Plant systems have gained increased attention as an alternative platform for producing heterologous proteins, particularly for industrially relevant proteins. The Cynara cardunculus L. flower extract is traditionally used in cheese production across Mediterranean countries due to its milk-clotting properties. To address the growing demand for plant-based milk-clotting enzymes, we investigated the heterologous production of cyprosin B (CYPB), a key milk-clotting enzyme, in Nicotiana benthamiana. We also examined the role of its plant-specific insert (PSI) domain in enzymatic activity, protein yield and subcellular localisation. Full-length CYPB and a PSI domain-deleted variant (CYPB Delta PSI) were transiently expressed in N. benthamiana leaves using agroinfiltration. Proteins were purified 9 days post-infiltration, yielding similar to 81 mg/kg (CYPB) and similar to 60 mg/kg (CYPB Delta PSI) fresh weight. CYPB Delta PSI showed higher proteolytic activity (similar to 168 IU/mg) than CYPB (similar to 57 IU/mg) and exhibited faster milk-clotting times, suggesting that PSI removal may contribute to enhanced enzymatic efficiency. However, additional factors such as altered glycosylation or localisation may also play a role. Subcellular localisation indicated that CYPB and its PSI domain targeted the vacuole and endocytic vesicles, while CYPB Delta PSI predominantly localised to the endoplasmic reticulum and tonoplast. This suggests that the PSI domain's vital role in vacuolar targeting and membrane permeabilisation ultimately influences protein yield. Our study shows N. benthamiana as a scalable platform for producing recombinant CYPB variants with improved enzymatic activity. It highlights the PSI domain's role in vacuolar sorting without impairing function. These findings contribute to the development of plant-based systems for milk-clotting enzymes for cheese-making.

Sesquiterpene coumarins are primarily produced by plants of the Apiaceae and Asteraceae families. Farnesylation of 7-hydroxycoumarins such as umbelliferone, scopoletin or isofraxidin yield linear 7-farnesyloxycoumarins that are converted to various cyclic sesquiterpene coumarins by sesquiterpene coumarin synthases (cyclases). The terminal double bond of the linear 7-farnesyloxycoumarins is epoxidized by a sesquiterpene coumarin epoxidase. The diverse 7-(10 ',11 '-oxidofarnesyloxy)-coumarins produced are protonated by various sesquiterpene coumarin synthases to generate a carbocation that initiates cyclization of the farnesyl moiety (A process analogous to the carbocation cascades observed with sesquiterpene synthases and other cyclases involved in the biosynthesis of additional terpene classes, such as the triterpenes). These reaction mechanisms typically include Wagner-Meerwein rearrangements, such as hydride, methyl, and other alkyl shifts, but can also involve more complex processes including Grob fragmentations. Around 260 sesquiterpene coumarins based on 7-farnesyloxycoumarins have been described, but essentially nothing is known about the biosynthetic enzymes involved, i.e., farnesyltransferase, sesquiterpene coumarin epoxidase and synthase. In this review, putative reaction pathways for formation of the carbon skeletons of all known 7-farnesyloxycoumarins-derived sesquiterpene coumarins are presented.

Self-healing carbohydrate polymers were synthesized by Diels-Alder reaction. Intermediate products and the carbohydrate matrices were characterized by Fourier transform infrared spectroscopy (FT-IR) and H-1 NMR, while the thermally reversible properties were assessed by FT-IR and differential scanning calorimetry. The mechanical properties, water absorption, and enzymatic degradation of starch/PVA/modified carbohydrate films were examined, as well as the relationship of the properties to the DA and rDA reactions. These results showed that DA bonds were introduced into the carbohydrate polymers successfully and endow the material with self-healing thermal recyclability. The mixed films exhibited alternating strong and weak mechanical properties upon cycling through the DA and rDA reactions. Water absorption was limited and the films demonstrated good water resistance. The status of the DA bonds was found not to affect the enzymatic degradation rates of the various carbohydrate films.

Verbascoside (VER) is the enzymatic hydrolysis product of echinacoside (ECH). The molecular structures of ECH and VER have different glucosyl groups so they bind to serum albumin in different ways, resulting in different pharmacological actions. In this report, we have examined the binding characteristics between human serum albumin (HSA) and ECH/VER by molecular modeling and spectroscopic approaches. Molecular modeling revealed that VER bound to HSA mainly through hydrogen bonds, van der Waals forces and hydrophobic forces. The spectroscopic results showed that the interactions between HSA and VER/ECH involved a static binding process, and the bonding strength of the VER-HSA complex was stronger than that of the ECH-HSA complex. The value of the binding distances (r) was low, which indicated the occurrence of energy transfer. The reaction conformational pattern of HSA-VER and HSA-ECH gave a "two-state model" based on fluorescent phase diagram analysis. According to the thermodynamic model, the main forces between interaction of VER and HSA were hydrogen bonds and van der Waals forces, whereas the interaction between ECH and HSA was hydrophobic force. The fluorescence polarization analysis demonstrated that the interaction between HSA and VER or ECH generated a non-covalent complex. Compared with ECH, VER was more likely to bind with HSA because of its smaller molecular size and low polarity. The results of the spectral analysis concurred with the molecular modeling data, which provides a helpful reference for the study of the molecular reaction mechanism of VER/ECH binding to HSA.

The sesquiterpene beta-caryophyllene is an ubiquitous component in many plants that has commercially been used as an aroma in cosmetics and perfumes. Recent studies have shown its potential use as a therapeutic agent and biofuel. Currently, beta-caryophyllene is isolated from large amounts of plant material. Molecular farming based on the Nicotiana benthamiana transient expression system may be used for a more sustainable production of beta-caryophyllene. In this study, a full-length cDNA of a new duplicated beta-caryophyllene synthase from Artemisia annua (AaCPS1) was isolated and functionally characterized. In order to produce beta-caryophyllene in vitro, the AaCPS1 was cloned into a plant viral-based vector pEAQ-HT. Subsequently, the plasmid was transferred into the Agrobacterium and agroinfiltrated into N. benthamiana leaves. The AaCPS1 expression was analyzed by quantitative PCR at different time points after agroinfiltration. The highest level of transcripts was observed at 9 days post infiltration (dpi). The AaCPS1 protein was extracted from the leaves at 9 dpi and purified by cobalt-nitrilotriacetate (Co-NTA) affinity chromatography using histidine tag with a yield of 89 mg kg(-1). fresh weight of leaves. The protein expression of AaCPS1 was also confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses. AaCPS1 protein uses farnesyl diphosphate (FPP) as a substrate to produce p-caryophyllene. Product identification and determination of the activity of purified AaCPS1 were done by gas chromatography-mass spectrometry (GC-MS). GC-MS results revealed that the AaCPS1 produced maximum 26.5 +/- 1 mg of P-caryophyllene per kilogram fresh weight of leaves after assaying with FPP for 6 h. Using AaCPS1 as a proof of concept, we demonstrate that N. benthamiana can be considered as an expression system for production of plant proteins that catalyze the formation of valuable chemicals for industrial applications.

New molecularly imprinted polymers (MIPs), which exhibit specific recognition of ethyl carbamate (EC) have been synthesized and studied. In this process, EC was the template molecule and -cyclodextrin derivatives were employed as functional monomers in the molecular imprinting technique (MIT). An EC molecularly imprinted sensor (EC-MIS) was prepared by using MIT surface modification. The EC-MIS was characterized by cyclic voltammetry, electrochemical impedance spectroscopy and differential pulse voltammetry. EC detection performance, binding parameters and dynamics mechanism were investigated. The result showed that the synthetic route designed was appropriate and that new MIP and EC-MIS were successfully prepared. The EC-MIS exhibited a good molecular recognition of EC. A linear relationship between current and EC concentration was observed using cyclic voltammetry and the detection limit was 5.86 g L-1. The binding constant (K = 4.75 x 10(6) L mol(-1)) between EC and the EC-MIS, as well as, the number of binding sites (n = 1.48) has been determined. The EC-MIS recognition mechanism for the EC is a two-step process. The sensor was applied for the determination of EC in Chinese yellow wines, and the results were in good agreement with the gas chromatography-mass spectrometry (GC-MS) method.

Artemisia annua, commonly known as sweet wormwood or Qinghao, is a shrub native to China and has long been used for medicinal purposes. A. annua is now cultivated globally as the only natural source of a potent anti-malarial compound, artemisinin. Here, we report a high-quality draft assembly of the 1.74-gigabase genome of A. annua, which is highly heterozygous, rich in repetitive sequences, and contains 63 226 protein-coding genes, one of the largest numbers among the sequenced plant species. We found that, as one of a few sequenced genomes in the Asteraceae, the A. annua genome contains a large number of genes specific to this large angiosperm clade. Notably, the expansion and functional diversification of genes encoding enzymes involved in terpene biosynthesis are consistent with the evolution of the artemisinin biosynthetic pathway. We further revealed by transcriptome profiling that A. annua has evolved the sophisticated transcriptional regulatory networks underlying artemisinin biosynthesis. Based on comprehensive genomic and transcriptomic analyses we generated transgenic A. annua lines producing high levels of artemisinin, which are now ready for large-scale production and thereby will help meet the challenge of increasing global demand of artemisinin.

The effective anti-malarial drug artemisinin (AN) isolated from Artemisia annua is relatively expensive due to the low AN content in the plant as AN is only synthesized within the glandular trichomes. Therefore, genetic engineering of A. annua is one of the most promising approaches for improving the yield of AN. In this work, the AaMYB1 transcription factor has been identified and characterized. When AaMYB1 is overexpressed in A. annua, either exclusively in trichomes or in the whole plant, essential AN biosynthetic genes are also overexpressed and consequently the amount of AN is significantly increased. Artemisia AaMYB1 constitutively overexpressing plants displayed a greater number of trichomes. In order to study the role of AaMYB1 on trichome development and other possibly connected biological processes, AaMYB1 was overexpressed in Arabidopsis thaliana. To support our findings in Arabidopsis thaliana, an AaMYB1 orthologue from this model plant, AtMYB61, was identified and atmyb61 mutants characterized. Both AaMYB1 and AtMYB61 affected trichome initiation, root development and stomatal aperture in A. thaliana. Molecular analyses indicated that two crucial trichome activator genes are misexpressed in atmyb61 mutant plants and in plants overexpressing AaMYB1. Furthermore, AaMYB1 and AtMYB61 are also essential for gibberellin (GA) biosynthesis and degradation in both species by positively affecting the expression of the enzymes that convert GA(9) into the bioactive GA(4) as well as the enzymes involved in the degradation of GA(4). Overall, these results identify AaMYB1/AtMYB61 as a key component of the molecular network that connects important biosynthetic processes, and reveal its potential value for AN production through genetic engineering.

The binding properties of pentacyclic triterpenoid isomeric drugs, i.e. ursolic acid (UA) and oleanolic acid (OA), to bovine lactoferrin (BLF) have been studied by molecule modeling, fluorescence spectroscopy, UV-visible absorbance spectroscopy and infrared spectroscopy (IR). Molecular docking, performed to reveal the possible binding mode or mechanism, suggested that hydrophobic interaction and hydrogen bonding play important roles to stabilize the complex. The results of spectroscopic measurements showed that the two isomeric drugs both strongly quenched the intrinsic fluorescence of BLF through a static quenching procedure although some differences between UM and OA binding strength and non-radiation energy transfer occurred within the molecules. The number of binding sites was 3.44 and 3.10 for UA and OA, respectively, and the efficiency of Forster energy transfer provided a distance of 0.77 and 1.21 nm for UA and OA, respectively. The conformation transformation of BLF affected by the drugs conformed to the "all-or-none" pattern. In addition, the changes of the ratios of alpha-helices, beta-sheets and beta-turns of BLF during the process of the interaction were obtained. The results of the experiments in combination with the calculations showed that there are two modes of pentacyclic triterpenoid binding to BLF instead of one binding mode only governed by the principle of the lowest bonding energy.