Biosynthesis of the Non-canonical C17 Sesquiterpenoids Chlororaphen A and B from Pseudomonas chlororaphis.

Chlororaphens A and B are structurally unique non-canonical C17 sesquiterpenoids from Pseudomonas chlororaphis that are made by two SAM-dependent methyltransferases and a type I terpene synthase. This study addresses the mechanism of their formation in isotopic labelling experiments and DFT calculations. The results demonstrate an astonishing complexity with distribution of labellings within a cyclopentane core that is reversely connected to two acyclic fragments in chlororaphen A and B. In addition, the uptake of up to 14 deuterium atoms from D2O was observed. These findings are explainable by a repeated late stage multistep rearrangement sequence. The absolute configurations of the chlororaphens and their biosynthetic intermediates were elucidated in stereoselective labelling experiments.

Terpenoids from quinoa reverse drug resistance of colon cancer by upregulating miR-495-3p.

BACKGROUND: Quinoa contains far more nutrients than any traditional grain crop. It is known that terpenoids in quinoa have anti-inflammatory and antitumor effects, but their role in reversing drug resistance remains unclear. RESULTS: Our previous studies showed that quinoa-derived terpenoid compounds (QBT) can inhibit the occurrence and development of colon cancer. This study further indicates that QBT markedly reverse drug resistance of colon cancer. The results showed that QBT combined with 5-fluorouracil (5-Fu) treatment significantly enhanced the chemotherapy sensitivity of HCT-8/Fu, compared with 5-Fu treatment alone. Moreover, we found that QBT significantly reduced the expression of drug-resistant proteins (P-gp, MRP1, BCRP), and increased the accumulation of chemotherapy drugs. Taking P-gp as the target for biogenesis prediction analysis, results showed that upregulation of miR-495-3p enhanced the chemosensitivity of drug-resistant HCT-8/Fu cells. Besides, the results showed that miR-495-3p was abnormally methylated in HCT-8/Fu compared with HCT-8 colon cancer cells. The expression of methyltransferases DNMT1, DNMT3a and DNMT3b was abnormal. After QBT treatment, the expression level of methyltransferases returned to normal. In addition, the QBT + 5Fu group showed inhibition of tumors in nude mice. CONCLUSION: QBT treatment downregulated the expression of drug-resistant protein P-gp by inhibiting the methylation of miR-495-3p, and enhanced the accumulation of 5-Fu in vivo, which in turn reversed its chemoresistance. This suggests that QBT has potential ability as a new drug-resistance reversal agent in colorectal cancer. © 2024 Society of Chemical Industry.

From grape to wine: A thorough compound specific isotopic, enantiomeric and quali-quantitative investigation by means of gas chromatographic analysis.

In this study, multiple analytical approaches, including simultaneous enantiomeric and isotopic analysis, were employed to thoroughly investigate the volatile fraction in Moscato giallo grape berries and wines. For the qualitative and quantitative profiling, a fast GC-QqQ/MS approach was successfully utilized. However, prior to isotopic analysis, the extracts underwent an additional concentration step, necessitating an assessment of isotopic fractionation during the concentration process. Once the absence of carbon isotopic fractionation was confirmed, this research aimed to develop a suitable gas chromatographic method for the simultaneous detection of both enantiomeric and isotopic ratios of target monoterpenoids in Moscato giallo samples. To address the limitations associated with a one-dimensional approach, multidimensional gas chromatography was employed to enhance separation before IRMS and qMS detections. Utilizing a Deans switch transfer device, the coupling of an apolar column in the first dimension and a chiral cyclodextrin-based stationary phase in the second dimension proved effective for this purpose. The data obtained from the analysis of Moscato giallo samples allowed for the assessment of natural isotopic and enantiomeric distributions in grapes and wines for the first time in the literature. Significant enantiomeric excesses were observed for the target terpenoids investigated. Regarding isotopic distribution, a consistent trend was observed for all detected target terpenols, including the linalool enantiomers. To date, this study represents the first investigation of simultaneous δ13C and chiral investigation of the main terpenoids in oenological products in the literature.

Unlocking Nature's Rhythms: Insights into Secondary Metabolite Modulation by the Circadian Clock.

Plants, like many other living organisms, have an internal timekeeper, the circadian clock, which allows them to anticipate photoperiod rhythms and environmental stimuli to optimally adjust plant growth, development, and fitness. These fine-tuned processes depend on the interaction between environmental signals and the internal interactive metabolic network regulated by the circadian clock. Although primary metabolites have received significant attention, the impact of the circadian clock on secondary metabolites remains less explored. Transcriptome analyses revealed that many genes involved in secondary metabolite biosynthesis exhibit diurnal expression patterns, potentially enhancing stress tolerance. Understanding the interaction mechanisms between the circadian clock and secondary metabolites, including plant defense mechanisms against stress, may facilitate the development of stress-resilient crops and enhance targeted management practices that integrate circadian agricultural strategies, particularly in the face of climate change. In this review, we will delve into the molecular mechanisms underlying circadian rhythms of phenolic compounds, terpenoids, and N-containing compounds.

Natural Bioactive Compounds in the Management of Periodontal Diseases: A Comprehensive Review.

Periodontal diseases, chronic inflammatory conditions affecting oral health, are primarily driven by microbial plaque biofilm and the body's inflammatory response, leading to tissue damage and potential tooth loss. These diseases have significant physical, psychological, social, and economic impacts, necessitating effective management strategies that include early diagnosis, comprehensive treatment, and innovative therapeutic approaches. Recent advancements in biomanufacturing have facilitated the development of natural bioactive compounds, such as polyphenols, terpenoids, alkaloids, saponins, and peptides, which exhibit antimicrobial, anti-inflammatory, and tissue regenerative properties. This review explores the biomanufacturing processes-microbial fermentation, plant cell cultures, and enzymatic synthesis-and their roles in producing these bioactive compounds for managing periodontal diseases. The integration of these natural compounds into periodontal therapy offers promising alternatives to traditional treatments, potentially overcoming issues like antibiotic resistance and the disruption of the natural microbiota, thereby improving patient outcomes.

Elucidating the Phytochemical Landscape of Leaves, Stems, and Tubers of Codonopsis convolvulacea through Integrated Metabolomics.

Codonopsis convolvulacea is a highly valued Chinese medicinal plant containing diverse bioactive compounds. While roots/tubers have been the main medicinal parts used in practice, leaves and stems may also harbor valuable phytochemicals. However, research comparing volatiles across tissues is lacking. This study performed metabolomic profiling of leaves, stems, and tubers of C. convolvulacea to elucidate tissue-specific accumulation patterns of volatile metabolites. Ultra-high performance liquid chromatography-tandem mass spectrometry identified 302 compounds, belonging to 14 classes. Multivariate analysis clearly differentiated the metabolic profiles of the three tissues. Numerous differentially accumulated metabolites (DAMs) were detected, especially terpenoids and esters. The leaves contained more terpenoids, ester, and alcohol. The stems accumulated higher levels of terpenoids, heterocyclics, and alkaloids with pharmaceutical potential. The tubers were enriched with carbohydrates like sugars and starch, befitting their storage role, but still retained reasonable amounts of valuable volatiles. The characterization of tissue-specific metabolic signatures provides a foundation for the selective utilization of C. convolvulacea parts. Key metabolites identified include niacinamide, p-cymene, tridecanal, benzeneacetic acid, benzene, and carveol. Leaves, stems, and tubers could be targeted for antioxidants, drug development, and tonics/nutraceuticals, respectively. The metabolomic insights can also guide breeding strategies to enhance the bioactive compound content in specific tissues. This study demonstrates the value of tissue-specific metabolite profiling for informing the phytochemical exploitation and genetic improvement of medicinal plants.

Antiviral and Cytotoxic Activities of Ilex aquifolium Silver Queen in the Context of Chemical Profiling of Two Ilex Species.

The leaves of Ilex paraguariensis (known as Yerba mate), used as a popular beverage, are a very well-recognized plant material with various biological activities, including analeptic (because of caffeine), anti-obesity (phenolics, saponins), antimicrobial, and antiviral (phenolics, saponins). Here, the chemical compositions of the leaves of two European Ilex species (× meserveae and aquifolium) with three varieties each were investigated. The terpenoid, saponin, and polyphenolic fractions were submitted for LC-MS or GC-MS analysis against a standard Mate leaf. In addition, the aroma profiles of all the species were analysed using HS-SPME-Arrow prior to GC-MS analysis. All fractions were subjected to antiviral and cytotoxic assays. We found 86 compounds in all accessions, with limonene, linalool, and p-cymene being predominant. There were minor similarities between the volatile compositions of the European and South American species. We found ursolic and oleanolic acid to be the main compounds in the terpenoid fraction. Mono-caffeoylquinic acids and di-caffeoylquinic acids were the main constituents of the polar fractions. About 180 compounds from the saponin group were tentatively identified, of which 9 and 3 were selected as distinctive markers for I. meserveae and I. aquifolium, respectively. Based on chemical screening, I. aquifolium Silver Queen was chosen as the source of terpenoid and saponin fractions and polyphenol extracts. The most substantial inhibition of cancer cell growth was observed with saponin in the case of the MCF7 (human breast cancer) cell line, while for LoVo and L929 cell lines (human colorectal cancer and reference mouse fibroblasts), it was slightly weaker. These results should be analysed further as a promising chemoprevention of colorectal and gastrointestinal cancers. Saponin and polyphenolic extracts exhibited similar activities against HSV-1 and HAdV-5, with 4-log reduction in virus titres. This study focuses our attention on a field of potential antiviral formulations derived from European holly.

Sequential Separation of Essential Oil Components during Hydrodistillation of Fresh Foliage from Azorean Cryptomeria japonica (Cupressaceae): Effects on Antibacterial, Antifungal, and Free Radical Scavenging Activities.

Cryptomeria japonica wood industry generates large amounts of foliage biomass residues. Due to the increasing applications and markets for essential oils (EOs), fresh Azorean C. japonica foliage (Az-CJF) residues are used for local EO production. Hydrodistillation (HD), a common process for obtaining EOs, also provides the possibility to fractionate them. Thus, this study evaluated the in vitro antimicrobial and antioxidant activities of six Az-CJF EO fractions (Frs. 1-6), collected at sequential HD timeframes (HDTs: 0-2, 2-10, 10-30, 30-60, 60-120, and 120-240 min), in comparison to the crude EO, obtained from a non-fractionated HD (0-240 min HDT). Antimicrobial activities were assessed via disc diffusion method against seven bacteria (foodborne and/or human pathogens) and two Penicillium spp. (phytopathogenic fungi), and antioxidant activity was estimated using DPPH and ABTS assays. Concerning the antibacterial activity, all the EO samples were effective only toward Gram-positive bacteria. Fractions 1-3 (<30 min HDT) were the most active, with growth inhibition zones (GIZ) of 7.0-23.3 mm (1.4-2.2 times higher than those of the crude EO), being Bacillus spp. (B. licheniformis and B. subtilis) the most sensitive, followed by Staphylococcus aureus and Micrococcus luteus. Regarding the antifungal activity, Frs. 1-3 also displayed the best activities, but only against P. italicum (GIZ around 9.0 mm), while the crude EO showed no antifungal activity. Overall, the best antimicrobial properties of Frs. 1-3 could be attributed, at least in part, to their highest content in α-pinene and bornyl acetate. On the other hand, Frs. 4-6 (>30 min HDT) exhibited the strongest antioxidant activities (EC50 values: 1.5-2.3 and 1.0-1.7 mg mL-1 for DPPH and ABTS, respectively), being at least 1.3-fold higher than those of the crude EO. The presence of nezukol, elemol, and eudesmol isomers could strongly contribute to the best free radical scavenging properties of Frs. 4-6. In conclusion, HD was found to be an efficient process for obtaining new Az-CJF EO fractions with variable and enhanced bioactivities due to their differential composition, as assessed using GC-MS. Hence, these findings could contribute to increasing the commercial potential of the C. japonica EO industry, namely, the Fr2 and Fr6, which presented the most significant activities and can have potential applications in the food, medical, and agriculture sectors.

Insecticidal Activity of Some Major Essential Oil Components against Metopolophium dirhodum and Its Predators.

Essential oils (EOs) are plant metabolites with important insecticidal effects. Nevertheless, information on the efficacy of the major substances on aphids and their natural enemies is still missing. The objective of this paper is, therefore, to identify the efficacy of selected EO majority substances-ß-citronellol, carvacrol, isoeugenol, and linalool, including their binary mixtures-on the mortality and fertility of the aphid Metopolophium dirhodum, an important cereal pest. The best efficacy was proven for the binary mixture of ß-citronellol and linalool (1:1 ratio), for which the estimated LC50(90) is 0.56(1.58) mL L-1. This binary mixture applied in sublethal concentrations significantly reduced aphid fertility. It was found that the phenomenon can be attributed to ß-citronellol, as the females treated with LC30 laid 45.9% fewer nymphs, on average, compared to the control. Although ß-citronellol and linalool, including their 1:1 mixture, showed very good efficacy on aphid mortality, they were, on the other hand, very friendly to the larvae of Aphidoletes aphidimyza and Chrysoperla carnea, which are important aphid predators. Based on our results, the newly discovered synergically acting binary mixture ß-citronellol/linalool can be recommended as an efficient substance suitable for the further development of botanical insecticides used against aphids.

A Functional Switch Between Asperfumene and Fusicoccadiene Synthase and Entrance to Asperfumene Biosynthesis through a Vicinal Deprotonation-Reprotonation Process.

The diterpene synthase AfAS was identified from Aspergillus fumigatiaffinis. Its amino acid sequence and - according to a structural model - active site architecture are highly similar to those of the fusicocca-2,10(14)-diene synthase PaFS, but AfAS produces a structurally much more complex diterpene with a novel 6-5-5-5 tetracyclic skeleton called asperfumene. The cyclisation mechanism of AfAS was elucidated through isotopic labelling experiments and DFT calculations. The reaction cascade proceeds in its initial steps through similar intermediates as for the PaFS cascade, but then diverges through an unusual vicinal deprotonation-reprotonation process that triggers a skeletal rearrangement at the entrance to the steps leading to the unique asperfumene skeleton. The structural model revealed only one major difference between the active sites: The PaFS residue F65 is substituted by I65 in AfAS. Intriguingly, site-directed mutagenesis experiments with both diterpene synthases revealed that position 65 serves as a bidirectional functional switch for the biosynthesis of tetracyclic asperfumene versus structurally less complex diterpenes.

Multi-omics analyses and botanical perfumer hypothesis provide insights into the formation and maintenance of aromatic characteristics of Dendrobium loddigesii flowers.

Dendrobium loddigesii, a member of the Orchidaceae family, is a valuable horticultural crop known for its aromatic qualities. However, the mechanisms responsible for the development of its aromatic characteristics remain poorly understood. To elucidate these underlying mechanisms, we assembled the first chromosome-level reference genome of D. loddigesii using PacBio HiFi-reads, Illumina short-reads, and Hi-C data. The assembly comprises 19 pseudochromosomes with N50 contig and N50 scaffold sizes of 55.15 and 89.94 Mb, respectively, estimating the genome size to be 1.68 Gb, larger than that of other sequenced Dendrobium species. During the flowering stages, we conducted a comprehensive analysis combining volatilomics and transcriptomics to understand the characteristics and biosynthetic mechanisms pathways of the floral scent. Our findings emphasize the significant contribution of aromatic terpenoids, especially monoterpenoids, in defining the floral aroma. Furthermore, we identified two crucial terpene synthase (TPS) genes that play a key role in maintaining the aroma during flowering. Through the integration volatilomics data with catalytic assays of DlTPSbs proteins, we identified specific compounds responsible for the aromatic characteristics of D. loddigesii. This integrated analysis of the genome, transcriptome, and volatilome, offers valuable insights into the development and preservation of D. loddigesii's aromatic characteristics, setting the stage for further exploration of the botanical perfumer hypothesis.

[Advances in the biosynthesis and metabolic regulation of terpenoids in Saccharomyces cerevisiae].

Terpenoids are the one of most abundant natural products. With diverse varieties and biological activities, they are widely used in the food, medicine, chemical industry, and novel fuels. However, the conventional methods such as plant extraction and chemical synthesis cannot meet the current market demand for terpenoids. Efficient microbial cell factories, especially engineered Saccharomyces cerevisiae strains, have been constructed for the industrial production of terpenoids. In recent years, researchers have constructed multiple S. cerevisiae strains with increased yield and productivity via approaches of synthetic biology and metabolic engineering. This paper reviews the recent progress in the biosynthesis of terpenoids in S. cerevisiae cells and summarizes a variety of metabolic engineering strategies for the production of terpenoids in S. cerevisiae. These strategies include the construction and optimization of metabolic pathways, the mining and modification of key enzymes, the regeneration of cofactors, the engineering of cell localization and cell efflux, and the improvement of cell tolerance. Our review will provide information and strategies for the effective biosynthesis of terpenoids in S. cerevisiae.

Production of non-natural terpenoids through chemoenzymatic synthesis using substrate analogs.

Chemoenzymatic synthesis of non-natural terpenes using the promiscuous activity of terpene synthases allows for the expansion of the chemical space of terpenoids with potentially new bioactivities. In this report, we describe protocols for the preparation of a novel aphid attractant, (S)-14,15-dimethylgermacrene D, by exploiting the promiscuity of (S)-germacrene D synthase from Solidago canadensis and using an engineered biocatalytic route to convert prenols to terpenoids. The method uses a combination of five enzymes to carry out the preparation of terpenoid semiochemicals in two steps: (1) diphosphorylation of five or six carbon precursors (prenol, isoprenol and methyl-isoprenol) catalyzed by Plasmodium falciparum choline kinase and Methanocaldococcus jannaschii isopentenyl phosphate kinase to form DMADP, IDP and methyl-IDP, and (2) chain elongation and cyclization catalyzed by Geobacillus stearothermophilus (2E,6E)-farnesyl diphosphate synthase and S. canadensis (S)-germacrene D synthase to produce (S)-germacrene D and (S)-14,15-dimethylgermacrene D. Using this method, new non-natural terpenoids are readily accessible and the approach can be adopted to produce different terpene analogs and terpenoid derivatives with potential novel applications.

Structural biology of terpene synthases.

Structural biology research of terpene synthases (TSs) has provided a useful basis to understand their catalytic mechanisms in producing diverse terpene products with polycyclic ring systems and multiple chiral centers. However, compared to the large numbers of>95,000 terpenoids discovered to date, few structures of TSs have been solved and the understanding of their catalytic mechanisms is lagging. We here (i) introduce the basic catalytic logic, the structural architectures, and the metal-binding conserved motifs of TSs; (ii) provide detailed experimental procedures, in gene cloning and plasmid construction, protein purification, crystallization, X-ray diffraction data collection and structural elucidation, for structural biology research of TSs; and (iii) discuss the prospects of structure-based engineering and de novo design of TSs in generating valuable terpene molecules, which cannot be easily achieved by chemical synthesis.

Common Biosynthesis of Non-canonical C16 Terpenes through a Fragmentation-Recombination Mechanism.

The biosynthesis of six recently reported non-canonical C16 sesquiterpenoids named after ancient Greek philosophers including archimedene, aristotelene, eratosthenene, pythagorene, a-democritene and anaximandrene was investigated through density functional theory (DFT) calculations and isotopic labeling experiments. The results revealed for all compounds except archimedene a unique fragmentation-recombination mechanism as previously demonstrated for sodorifen biosynthesis, in addition to a remarkable "dancing" mechanism for anaximandrene biosynthesis.

Ginkgo biloba: A Leaf of Hope in the Fight against Alzheimer's Dementia: Clinical Trial Systematic Review.

Alzheimer's disease (AD) is a stealthy and progressive neurological disorder that is a leading cause of dementia in the global elderly population, imposing a significant burden on both the elderly and society. Currently, the condition is treated with medications that alleviate symptoms. Nonetheless, these drugs may not consistently produce the desired results and can cause serious side effects. Hence, there is a vigorous pursuit of alternative options to enhance the quality of life for patients. Ginkgo biloba (GB), an herb with historical use in traditional medicine, contains bioactive compounds such as terpenoids (Ginkgolides A, B, and C), polyphenols, organic acids, and flavonoids (quercetin, kaempferol, and isorhamnetin). These compounds are associated with anti-inflammatory, antioxidant, and neuroprotective properties, making them valuable for cognitive health. A systematic search across three databases using specific keywords-GB in AD and dementia-yielded 1702 documents, leading to the selection of 15 clinical trials for synthesis. In eleven studies, GB extract/EGb 761® was shown to improve cognitive function, neuropsychiatric symptoms, and functional abilities in both dementia types. In four studies, however, there were no significant differences between the GB-treated and placebo groups. Significant improvements were observed in scores obtained from the Mini-Mental State Examination (MMSE), Short Cognitive Performance Test (SKT), and Neuropsychiatric Inventory (NPI). While the majority of synthesized clinical trials show that Ginkgo biloba has promising potential for the treatment of these conditions, more research is needed to determine optimal dosages, effective delivery methods, and appropriate pharmaceutical formulations. Furthermore, a thorough assessment of adverse effects, exploration of long-term use implications, and investigation into potential drug interactions are critical aspects that must be carefully evaluated in future studies.

The Genus Hyssopus: Traditional Use, Phytochemicals and Pharmacological Properties.

According to modern concepts, the genus Hyssopus L. includes seven plant species (Hyssopus ambiguus (Trautv.) Iljin ex Prochorov. & Lebel; Hyssopus cuspidatus Boriss; Hyssopus latilabiatus C.Y.Wu & H.W. Li; Hyssopus macranthus Boriss.; Hyssopus officinalis L.; Hyssopus seravschanicus (Dubj.) Pazij; Hyssopus subulifolius (Rech.f.) Rech.f.). The plants are rich in various groups of biologically active substances with a wide spectrum of pharmacological action. This review presents a modern comprehensive overview of the botanical research, extraction methods, chemical composition and pharmacological activity of plants of the genus Hyssopus L. As a result of the review, it was established that the chemical composition of plant extracts of the genus Hyssopus L. depends on various factors (place of growth, weather conditions, chemotypes, extraction methods, etc.). For the further use of the plants, the extraction methods and low-molecular metabolites isolated from them (mono- and sesquiterpenoids, flavonoids, alkaloids, etc.) are discussed. The data from the review provide an assessment of the relevance.

Aerobiology of the Family Lamiaceae: Novel Perspectives with Special Reference to Volatiles Emission.

Lamiaceae is a botanical family rich in aromatic species that are in high demand such as basil, lavender, mint, oregano, sage, and thyme. It has great economical, ecological, ethnobotanical, and floristic importance. The aim of this work is to provide an updated view on the aerobiology of species from the family Lamiaceae, with an emphasis on novelties and emerging applications. From the aerobiology point of view, the greatest interest in this botanical family is related to the volatile organic compounds emitted by the plants and, to a much lesser extent, their pollen. Research has shown that the major volatile organic compounds emitted by the plants from this botanical family are monoterpenes and sesquiterpenes. The most important monoterpenes reported across studies include α-pinene, ß-pinene, 1,8-cineole, menthol, limonene, and γ-terpinene. Most reports tend to cover species from the subfamily Nepetoideae. Volatile oils are produced by glandular trichomes found on aerial organs. Based on general morphology, two main types are found in the family Lamiaceae, namely peltate and capitate trichomes. As a result of pollinator-mediated transfer of pollen, Lamiaceae species present a reduced number of stamens and quantity of pollen. This might explain the low probability of pollen presence in the air from these species. A preliminary synopsis of the experimental evidence presented in this work suggests that the interplay of the organic particles and molecules released by these plants and their environment could be leveraged for beneficial outcomes in agriculture and landscaping. Emerging reports propose their use for intercropping to ensure the success of fructification, increased yield of entomophilous crops, as well as in sensory gardens due to the therapeutic effect of volatiles.

Identification and functional analysis of floral terpene synthase genes in Curcuma alismatifolia.

MAIN CONCLUSION: CaTPS2 and CaTPS3 were significantly expressed in flowers of Curcuma alismatifolia 'Shadow' and demonstrated bifunctional enzyme activity, CaTPS2 generated linalool and nerolidol as products, and CaTPS3 catalyzed ß-myrcene and ß-farnesene formation. This study presents the discovery and functional characterization of floral terpene synthase (TPS) genes in Curcuma alismatifolia 'Shadow', a cultivar renowned for its unique fragrance. Addressing the gap in understanding the genetic basis of floral scent in this species, we identified eight TPS genes through comprehensive transcriptome sequencing. Among these, CaTPS2 and CaTPS3 were significantly expressed in floral tissues and demonstrated bifunctional enzyme activity corresponding to the major volatile compounds detected in 'Shadow'. Functional analyses, including in vitro assays complemented with rigorous controls and alternative identification methods, elucidated the roles of these TPS genes in terpenoid biosynthesis. In vitro studies were conducted via heterologous expression in E. coli, followed by purification of the recombinant protein using affinity chromatography, enzyme assays were performed with GPP/FPP as the substrate, and volatile products were inserted into the GC-MS for analysis. Partially purified recombinant protein of CaTPS2 catalyzed GPP and FPP to produce linalool and nerolidol, respectively, while partially purified recombinant protein of CaTPS3 generated ß-myrcene and ß-farnesene with GPP and FPP as substrates, respectively. Real-time quantitative PCR further validated the expression patterns of these genes, correlating with terpenoid accumulation in different plant tissues. Our findings illuminate the molecular mechanisms underpinning floral fragrance in C. alismatifolia and provide a foundation for future genetic enhancements of floral scent in ornamental plants. This study, therefore, contributes to the broader understanding of terpenoid biosynthesis in plant fragrances, paving the way for biotechnological applications in horticulture plant breeding.

Development and Validation of a Liquid Chromatography-Tandem Mass Spectrometry Method for Screening Potential Citrate Lyase Inhibitors from a Library of Marine Compounds.

Tuberculosis, a persistent illness caused by Mycobacterium tuberculosis, remains a significant global public health challenge. The widespread use of anti-tuberculosis drugs has resulted in the emergence of drug-resistant strains, which complicates treatment efforts. Addressing this issue is crucial and hinges on the development of new drugs that can effectively target the disease. This involves identifying novel therapeutic targets that can disrupt the bacterium's survival mechanisms in various environments such as granulomas and lesions. Citrate lyase, essential for the survival of Mycobacterium species at lesion sites and in granulomatous conditions, is a potential target for the treatment of tuberculosis. This manuscript aimed to construct an efficient enzyme inhibitor screening platform using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF MS). This system can accurately identify compounds with enzyme inhibitory activity from a library of marine terpenoids and phenolic compounds. Utilizing the screened herbal enzyme inhibitors as a starting point, we analyzed their chemical structures and skillfully built a library of marine compounds based on these structures. The results showed that all of the tested compounds from the phenolics library inhibited citrate lyase by more than 50%, and a significant portion of terpenoids also demonstrated inhibition, with these active terpenoids comprising over half of the terpenoids tested. The study underscores the potential of marine-derived phenolic and terpenoid compounds as potent inhibitors of citrate lyase, indicating a promising direction for future investigations in treating tuberculosis and associated disorders.