Impact of three decades of warming, increased nutrient availability, and increased cloudiness on the fluxes of greenhouse gases and biogenic volatile organic compounds in a subarctic tundra heath.

Climate change is exposing subarctic ecosystems to higher temperatures, increased nutrient availability, and increasing cloud cover. In this study, we assessed how these factors affect the fluxes of greenhouse gases (GHGs) (i.e., methane (CH4), nitrous oxide (N2O), and carbon dioxide (CO2)), and biogenic volatile organic compounds (BVOCs) in a subarctic mesic heath subjected to 34 years of climate change related manipulations of temperature, nutrient availability, and light. GHGs were sampled from static chambers and gases analyzed with gas chromatograph. BVOCs were measured using the push-pull method and gases analyzed with chromatography-mass spectrometry. The soil temperature and moisture content in the warmed and shaded plots did not differ significantly from that in the controls during GHG and BVOC measurements. Also, the enclosure temperatures during BVOC measurements in the warmed and shaded plots did not differ significantly from temperatures in the controls. Hence, this allowed for assessment of long-term effects of the climate treatment manipulations without interference of temperature and moisture differences at the time of measurements. Warming enhanced CH4 uptake and the emissions of CO2, N2O, and isoprene. Increased nutrient availability increased the emissions of CO2 and N2O but caused no significant changes in the fluxes of CH4 and BVOCs. Shading (simulating increased cloudiness) enhanced CH4 uptake but caused no significant changes in the fluxes of other gases compared to the controls. The results show that climate warming and increased cloudiness will enhance CH4 sink strength of subarctic mesic heath ecosystems, providing negative climate feedback, while climate warming and enhanced nutrient availability will provide positive climate feedback through increased emissions of CO2 and N2O. Climate warming will also indirectly, through vegetation changes, increase the amount of carbon lost as isoprene from subarctic ecosystems.

Coupling the Isopentenol Utilization Pathway and Prenyltransferase for the Biosynthesis of Licoflavanone in Recombinant Escherichia coli.

Prenylflavonoids are promising candidates for food additives and functional foods due to their diverse biological activities and potential health benefits. However, natural prenylflavonoids are generally present in low abundance and are limited to specific plant species. Here, we report the biosynthesis of licoflavanone from naringenin and prenol by recombinant Escherichia coli. By investigating the activities of seven different sources of prenyltransferases overexpressed in E. coli toward various flavonoid substrates, the prenyltransferase AnaPT exhibits substrate preference when naringenin serves as the prenyl acceptor. Furthermore, licoflavanone production was successfully achieved by coupling the isopentenol utilization pathway and AnaPT in recombinant E. coli. In addition, the effects of fermentation temperatures, induction temperatures, naringenin concentrations, and substrate feeding strategies were investigated on the biosynthesis of licoflavanone in recombinant E. coli. Consequently, the recombinant E. coli strain capable of improved dimethylallyl diphosphate (DMAPP) supply and suitable for prenylflavonoid biosynthesis increased licoflavanone titers to 142.1 mg/L in a shake flask and to 537.8 mg/L in a 1.3 L fermentor, which is the highest yield for any prenylflavonoids reported to date. These strategies proposed in this study provide a reference for initiating the production of high-value prenylflavonoids.

High-Resolution Modeling of Summertime Biogenic Isoprene Emissions in New York City.

As cities strive for ambitious increases in tree canopy cover and reductions in anthropogenic volatile organic compound (AVOC) emissions, accurate assessments of the impacts of biogenic VOCs (BVOCs) on air quality become more important. In this study, we aim to quantify the impact of future urban greening on ozone production. BVOC emissions in dense urban areas are often coarsely represented in regional models. We set up a high-resolution (30 m) MEGAN (The Model of Emissions of Gases and Aerosols from Nature version 3.2) to estimate summertime biogenic isoprene emissions in the New York City metro area (NYC-MEGAN). Coupling an observation-constrained box model with NYC-MEGAN isoprene emissions successfully reproduced the observed isoprene concentrations in the city core. We then estimated future isoprene emissions from likely urban greening scenarios and evaluated the potential impact on future ozone production. NYC-MEGAN predicts up to twice as much isoprene emissions in NYC as the coarse-resolution (1.33 km) Biogenic Emission Inventory System version 3.61 (BEIS) on hot summer days. We find that BVOCs drive ozone production on hot summer days, even in the city core, despite large AVOC emissions. If high isoprene emitting species (e.g., oak trees) are planted, future isoprene emissions could increase by 1.4-2.2 times in the city core, which would result in 8-19 ppbv increases in peak ozone on ozone exceedance days with current NOx concentrations. We recommend planting non- or low-isoprene emitting trees in cities with high NOx concentrations to avoid an increase in the frequency and severity of future ozone exceedance events.

A picomolar inhibitor of the Plasmodium falciparum IPP pathway.

We identified MMV026468 as a picomolar inhibitor of blood-stage Plasmodium falciparum. Phenotyping assays, including isopentenyl diphosphate rescue of parasite growth inhibition, demonstrated that it targets MEP isoprenoid precursor biosynthesis. MMV026468-treated parasites showed an overall decrease in MEP pathway intermediates, which could result from inhibition of the first MEP enzyme DXS or steps prior to DXS such as regulation of the MEP pathway. Selection of MMV026468-resistant parasites lacking DXS mutations suggested that other targets are possible. The identification of MMV026468 could lead to a new class of antimalarial isoprenoid inhibitors.

Sensitive quantification of mevalonate pathway intermediates and prediction of relative novel analogs by chemical derivatization-based LC-MS/MS.

The mevalonate (MVA) pathway plays a crucial role in the occurrence and progression of various diseases, such as osteoporosis, breast cancer, and lung cancer, etc. However, determining all the MVA pathway intermediates is still challenging due to their high polarity, low concentration, chelation effect with metal compartments, and poor mass spectrometric response. In this study, we established a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method coupled with N2, N2, N4, N4-tetramethyl-6-(4-(piperazin-1-ylsulfonyl) phenyl)-1,3,5-triazine-2,4-diamine (Tmt-PP) labeling for the simultaneous analysis of all MVA intermediates in biospecimens. Chemical derivatization significantly improved the chromatographic retention, peak shape, and detection sensitivity of the analytes. Moreover, we employed a method named mass spectrum calculation to achieve the absolute quantification of the isomers, i.e., isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The established method was fully qualified and applied to explore the difference of these metabolites in cisplatin-resistant non-small cell lung cancer (NSCLC) cells. Additionally, several MVA intermediate analogs, including isopentenyl monophosphate or dimethylallyl monophosphate (IMP/DMAMP), geranyl monophosphate (GMP), 5-triphosphomevalonate (MTP), and isopentenyl triphosphate or dimethylallyl triphosphate (ITP/DMATP), were identified for the first time using a knowledge-driven prediction strategy. We further explored the tissue distribution of these novel metabolites. Overall, this work developed a sensitive quantification method for all MVA intermediates, which will enhance our understanding of the role of this pathway in various health and disease conditions. The novel metabolites we discovered warrant further investigations into their biosynthesis and biological functions.

Kinetic study of isoprene hydroxy hydroperoxide radicals reacting with sulphur dioxide and their global-scale impact on sulphate formation.

Isoprene is the most relevant volatile organic compound emitted during the biosynthesis of metabolism processes. The oxidation of isoprene by a hydroxy radical (OH) is one of the main consumption schemes that generate six isomers of isoprene hydroxy hydroperoxide radicals (ISOPOOs). In this study, the rate constants of ISOPOOs + sulphur dioxide (SO2) reactions that eventually generate sulphur trioxide (SO3), the precursor of sulphate aerosol (SO42-(p)), are determined using microcanonical kinetic theories coupled with molecular structures and energies estimated by quantum chemical calculations. The results show that the reaction rates range from 10-27 to 10-20 cm3 molecule-1 s-1, depending on the atmospheric temperature and structure of the six ISOPOO isomers. The effect of SO3 formation from SO2 oxidation by ISOPOOs on the atmosphere is evaluated by a global chemical transport model, along with the rate constants obtained from microcanonical kinetic theories. The results show that SO3 formation is enhanced in regions with high SO2 or low nitrogen oxide (NO), such as China, the Middle East, and Amazon rainforests. However, the production rates of SO3 formation by ISOPOOs + SO2 reactions are eight orders of magnitude lower than that from the OH + SO2 reaction. This is indicative of SO42-(p) formation from the direct oxidation of SO2 by ISOPOOs, which is almost negligible in the atmosphere. The results of this study entail a detailed analysis of SO3 formation from gas-phase reactions of isoprene-derived products.

Natural transformation of Vibrio natriegens with large genetic cluster enables alginate assimilation for isopentenol production.

Alginate is a major component of brown macroalgae, and its efficient utilization is critical for developing sustainable technologies. Vibrio natriegens is a fast-growing marine bacterium that has gained massive attention due to its potential as an alternative industrial chassis. However, V. natriegens cannot naturally metabolize alginate, limiting its usage in marine biomass conversion. In this study, V. natriegens was engineered to utilize marine biomass, kelp, as a carbon source. A total of 33.8 kb of the genetic cluster for alginate assimilation from Vibrio sp. dhg was integrated into V. natriegens by natural transformation. Engineered V. natriegens was further modified to produce 1.8 mg/L of isopentenol from 16 g/L of crude kelp powder. This study not only presents the very first case in which V. natriegens can be naturally transformed with large DNA fragments but also highlights the potential of this strain for converting marine biomass into valuable products.

Effects of aluminum (Al) stress on the isoprenoid metabolism of two Citrus species differing in Al-tolerance.

Isoprenoid metabolism and its derivatives took part in photosynthesis, growth regulation, signal transduction, and plant defense to biotic and abiotic stresses. However, how aluminum (Al) stress affects the isoprenoid metabolism and whether isoprenoid metabolism plays a vital role in the Citrus plants in coping with Al stress remain unclear. In this study, we reported that Al-treatment-induced alternation in the volatilization rate of monoterpenes (α-pinene, ß-pinene, limonene, α-terpinene, γ-terpinene and 3-carene) and isoprene were different between Citrus sinensis (Al-tolerant) and C. grandis (Al-sensitive) leaves. The Al-induced decrease of CO2 assimilation, maximum quantum yield of primary PSII photochemistry (Fv/Fm), the lower contents of glucose and starch, and the lowered activities of enzymes involved in the mevalonic acid (MVA) pathway and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway might account for the different volatilization rate of isoprenoids. Furthermore, the altered transcript levels of genes related to isoprenoid precursors and/or derivatives metabolism, such as geranyl diphosphate (GPP) synthase (GPPS) in GPP biosynthesis, geranylgeranyl diphosphate synthase (GGPPS), chlorophyll synthase (CHS) and GGPP reductase (GGPPR) in chlorophyll biosynthesis, limonene synthase (LS) and α-pinene synthase (APS) in limonene and α-pinene synthesis, respectively, might be responsible for the different contents of corresponding products in C. grandis and C. sinensis. Our data suggested that isoprenoid metabolism was involved in Al tolerance response in Citrus, and the alternation of some branches of isoprenoid metabolism could confer different Al-tolerance to Citrus species.

EuHDZ25 positively affects rubber biosynthesis by targeting EuFPS1 in Eucommia leaves.

Eucommia ulmoides is a temperate gum source plant that produces trans-polyisoprene (TPI), also known as Eucommia rubber. The structural configuration and function of TPI offer a new material with important potential for industrial development. In this study, we detected the TPI content in the leaves of diploid and triploid E. ulmoides plants. The average TPI content in the leaves of triploid E. ulmoides was significantly higher than that of diploid. Transcriptome data and weighted gene co-expression network analyses identified a significant positive correlation between the EuFPS1 gene and TPI content. Overexpression of EuFPS1 increased the density of rubber particles and TPI content, indicating its crucial role in TPI biosynthesis. In addition, the expression of EuHDZ25 in E. ulmoides was significantly positively correlated with EuFPS1 expression. Yeast one-hybrid and dual-luciferase assays demonstrated that EuHDZ25 mainly promotes TPI biosynthesis through positive regulation of EuFPS1 expression. The significantly up-regulated expression of EuHDZ25 and its consequent upregulation of EuFPS1 during the biosynthesis of TPI may partially explain the increased TPI content of triploids. This study provides an important theoretical foundation for further exploring the molecular mechanism of secondary metabolites content variation in polyploids and can help to promote the development and utilization of rubber resources.

Transcriptomic and free monoterpene analyses of aroma reveal that isopentenyl diphosphate isomerase inhibits monoterpene biosynthesis in grape (Vitis vinifera L.).

BACKGROUND: Monoterpenes are among the most important volatile aromatic compounds contributing to the flavor and aroma of grapes and wine. However, the molecular basis of monoterpene biosynthesis has not yet been fully elucidated. RESULTS: In our study, transcriptomics and gas chromatography-mass spectrometry (GC-MS) were used to mine candidate genes and transcription factors involved in monoterpene biosynthesis between high-monoterpene and zero-monoterpene table grape cultivars. We found that monoterpene biosynthesis was positively correlated by the expression of five genes encoding 1-deoxy-D-xylulose-5-phosphate synthase (VvDXSs), one encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (VvHDR), three hydroxy-3-methylglutaryl-CoA synthases (VvHMGSs) and one mevalonate kinase (VvMVK), whereas the expression of one isopentenyl diphosphate isomerase (VvIDI) and one 3-hydroxy-3-methylglutaryl-CoA reductase (VvHMGR) negatively correlated monoterpene biosynthesis. Of these genes, VvIDI was selected to validate its function in monoterpene accumulation through a transient overexpression experiment, and was shown to inhibit the biosynthesis of grape linalool and α-terpineol. Meanwhile, we found that a 64-amino acid extension sequence at the N-terminus can guide the VvIDI protein to target the chloroplast. CONCLUSIONS: The findings of this study should help to guide future functional analysis of key genes as well as mining the potential regulatory mechanism of monoterpene biosynthesis in grapes and grape products.

Chemical Composition, Anti-bacterial Activity and Molecular Docking Studies of Essential Oil Isolated from Sa Sam Nam (Launaea sarmentosa).

Launaea sarmentosa, also known as Sa Sam Nam, is a widely used remedy in Vietnamese traditional medicine and cuisine. However, the chemical composition and bioactivity of its essential oil have not been elucidated yet. In this study, we identified 40 compounds (98.6% of total peak area) in the essential oil via GC-MS analysis at the first time. Among them, five main compounds including Thymohydroquinone dimethyl ether (52.4%), (E)-α-Atlantone (9.0%), Neryl isovalerate (6.6%), Davanol D2 (isomer 2) (3.9%), and trans-Sesquisabinene hydrate (3.9%) have accounted for 75.8% of total peak area. The anti-bacterial activity of the essential oil against 4 microorganisms including Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa has also investigated via agar well diffusion assay. The results showed that the essential oil exhibited a strong antibacterial activity against Bacillus subtilis with the inhibition zones ranging from 8.2 to 18.7 mm. To elucidate the anti-bacterial effect mechanism of the essential oil, docking study of five main compounds of the essential oil (Thymohydroquinone dimethyl ether, (E)-α-Atlantone, Neryl isovalerate, Davanol D2 (isomer 2), and trans-Sesquisabinene hydrate) against some key proteins for bacterial growth such as DNA gyrase B, penicillin binding protein 2A, tyrosyl-tRNA synthetase, and dihydrofolate reductase were performed. The results showed that the main constituents of essential oil were highly bound with penicillin binding protein 2A with the free energies ranging -27.7 to -44.8 kcal/mol, which suggests the relationship between the antibacterial effect of essential oil and the affinity of main compounds with penicillin binding protein. In addition, the free energies of main compounds of the essential oil with human cyclooxygenase 1, cyclooxygenase 2, and phospholipase A2, the crucial proteins related with inflammatory response were less than diclofenac, a non-steroidal antiinflammatory drug. These findings propose the essential oil as a novel and promising anti-bacterial and anti-inflammatory medicine or cosmetic products.

Histochemical studies on the distribution and accumulation of trans-polyisoprene in the rubber-producing plant Eucommia ulmoides.

Eucommia ulmoides rubber (EUR) is a high-quality natural rubber resource, which can be extracted from different organs of the Eucommia ulmoides tree. In this study, EUR was isolated from the leaves, barks, and pericarps, and the structural characteristics and physicochemical properties of EUR were systematically determined. The accumulation and distribution of EUR in different tissues were assessed through in situ observations combined with cellular and subcellular scales. The preliminary analyses indicated that the variations in the physicochemical properties of EUR across different tissues were associated with its accumulation microstructure. Further analyses by SEM and TEM showed that the initial cell differentiation and fusion resulted in the formation of tubular structures without any nucleus. A limited number of rubber particles were generated within the cytoplasm, concurrent with aggregation and fusion. Eventually, rubber particles filled the entire cytoplasm, and organelles disappeared to form highly aggregated filamentous structures. In addition, the number and area of EUR-containing cells were closely related to the organization sizes of barks and leaves. This study provided valuable insights into Eucommia ulmoides histology and the rubber industry.

GAPDH inhibition mediated by thiol oxidation in human airway epithelial cells exposed to an environmental peroxide.

Intracellular redox homeostasis in the airway epithelium is closely regulated through adaptive signaling and metabolic pathways. However, inhalational exposure to xenobiotic stressors such as secondary organic aerosols (SOA) can alter intracellular redox homeostasis. Isoprene hydroxy hydroperoxide (ISOPOOH), a ubiquitous volatile organic compound derived from the atmospheric photooxidation of biogenic isoprene, is a major contributor to SOA. We have previously demonstrated that exposure of human airway epithelial cells (HAEC) to ISOPOOH induces oxidative stress through multiple mechanisms including lipid peroxidation, glutathione oxidation, and alterations of glycolytic metabolism. Using dimedone-based reagents and copper catalyzed azo-alkynyl cycloaddition to tag intracellular protein thiol oxidation, we demonstrate that exposure of HAEC to micromolar levels of ISOPOOH induces reversible oxidation of cysteinyl thiols in multiple intracellular proteins, including GAPDH, that was accompanied by a dose-dependent loss of GAPDH enzymatic activity. These results demonstrate that ISOPOOH induces an oxidative modification of intracellular proteins that results in loss of GAPDH activity, which ultimately impacts the dynamic regulation of the intracellular redox homeostatic landscape in HAEC.

Optimal seasonal schedule for the production of isoprene, a highly volatile biogenic VOC.

The leaves of many trees emit volatile organic compounds (abbreviated as BVOCs), which protect them from various damages, such as herbivory, pathogens, and heat stress. For example, isoprene is highly volatile and is known to enhance the resistance to heat stress. In this study, we analyze the optimal seasonal schedule for producing isoprene in leaves to mitigate damage. We assume that photosynthetic rate, heat stress, and the stress-suppressing effect of isoprene may vary throughout the season. We seek the seasonal schedule of isoprene production that maximizes the total net photosynthesis using Pontryagin's maximum principle. The isoprene production rate is determined by the changing balance between the cost and benefit of enhanced leaf protection over time. If heat stress peaks in midsummer, isoprene production can reach its highest levels during the summer. However, if a large portion of leaves is lost due to heat stress in a short period, the optimal schedule involves peaking isoprene production after the peak of heat stress. Both high photosynthetic rate and high isoprene volatility in midsummer make the peak of isoprene production in spring. These results can be clearly understood by distinguishing immediate impacts and the impacts of future expectations.

Computational investigation of cis-1,4-polyisoprene binding to the latex-clearing protein LcpK30.

Latex clearing proteins (Lcps) catalyze the oxidative cleavage of the C = C bonds in cis-1,4-polyisoprene (natural rubber), producing oligomeric compounds that can be repurposed to other materials. The active catalytic site of Lcps is buried inside the protein structure, thus raising the question of how the large hydrophobic rubber chains can access the catalytic center. To improve our understanding of hydrophobic polymeric substrate binding to Lcps and subsequent catalysis, we investigated the interaction of a substrate model containing ten carbon-carbon double bonds with the structurally characterized LcpK30, using multiple computational tools. Prediction of the putative tunnels and cavities in the LcpK30 structure, using CAVER-Pymol plugin 3.0.3, fpocket and Molecular Dynamic (MD) simulations provided valuable insights on how substrate enters from the surface to the buried active site. Two dominant tunnels were discovered that provided feasible routes for substrate binding, and the presence of two hydrophobic pockets was predicted near the heme cofactor. The larger of these pockets is likely to accommodate the substrate and to determine the size distribution of the oligomers. Protein-ligand docking was carried out using GOLD software to predict the conformations and interactions of the substrate within the protein active site. Deeper insight into the protein-substrate interactions, including close-contacts, binding energies and potential cleavage sites in the cis-1,4-polyisoprene, were obtained from MD simulations. Our findings provide further justification that the protein-substrate complexation in LcpK30 is mainly driven by the hydrophobic interactions accompanied by mutual conformational changes of both molecules. Two potential binding modes were identified, with the substrate in either extended or folded conformations. Whilst binding in the extended conformation was most favorable, the folded conformation suggested a preference for cleavage of a central double bond, leading to a preference for oligomers with 5 to 6 C = C bonds. The results provide insight into further enzyme engineering studies to improve catalytic activity and diversify the substrate and product scope of Lcps.

Deoxyxylulose 5-Phosphate Synthase Does Not Play a Major Role in Regulating the Methylerythritol 4-Phosphate Pathway in Poplar.

The plastidic 2-C-methylerythritol 4-phosphate (MEP) pathway supplies the precursors of a large variety of essential plant isoprenoids, but its regulation is still not well understood. Using metabolic control analysis (MCA), we examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), in multiple grey poplar (Populus × canescens) lines modified in their DXS activity. Single leaves were dynamically labeled with 13CO2 in an illuminated, climate-controlled gas exchange cuvette coupled to a proton transfer reaction mass spectrometer, and the carbon flux through the MEP pathway was calculated. Carbon was rapidly assimilated into MEP pathway intermediates and labeled both the isoprene released and the IDP+DMADP pool by up to 90%. DXS activity was increased by 25% in lines overexpressing the DXS gene and reduced by 50% in RNA interference lines, while the carbon flux in the MEP pathway was 25-35% greater in overexpressing lines and unchanged in RNA interference lines. Isoprene emission was also not altered in these different genetic backgrounds. By correlating absolute flux to DXS activity under different conditions of light and temperature, the flux control coefficient was found to be low. Among isoprenoid end products, isoprene itself was unchanged in DXS transgenic lines, but the levels of the chlorophylls and most carotenoids measured were 20-30% less in RNA interference lines than in overexpression lines. Our data thus demonstrate that DXS in the isoprene-emitting grey poplar plays only a minor part in controlling flux through the MEP pathway.

Isoprenoid emissions from Schima superba and Cunninghamia lanceolata: Their responses to elevated temperature by two warming facilities.

Isoprenoids (including isoprene (ISO) and monoterpenes (MTs)) are the majority of biogenic volatile organic compounds (BVOCs) which are important carbon-containing secondary metabolites biosynthesized by organisms, especially plant in terrestrial ecosystem. Results of the warming effects on isoprenoid emissions vary within species and warming facilities, and thus conclusions remain controversial. In this study, two typical subtropical tree species seedlings of Schima superba and Cunninghamia lanceolata were cultivated under three conditions, namely no warming (CK) and two warming facilities (with infrared radiators (IR) and heating wires (HW)) in open top chamber (OTC), and the isoprenoid emissions were measured with preconcentor-GC-MS system after warming for one, two and four months. The results showed that the isoprenoid emissions from S. superba and C. lanceolata exhibited uniformity in response to two warming facilities. IR and HW both stimulated isoprenoid emissions in two plants after one month of treatment, with increased ratios of 16.3 % and 72.5 % for S. superba, and 2.47 and 5.96 times for C. lanceolata. However, the emissions were suppressed after four months, with more pronounced effect for HW. The variation in isoprenoid emissions was primarily associated with the levels of Pn, Tr, monoterpene synthase (MTPS) activity. C. lanceolata predominantly released MTs (mainly α-pinene, α-terpene, γ-terpene, and limonene), with 39.7 % to 99.6 % of the total isoprenoid but ISO was only a very minor constituent. For S. superba, MTs constituted 24.7 % to 96.1 % of total isoprenoid. It is noteworthy that HW generated a greater disturbance to physiology activity in plants. Our study provided more comprehensive and more convincing support for integrating temperature-elevation experiments of different ecosystems and assessing response and adaptation of forest carbon cycle to global warming.

Unravelling the origin of isoprene in the human body-a forty year Odyssey.

In the breath research community's search for volatile organic compounds that can act as non-invasive biomarkers for various diseases, hundreds of endogenous volatiles have been discovered. Whilst these systemic chemicals result from normal and abnormal metabolic activities or pathological disorders, to date very few are of any use for the development of clinical breath tests that could be used for disease diagnosis or to monitor therapeutic treatments. The reasons for this lack of application are manifold and complex, and these complications either limit or ultimately inhibit the analytical application of endogenous volatiles for use in the medical sciences. One such complication is a lack of knowledge on the biological origins of the endogenous volatiles. A major exception to this is isoprene. Since 1984, i.e. for 40 years, it has been generally accepted that the pathway to the production of human isoprene, and hence the origin of isoprene in exhaled breath, is through cholesterol biosynthesis via the mevalonate (MVA) pathway within the liver. However, various studies between 2001 and 2012 provide compelling evidence that human isoprene is produced in skeletal muscle tissue. A recent multi-omic investigation of genes and metabolites has revealed that this proposal is correct by showing that human isoprene predominantly results from muscular lipolytic cholesterol metabolism. Despite the overwhelming proof for a muscular pathway to isoprene production in the human body, breath research papers still reference the hepatic MVA pathway. The major aim of this perspective is to review the evidence that leads to a correct interpretation for the origins of human isoprene, so that the major pathway to human isoprene production is understood and appropriately disseminated. This is important, because an accurate attribution to the endogenous origins of isoprene is needed if exhaled isoprene levels are to be correctly interpreted and for assessing isoprene as a clinical biomarker.

Biological Demands and Toxicity of Isoprenoid Precursors in Bacillus Subtilis Through Cell-Permeant Analogs of Isopentenyl Pyrophosphate and Dimethylallyl Pyrophosphate.

Bacterial isoprenoids are necessary for many biological processes, including maintaining membrane integrity, facilitating intercellular communication, and preventing oxidative damage. All bacterial isoprenoids are biosynthesized from two five carbon structural isomers, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are cell impermeant. Herein, we demonstrate exogenous delivery of IPP and DMAPP into Bacillus subtilis by utilizing a self-immolative ester (SIE)-caging approach. We initially evaluated native B. subtilis esterase activity, which revealed a preference for short straight chain esters. We then examined the viability of the SIE-caging approach in B. subtilis and demonstrate that the released caging groups are well tolerated and the released IPP and DMAPP are bioavailable, such that isoprenoid biosynthesis can be rescued in the presence of pathway inhibitors. We further show that IPP and DMAPP are both toxic and inhibit growth of B. subtilis at the same concentration. Lastly, we establish the optimal ratio of IPP to DMAPP (5 : 1) for B. subtilis growth and find that, surprisingly, DMAPP alone is insufficient to rescue isoprenoid biosynthesis under high concentrations of fosmidomycin. These findings showcase the potential of the SIE-caging approach in B. subtilis and promise to both aid in novel isoprenoid discovery and to inform metabolic engineering efforts in bacteria.

Molecular cloning and characterization of farnesyl diphosphate synthase from Rosa rugosa Thunb associated with salinity stress.

Rosa rugosa, a renowned ornamental plant, is cultivated for its essential oil containing valuable monoterpenes, sesquiterpenes, and other compounds widely used in the floriculture industry. Farnesyl diphosphate synthase (FPPS) is a key enzyme involved in the biosynthesis of sesquiterpenes and triterpenes for abiotic or biotic stress. In this study, we successfully cloned and characterized a full-length FPPS- encoding cDNA identified as RrFPPS1 using RT-PCR from R. rugosa. Phylogenetic analysis showed that RrFPPS1 belonged to the angiosperm-FPPS clade. Transcriptomic and RT-qPCR analyses revealed that the RrFPPS1 gene had tissue-specific expression patterns. Subcellular localization analysis using Nicotiana benthamiana leaves showed that RrFPPS1 was a cytoplasmic protein. In vitro enzymatic assays combined with GC-MS analysis showed that RrFPPS1 produced farnesyl diphosphate (FPP) using isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) as substrates to provide a precursor for sesquiterpene and triterpene biosynthesis in the plant. Additionally, our research found that RrFPPS1 was upregulated under salt treatment. These substantial findings contribute to an improved understanding of terpene biosynthesis in R. rugosa and open new opportunities for advancements in horticultural practices and fragrance industries by overexpression of the RrFPPS1 gene in vivo increased FPP production and subsequently led to elevated sesquiterpene yields in the future. The knowledge gained from this study can potentially lead to the development of enhanced varieties of R. rugosa with improved aroma, medicinal properties, and resilience to environmental stressors.