM: CCE with pet28a-CobJM; FK: CCE with pet28a-CobFK; LH: CCE with pet28a-CobLH. Unpaired t -test of data: AIG 30 OD600 to Control in Urogen III titer, ∗∗∗, P = 0.0009 (t = 8.801); JM 30 OD600 to Control in Urogen III titer, ∗∗∗∗, P < 0.0001 (t = 24.39); FK 30 OD600 to Control in Urogen III titer, ∗, P = 0.0304 (t = 3.285); LH 30 OD600 to Control in Urogen III titer, ∗∗∗∗, P < 0.0001 (t = 15.93); g, Screening of reactions supplemented with CCE from strains heterologously expressing a single Cob enzyme. Unpaired t -test of data: CobA + to ori, ∗∗∗, P = 0.0002 (t = 13.95); CobI + to ori, ∗∗∗, P = 0.0005 (t = 10.57); CobG + to ori, ∗∗∗, P = 0.0003 (t = 12.00); CobJ + to ori, ∗∗∗, P = 0.0001 (t = 15.48); CobM + to ori, ∗∗∗, P = 0.0005 (t = 10.46); CobF + to ori, ∗∗∗∗, P < 0.0001 (t = 16.42); CobK + to ori, ∗∗∗, P = 0.0006 (t = 9.873); CobL + to ori, ∗∗, P = 0.0042 (t = 5.882); CobH + to ori, ∗∗∗, P = 0.001 (t = 8.675).

Article Snippet: A representative example is the HBA synthetic pathway: after the porphyrin ring compound Urogen III is assembled, nine successive enzymatic reactions modify the macrocycle by introducing eight methyl groups and removing a methylene bridge, thereby altering its symmetry.

Techniques: In Vitro, Plasmid Preparation, Expressing, Comparison, Control

Journal: Synthetic and Systems Biotechnology

Article Title: Reconstructing the hydrogenobyrinic acid synthetic toolkit by combining cell-free systems and metabolic engineering

doi: 10.1016/j.synbio.2026.01.012

Figure Lengend Snippet: Optimization of the artificial HBA operon. a, In vitro HBA production using CCE from various strains under different modifications. promoter replacement (H2 vs. H46 and H47, trc or tac promoters): Two-sided unpaired t -test is carried out between H2 to H46 and H47. Unpaired t -test of data: H46 to H2, ∗∗∗∗, P < 0.0001 (t = 23.77); H47 to H2, ∗∗∗∗, P < 0.0001 (t = 33.93); chassis variation (H2 vs. H48–H52, different E. coli strains harboring the SmHBA plasmid): Two-sided unpaired t -test is carried out between H2 to H48-52, but no significant differences were observed; culture medium effects (H2 grown in different media): Unpaired t -test of data: 2YT to LB, ∗, P = 0.0168 (t = 3.954); and operon composition (H2 vs. H53 and H54, carrying the FKLHIGJM or FKLH + operon): Two-sided unpaired t -test is carried out between H2 to H53 and H54. Unpaired t -test of data: H53 to H2, ∗∗∗∗, P < 0.0001 (t = 44.32). b, Schematic illustration of the ASmHBA (H2 strain), FKLHIGJM (H53 strain), and FKLH + (H54 strain) operons. c, In vitro HBA synthesis using CCEs from H57 cultivated in 2YT medium, supplemented with gradient amounts of either CCE or CobA enzyme. d. Chromatogram of synthetic HBA in CCE3 group, CCE1 group in (c) with standards. UroIII-STD, uroporphyrinogen III standard; HBA-STD, hydrogenobyrinate acid standard; CCE3 and CCE1, reactants using 1-fold H57 CCE and 3-fold H57 CCE in (c).

Article Snippet: A representative example is the HBA synthetic pathway: after the porphyrin ring compound Urogen III is assembled, nine successive enzymatic reactions modify the macrocycle by introducing eight methyl groups and removing a methylene bridge, thereby altering its symmetry.

Techniques: In Vitro, Plasmid Preparation

Journal: Synthetic and Systems Biotechnology

Article Title: Reconstructing the hydrogenobyrinic acid synthetic toolkit by combining cell-free systems and metabolic engineering

doi: 10.1016/j.synbio.2026.01.012

Figure Lengend Snippet: Optimization of the HBA synthetic system. a, HBA titers obtained from screening single Cob enzyme supplementation in the H53∗ CCE reaction: the control H53 CCE reaction, H53–CobA + (H53 CCE with 4 g/L CobA added), and H53–CCE + (H53 CCE with threefold CCE input). ∗ indicates that H53 CCE was prepared from cultures grown in LB medium, whereas unmarked H53 CCE was prepared from cultures grown in 2YT medium. Statistical Significance without bracket denotes unpaired t -test comparisons between each supplemented group (A+, I+, G+, J+, M+, F+, K+, L+, H+) and the corresponding Ori group under the same CCE introduction condition. Unpaired t -test of data: H53-CobA + to H53∗, ∗∗∗, P = 0.0004 (t = 10.67), H53-CCE + to H53∗, ∗∗∗∗, P < 0.0001 (t = 31.05). b, Urogen III accumulation under the same conditions as in (a). Unpaired t -test of data: H53-CobA + to H53∗, ns, P = 0.2509 (t = 1.341), H53-CCE + to H53∗, ∗∗∗∗, P < 0.0001 (t = 61.82). c, Schematic illustration of HBA biosynthesis with SAM supplementation. Abbreviation: 5-ALA, 5-Aminolevulinate; PBG, porphobilinogen; HMB, hydroxymethylbilane; L-Met, l -methionine; SAM, S-adenosyl- l -methionine; SAH, S-adenosyl- l -homocysteine; SRH, S-ribosyl- l -homocysteine. d, Orthogonal combinations of PpK, MetK, and MtnN enzymes used to enhance HBA production via SAM supplementation. The color intensity of the bar corresponds to the magnitude of the values for enhanced visual clarity. e, HBA titers in the optimized SAM supplementation system by adjusting ATP synthesis through varying AMP and SHMP inputs.

Article Snippet: A representative example is the HBA synthetic pathway: after the porphyrin ring compound Urogen III is assembled, nine successive enzymatic reactions modify the macrocycle by introducing eight methyl groups and removing a methylene bridge, thereby altering its symmetry.

Techniques: Control

Journal: International Journal for Parasitology: Drugs and Drug Resistance

Article Title: In vitro and in vivo anti-piroplasm activity of PBT2 against Theileria annulata and Babesia microti

doi: 10.1016/j.ijpddr.2026.100646

Figure Lengend Snippet: Structures of PBT2 (A) and PBT2 reduce the mRNA levels of TaSP and Tap104 (B). Error bars represent the SD of the mean from three independent biological replicates. ns, P > 0.05; ∗∗∗∗, P < 0.0001 by two-way ANOVA followed by Dunnett's multiple comparisons test.

Article Snippet: The compounds PBT2 ( A), Bup and Diminazene aceturate (DA) were purchased from MedChemExpress (MCE) and stored as a 10 mM stock solution in DMSO (Macklin).

Techniques:

Journal: International Journal for Parasitology: Drugs and Drug Resistance

Article Title: In vitro and in vivo anti-piroplasm activity of PBT2 against Theileria annulata and Babesia microti

doi: 10.1016/j.ijpddr.2026.100646

Figure Lengend Snippet: PBT2 reduces the schizont burden in T. annulata -infected cells. Representative immunofluorescence images of TaNM and TaXJS cells in each treatment group, schizont nuclei are shown in green and host cell nuclei in blue (A). Quantification of the number of schizonts per host cell in each treatment group (B). Error bars represent the SD of the mean from three independent biological replicates. ns, P > 0.05; ∗∗∗∗, P < 0.0001 by two-way ANOVA followed by Dunnett's multiple comparisons test.

Article Snippet: The compounds PBT2 ( A), Bup and Diminazene aceturate (DA) were purchased from MedChemExpress (MCE) and stored as a 10 mM stock solution in DMSO (Macklin).

Techniques: Infection, Immunofluorescence

Journal: International Journal for Parasitology: Drugs and Drug Resistance

Article Title: In vitro and in vivo anti-piroplasm activity of PBT2 against Theileria annulata and Babesia microti

doi: 10.1016/j.ijpddr.2026.100646

Figure Lengend Snippet: PBT2 inhibits the proliferation of TaNM and TaXJS cells in vitro. The IC 50 values of PBT2 on TaNM and TaXJS cells (A). The cell viability of BoMac cells with different concentration of PBT2 (B). Colony formation assay demonstrated that PBT2 completely abolished clonogenic growth, representative images of colony formation are shown (C). Quantification of colony numbers before and after treatment with PBT2 or Bup (D). The values obtained were used to determine the IC 50 using nonlinear regression (curve fitting analysis) in GraphPad Prism software. Error bars represent the SD of the mean from three independent biological replicates. ns, P > 0.05; ∗∗∗∗, P < 0.0001 by two-way ANOVA followed by Dunnett's multiple comparisons test.

Article Snippet: The compounds PBT2 ( A), Bup and Diminazene aceturate (DA) were purchased from MedChemExpress (MCE) and stored as a 10 mM stock solution in DMSO (Macklin).

Techniques: In Vitro, Concentration Assay, Colony Assay, Software

Journal: International Journal for Parasitology: Drugs and Drug Resistance

Article Title: In vitro and in vivo anti-piroplasm activity of PBT2 against Theileria annulata and Babesia microti

doi: 10.1016/j.ijpddr.2026.100646

Figure Lengend Snippet: Metal ion content in T. annulata schizonts following PBT2 treatment. Error bars represent the SD of the mean from three independent biological replicates. ns, P > 0.05; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001 by one-way ANOVA followed by Dunnett's multiple comparisons test.

Article Snippet: The compounds PBT2 ( A), Bup and Diminazene aceturate (DA) were purchased from MedChemExpress (MCE) and stored as a 10 mM stock solution in DMSO (Macklin).

Techniques:

Journal: International Journal for Parasitology: Drugs and Drug Resistance

Article Title: In vitro and in vivo anti-piroplasm activity of PBT2 against Theileria annulata and Babesia microti

doi: 10.1016/j.ijpddr.2026.100646

Figure Lengend Snippet: PBT2 disrupts redox homeostasis and reduces SOD activity in infected cells. ROS levels in TaNM cells following PBT2 treatment were quantified by flow cytometry, representative flow cytometry histograms are shown (A). Quantitative analysis of ROS levels (B). SOD activity in TaNM cells after PBT2 exposure, showing a significant reduction compared with untreated controls (C). Manganese supplementation partially rescued PBT2-induced growth inhibition in TaNM cells, indicating functional involvement of Mn depletion in redox imbalance (D). Error bars represent the SD of the mean from three independent biological replicates. ns, P > 0.05; ∗, P < 0.05; ∗∗, P < 0.005; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001 by one-way ANOVA followed by Tukey's multiple comparisons test.

Article Snippet: The compounds PBT2 ( A), Bup and Diminazene aceturate (DA) were purchased from MedChemExpress (MCE) and stored as a 10 mM stock solution in DMSO (Macklin).

Techniques: Activity Assay, Infection, Flow Cytometry, Inhibition, Functional Assay

Journal: International Journal for Parasitology: Drugs and Drug Resistance

Article Title: In vitro and in vivo anti-piroplasm activity of PBT2 against Theileria annulata and Babesia microti

doi: 10.1016/j.ijpddr.2026.100646

Figure Lengend Snippet: The growth inhibition of PBT2 on B. microti in vivo. Parasitemia was calculated by counting infected RBCs among 2000 R BCs using Giemsa-stained thin blood smears. The arrow indicates 5 consecutive days of treatment. Error bars represent the SD of the mean from five independent biological replicates, ∗, P < 0.05 by two-way ANOVA followed by Dunnett's multiple comparisons test (A). Representative Giemsa-stained blood smear image obtained at peak parasitemia on the 8th day (B).

Article Snippet: The compounds PBT2 ( A), Bup and Diminazene aceturate (DA) were purchased from MedChemExpress (MCE) and stored as a 10 mM stock solution in DMSO (Macklin).

Techniques: Inhibition, In Vivo, Infection, Staining

Journal: International Journal for Parasitology: Drugs and Drug Resistance

Article Title: In vitro and in vivo anti-piroplasm activity of PBT2 against Theileria annulata and Babesia microti

doi: 10.1016/j.ijpddr.2026.100646

Figure Lengend Snippet: Hematology profiles of PBT2-treated mice in vivo. The changes of RBCs (A), HGB (B), and HCT (C) in mice treated with PBT2 and DA. Error bars represent the SD of the mean from five independent biological replicates. Different colored asterisks indicate that the differences between the various groups are statistically significant. ∗, P < 0.05; ∗∗, P < 0.005; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001 by two-way ANOVA followed by Dunnett's multiple comparisons test.

Article Snippet: The compounds PBT2 ( A), Bup and Diminazene aceturate (DA) were purchased from MedChemExpress (MCE) and stored as a 10 mM stock solution in DMSO (Macklin).

Techniques: In Vivo