Isoprenoid Precursors Research Articles

1-Deoxy-d-xylulose 5-phosphate reductoisomerase as target for anti Toxoplasma gondii agents: crystal structure, biochemical characterization and biological evaluation of inhibitors.

Toxoplasma gondii is a widely distributed apicomplexan parasite causing toxoplasmosis, a critical health issue for immunocompromised individuals and for congenitally infected foetuses. Current treatment options are limited in number and associated with severe side effects. Thus, novel anti-toxoplasma agents need to be identified and developed. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is considered the rate-limiting enzyme in the non-mevalonate pathway for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate in the parasite, and has been previously investigated for its key role as a novel drug target in some species, encompassing Plasmodia, Mycobacteria and Escherichia coli. In this study, we present the first crystal structure of T. gondii DXR (TgDXR) in a tertiary complex with the inhibitor fosmidomycin and the cofactor NADPH in dimeric conformation at 2.5 Å resolution revealing the inhibitor binding mode. In addition, we biologically characterize reverse α-phenyl-β-thia and β-oxa fosmidomycin analogues and show that some derivatives are strong inhibitors of TgDXR which also, in contrast with fosmidomycin, inhibit the growth of T. gondii in vitro. Here, ((3,4-dichlorophenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)thio)methyl)phosphonic acid was identified as the most potent anti T. gondii compound. These findings will enable the future design and development of more potent anti-toxoplasma DXR inhibitors.

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.

Effects of producing high levels of hyperthermophile-specific C25,C25-archaeal membrane lipids in Escherichia coli

A hyperthermophilic archaeon, Aeropyrum pernix, synthesizes C25,C25-archaeal membrane lipids, or extended archaeal membrane lipids, which contain two C25 isoprenoid chains that are linked to glycerol-1-phosphate via ether bonds and are longer than the usual C20,C20-archaeal membrane lipids. The C25,C25-archaeal membrane lipids are believed to allow the archaeon to survive under harsh conditions, because they are able to form lipid membranes that are impermeable at temperatures approaching the boiling point. The effect that C25,C25-archaeal membrane lipids exert on living cells, however, remains unproven along with an explanation for why the hyperthermophilic archaeon synthesizes these specific lipids instead of the more common C20,C20-archaeal lipids or double-headed tetraether lipids. To shed light on the effects that these hyperthermophile-specific membrane lipids exert on living cells, we have constructed an E. coli strain that produces C25,C25-archaeal membrane lipids. However, a resultant low level of productivity would not allow us to assess the effects of their production in E. coli cells. Herein, we report an enhancement of the productivity of C25,C25-archaeal membrane lipids in engineered E. coli strains via the introduction of metabolic pathways such as an artificial isoprenol utilization pathway where the precursors of isoprenoids are synthesized via a two-step phosphorylation of prenol and isoprenol supplemented to a growth medium. In the strain with the highest titer, a major component of C25,C25-archaeal membrane lipids reached ∼11 % of total lipids of E. coli. It is noteworthy that the high production of the extended archaeal lipids did not significantly affect the growth of the bacterial cells. The permeability of the cell membrane of the strain became slightly lower in the presence of the exogenous membrane lipids with longer hydrocarbon chains, which demonstrated the possibility to enhance bacterial cell membranes by the hyperthermophile-specific lipids, along with the surprising robustness of the E. coli cell membrane.

A pyruvate transporter in the apicoplast of apicomplexan parasites

Pyruvate lies at a pivotal node of carbon metabolism in eukaryotes. It is involved in diverse metabolic pathways in multiple organelles, and its interorganelle shuttling is crucial for cell fitness. Many apicomplexan parasites harbor a unique organelle called the apicoplast that houses metabolic pathways like fatty acid and isoprenoid precursor biosyntheses, requiring pyruvate as a substrate. However, how pyruvate is supplied in the apicoplast remains enigmatic. Here, deploying the zoonotic parasite Toxoplasma gondii as a model apicomplexan, we identified two proteins residing in the apicoplast membranes that together constitute a functional apicoplast pyruvate carrier (APC) to mediate the import of cytosolic pyruvate. Depletion of APC results in reduced activities of metabolic pathways in the apicoplast and impaired integrity of this organelle, leading to parasite growth arrest. APC is a pyruvate transporter in diverse apicomplexan parasites, suggesting a common strategy for pyruvate acquisition by the apicoplast in these clinically relevant intracellular pathogens.

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, β–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.

Functional analysis of the methylerythritol phosphate pathway terminal enzymes IspG and IspH from Zymomonas mobilis.

Isoprenoids are one of the largest classes of natural products, exhibiting diversity in structure and function. They also include compounds that are essential for cellular life across the biological world. In bacteria, isoprenoids are derived from two precursors, isopentenyl diphosphate and dimethylallyl diphosphate, synthesized primarily by the methylerythritol phosphate pathway. The aerotolerant Z. mobilis has the potential for methylerythritol phosphate pathway engineering by diverting some of the glucose that is typically efficiently converted into ethanol to produce isoprenoid precursors to make bioproducts and biofuels. Our data revealed the surprising finding that Z. mobilis IspG and IspH need to be co-optimized to improve flux via the methyl erythritol phosphate pathway in part to evade the oxygen sensitivity of IspH.

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.

Two natural compounds as potential inhibitors against the Helicobacter pylori and Acinetobacter baumannii IspD enzymes

In a vast majority of bacteria, protozoa and plants, the methylerythritol phosphate (MEP) pathway is utilized for the synthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), which are precursors for isoprenoids. Isoprenoids, such as cholesterol and coenzyme Q, play a variety of crucial roles in physiological activities, including cell-membrane formation, protein degradation, cell apoptosis, and transcription regulation. In contrast, humans employ the mevalonate (MVA) pathway for the production of IDP and DMADP, rendering proteins in the MEP pathway appealing targets for antimicrobial agents. This pathway consists of seven consecutive enzymatic reactions, of which 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD) and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) catalyze the third and fifth steps, respectively. In this study, we characterized the enzymatic activities and protein structures of Helicobacter pylori IspDF and Acinetobacter baumannii IspD. Then, using the direct interaction-based thermal shift assay, we conducted a compound screening of an approved drug library and identified 27 hit compounds potentially binding to AbIspD. Among them, two natural products, rosmarinic acid and tanshinone IIA sodium sulfonate, exhibited inhibitory activities against HpIspDF and AbIspD, by competing with one of the substrates, MEP. Moreover, tanshinone IIA sodium sulfonate also demonstrated certain antibacterial effects against H. pylori. In summary, we identified two IspD inhibitors from approved ingredients, broadening the scope for antibiotic discovery targeting the MEP pathway.

Ternary complexes of isopentenyl phosphate kinase from Thermococcus paralvinellae reveal molecular determinants of non-natural substrate specificity.

Isopentenyl phosphate kinases (IPKs) have recently garnered attention for their central role in biocatalytic "isoprenol pathways," which seek to reduce the synthesis of the isoprenoid precursors to two enzymatic steps. Furthermore, the natural promiscuity of IPKs toward non-natural alkyl-monophosphates (alkyl-Ps) as substrates has hinted at the isoprenol pathways' potential to access novel isoprenoids with potentially useful activities. However, only a handful of IPK crystal structures have been solved to date, and even fewer of these contain non-natural substrates bound in the active site. The current study sought to elucidate additional ternary complexes bound to non-natural substrates using the IPK homolog from Thermococcus paralvinellae (TcpIPK). Four such structures were solved, each bound to a different non-natural alkyl-P and the phosphoryl donor substrate/product adenosine triphosphate (ATP)/adenosine diphosphate (ADP). As expected, the quaternary, tertiary, and secondary structures of TcpIPK closely resembled those of IPKs published previously, and kinetic analysis of a novel alkyl-P substrate highlighted the potentially dramatic effects of altering the core scaffold of the natural substrate. Even more interesting, though, was the discovery of a trend correlating the position of two α helices in the active site with the magnitude of an IPK homolog's reaction rate for the natural reaction. Overall, the current structures of TcpIPK highlight the importance of continued structural analysis of the IPKs to better understand and optimize their activity with both natural and non-natural substrates.

YGL3 Encoding an IPP and DMAPP Synthase Interacts with OsPIL11 to Regulate Chloroplast Development in Rice

As the source of isoprenoid precursors, the plastidial methylerythritol phosphate (MEP) pathway plays an essential role in plant development. Here, we report a novel rice (Oryza sativa L.) mutant ygl3 (yellow-green leaf3) that exhibits yellow-green leaves and lower photosynthetic efficiency compared to the wild type due to abnormal chloroplast ultrastructure and reduced chlorophyll content. Map-based cloning showed that YGL3, one of the major genes involved in the MEP pathway, encodes 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, which is localized in the thylakoid membrane. A single base substitution in ygl3 plants resulted in lower 4-hydroxy-3-methylbut-2-enyl diphosphate reductase activity and lower contents of isopentenyl diphosphate (IPP) compared to the wild type. The transcript levels of genes involved in the syntheses of chlorophyll and thylakoid membrane proteins were significantly reduced in the ygl3 mutant compared to the wild type. The phytochrome interacting factor-like gene OsPIL11 regulated chlorophyll synthesis during the de-etiolation process by directly binding to the promoter of YGL3 to activate its expression. The findings provides a theoretical basis for understanding the molecular mechanisms by which the MEP pathway regulate chloroplast development in rice.

Evaluation of ketoclomazone and its analogues as inhibitors of 1-deoxy-d-xylulose 5-phosphate synthases and other thiamine diphosphate (ThDP)-dependent enzymes.

Most pathogenic bacteria, apicomplexan parasites and plants rely on the methylerythritol phosphate (MEP) pathway to obtain precursors of isoprenoids. 1-Deoxy-d-xylulose 5-phosphate synthase (DXPS), a thiamine diphosphate (ThDP)-dependent enzyme, catalyses the first and rate-limiting step of the MEP pathway. Due to its absence in humans, DXPS is considered as an attractive target for the development of anti-infectious agents and herbicides. Ketoclomazone is one of the earliest reported inhibitors of DXPS and antibacterial and herbicidal activities have been documented. This study investigated the activity of ketoclomazone on DXPS from various species, as well as the broader ThDP-dependent enzyme family. To gain further insights into the inhibition, we have prepared analogues of ketoclomazone and evaluated their activity in biochemical and computational studies. Our findings support the potential of ketoclomazone as a selective antibacterial agent.

Investigating Novel IspE Inhibitors of the MEP Pathway in Mycobacterium.

In a recent effort to mitigate harm from human pathogens, many biosynthetic pathways have been extensively evaluated for their ability to inhibit pathogen growth and to determine drug targets. One of the important products/targets of such pathways is isopentenyl diphosphate. Isopentenyl diphosphate is the universal precursor of isoprenoids, which are essential for the normal functioning of microorganisms. In general, two biosynthetic pathways lead to the formation of isopentenyl diphosphate: (1) the mevalonate pathway in animals; and (2) the non-mevalonate or methylerythritol phosphate (MEP) in many bacteria, and some protozoa and plants. Because the MEP pathway is not found in mammalian cells, it is considered an attractive target for the development of antimicrobials against a variety of human pathogens, including Mycobacterium tuberculosis (M.tb). In the MEP pathway, 4-diphosphocytidyl-2-c-methyl-d-erythritol kinase (IspE) phosphorylates 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDPME) to form 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDPME2P). A virtual high-throughput screening against 15 million compounds was carried out by docking IspE protein. We identified an active heterotricyclic compound which showed enzymatic activity; namely, IC50 of 6 µg/mL against M.tb IspE and a MIC of 12 µg/mL against M.tb (H37Rv). Hence, we designed and synthesized similar new heterotricyclic compounds and tested them against mycobacterium, observing a MIC of 5 µg/mL against M. avium. This study will provide the critical insight necessary for developing novel antimicrobials that target the MEP pathways in pathogens.

Miltiradiene Production by Cytoplasmic Metabolic Engineering in Nicotiana benthamiana.

Plant natural products are important sources of innovative drugs, but the extraction and isolation of medicinal natural products from plants is challenging as these compounds have complex structures that are difficult to synthesize chemically. Therefore, utilizing heterologous expression systems to produce medicinal natural products in plants is a novel, environmentally friendly, and sustainable method. In this study, Nicotiana benthamiana was used as the plant platform to successfully produce miltiradiene, the key intermediate of tanshinones, which are the bioactive constituents of the Chinese medicinal plant Salvia miltiorrhiza. The yield of miltiradiene was increased through cytoplasmic engineering strategies combined with the enhancement of isoprenoid precursors. Additionally, we discovered that overexpressing SmHMGR alone accelerated apoptosis in tobacco leaves. Due to the richer membrane systems and cofactors in tobacco compared to yeast, tobacco is more conducive to the expression of plant enzymes. Therefore, this study lays the foundation for dissecting the tanshinone biosynthetic pathway in tobacco, which is essential for subsequent research. Additionally, it highlights the potential of N. benthamiana as an alternative platform for the production of natural products in plants.

Development of Corynebacterium glutamicum as a monoterpene production platform

Monoterpenes are commonly known for their role in the flavors and fragrances industry and are also gaining attention for other uses like insect repellant and as potential renewable fuels for aviation. Corynebacterium glutamicum, a Generally Recognized as Safe microbe, has been a choice organism in industry for the annual million ton-scale bioproduction of amino acids for more than 50 years; however, efforts to produce monoterpenes in C. glutamicum have remained relatively limited. In this study, we report a further expansion of the C. glutamicum biosynthetic repertoire through the development and optimization of a mevalonate-based monoterpene platform. In the course of our plasmid design iterations, we increased flux through the mevalonate-based bypass pathway, measuring isoprenol production as a proxy for monoterpene precursor abundance and demonstrating the highest reported titers in C. glutamicum to date at 1504.6 mg/L. Our designs also evaluated the effects of backbone, promoter, and GPP synthase homolog origin on monoterpene product titers. Monoterpene production was further improved by disrupting competing pathways for isoprenoid precursor supply and by implementing a biphasic production system to prevent volatilization. With this platform, we achieved 321.1 mg/L of geranoids, 723.6 mg/L of 1,8-cineole, and 227.8 mg/L of linalool. Furthermore, we determined that C. glutamicum first oxidizes geraniol through an aldehyde intermediate before it is asymmetrically reduced to citronellol. Additionally, we demonstrate that the aldehyde reductase, AdhC, possesses additional substrate promiscuity for acyclic monoterpene aldehydes.

Exploring the Deoxy-D-xylulose-5-phosphate Synthase Gene Family in Tomato (Solanum lycopersicum).

Isoprenoids are a wide family of metabolites including high-value chemicals, flavors, pigments, and drugs. Isoprenoids are particularly abundant and diverse in plants. The methyl-D-erythritol 4-phosphate (MEP) pathway produces the universal isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate in plant plastids for the downstream production of monoterpenes, diterpenes, and photosynthesis-related isoprenoids such as carotenoids, chlorophylls, tocopherols, phylloquinone, and plastoquinone. The enzyme deoxy-D-xylulose 5-phosphate synthase (DXS) is the first and main rate-determining enzyme of the MEP pathway. In tomato (Solanum lycopersicum), a plant with an active isoprenoid metabolism in several tissues, three genes encode DXS-like proteins (SlDXS1 to 3). Here, we show that the expression patterns of the three genes suggest distinct physiological roles without excluding that they might function together in some tissues. We also confirm that SlDXS1 and 2 are true DXS enzymes, whereas SlDXS3 lacks DXS activity. We further show that SlDXS1 and 2 co-localize in plastidial speckles and that they can be immunoprecipitated together, suggesting that they might form heterodimers in vivo in at least some tissues. These results provide novel insights for the biotechnological use of DXS isoforms in metabolic engineering strategies to up-regulate the MEP pathway flux.

Medium-chain-length polyprenol (C45–C55) formation in chloroplasts of Arabidopsis is brassinosteroid-dependent

Brassinosteroids are important plant hormones influencing, among other processes, chloroplast development, the electron transport chain during light reactions of photosynthesis, and the Calvin-Benson cycle. Medium-chain-length polyprenols built of 9–11 isoprenoid units (C45–C55 carbons) are a class of isoprenoid compounds present in abundance in thylakoid membranes. They are synthetized in chloroplast by CPT7 gene from Calvin cycle derived precursors on MEP (methylerythritol 4-phosphate) isoprenoid biosynthesis pathway. C45–C55 polyprenols affect thylakoid membrane ultra-structure and hence influence photosynthetic apparatus performance in plants such as Arabidopsis and tomato. So far nothing is known about the hormonal or environmental regulation of CPT7 gene expression. The aim of our study was to find out if medium-chain-length polyprenol biosynthesis in plants may be regulated by hormonal cues.We found that the CPT7 gene in Arabidopsis has a BZR1 binding element (brassinosteroid dependent) in its promoter. Brassinosteroid signaling mutants in Arabidopsis accumulate a lower amount of medium-chain-length C45–C55 polyprenols than control plants. At the same time carotenoid and chlorophyll content is increased, and the amount of PsbD1A protein coming from photosystem II does not undergo a significant change. On contrary, treatment of WT plants with epi-brassinolide increases C45–C55 polyprenols content. We also report decreased transcription of MEP enzymes (besides C45–C55 polyprenols, precursors of numerous isoprenoids, e.g. phytol, carotenoids are derived from this pathway) and genes encoding biosynthesis of medium-chain-length polyprenol enzymes in brassinosteroid perception mutant bri1-116. Taken together, we document that brassinosteroids affect biosynthetic pathway of C45–C55 polyprenols.

The interdependence of isoprenoid synthesis and apicoplast biogenesis in malaria parasites.

Isoprenoid precursor synthesis is an ancient and fundamental function of plastid organelles and a critical metabolic activity of the apicoplast in Plasmodium malaria parasites [1-3]. Over the past decade, our understanding of apicoplast properties and functions has increased enormously [4], due in large part to our ability to rescue blood-stage parasites from apicoplast-specific dysfunctions by supplementing cultures with isopentenyl pyrophosphate (IPP), a key output of this organelle [5,6]. In this Pearl, we explore the interdependence between isoprenoid metabolism and apicoplast biogenesis in P. falciparum and highlight critical future questions to answer.

Overexpression of a pseudo-etiolated-in-light-like protein in Taraxacum koksaghyz leads to a pale green phenotype and enables transcriptome-based network analysis of photomorphogenesis and isoprenoid biosynthesis.

Plant growth and greening in response to light require the synthesis of photosynthetic pigments such as chlorophylls and carotenoids, which are derived from isoprenoid precursors. In Arabidopsis, the pseudo-etiolated-in-light phenotype is caused by the overexpression of repressor of photosynthetic genes 2 (RPGE2), which regulates chlorophyll synthesis and photosynthetic genes. We investigated a homologous protein in the Russian dandelion (Taraxacum koksaghyz) to determine its influence on the rich isoprenoid network in this species, using a combination of in silico analysis, gene overexpression, transcriptomics and metabolic profiling. Homology-based screening revealed a gene designated pseudo-etiolated-in-light-like (TkPEL-like), and in silico analysis identified a light-responsive G-box element in its promoter. TkPEL-like overexpression in dandelion plants and other systems reduced the levels of chlorophylls and carotenoids, but this was ameliorated by the mutation of one or both conserved cysteine residues. Comparative transcriptomics in dandelions overexpressing TkPEL-like showed that genes responsible for the synthesis of isoprenoid precursors and chlorophyll were downregulated, probably explaining the observed pale green leaf phenotype. In contrast, genes responsible for carotenoid synthesis were upregulated, possibly in response to feedback signaling. The evaluation of additional differentially expressed genes revealed interactions between pathways. We propose that TkPEL-like negatively regulates chlorophyll- and photosynthesis-related genes in a light-dependent manner, which appears to be conserved across species. Our data will inform future studies addressing the regulation of leaf isoprenoid biosynthesis and photomorphogenesis and could be used in future breeding strategies to optimize selected plant isoprenoid profiles and generate suitable plant-based production platforms.

Biosynthetic Pathway and Medical Role of Cannabigerolic Acid

Nowadays, the application of cannabinoids in various fields is gradually increasing, and more and more studies have been conducted on cannabigerolic acid, the basic compound produced by cannabis plants, which is produced by the reclamation of olivine acid and isopentenyl catalyzed by cannabis isopentenyl transferase. They are isoprenylated polyketone compounds derived from fatty acids and isoprenoid precursors. They are common receptors for all cannabinoid synthetases and have important effects on the production of cannabinoids. At the same time, CBGA also has certain medicinal value, which has certain efficacy in the treatment of diabetes, and glioblastoma, reducing neuroinflammation of neurodegenerative diseases and treating the side effects of chemotherapy, and will not produce psychotoxic side effects. This paper will describe the synthesis pathway and medical effects of cannabigerolic acid.

Specific pathway abundances in the neonatal calf faecal microbiome are associated with susceptibility to Cryptosporidium parvum infection: a metagenomic analysis

BackgroundCryptosporidium parvum is the main cause of calf scour worldwide. With limited therapeutic options and research compared to other Apicomplexa, it is important to understand the parasites’ biology and interactions with the host and microbiome in order to develop novel strategies against this infection. The age-dependent nature of symptomatic cryptosporidiosis suggests a link to the undeveloped immune response, the immature intestinal epithelium, and its associated microbiota. This led us to hypothesise that specific features of the early life microbiome could predict calf susceptibility to C. parvum infection.ResultsIn this study, a single faecal swab sample was collected from each calf within the first week of life in a cohort of 346 animals. All 346 calves were subsequently monitored for clinical signs of cryptosporidiosis, and calves that developed diarrhoea were tested for Rotavirus, Coronavirus, E. coli F5 (K99) and C. parvum by lateral flow test (LFT). A retrospective case–control approach was taken whereby a subset of healthy calves (Control group; n = 33) and calves that went on to develop clinical signs of infectious diarrhoea and test positive for C. parvum infection via LFT (Cryptosporidium-positive group; n = 32) were selected from this cohort, five of which were excluded due to low DNA quality. A metagenomic analysis was conducted on the faecal microbiomes of the control group (n = 30) and the Cryptosporidium-positive group (n = 30) prior to infection, to determine features predictive of cryptosporidiosis. Taxonomic analysis showed no significant differences in alpha diversity, beta diversity, and taxa relative abundance between controls and Cryptosporidium-positive groups. Analysis of functional potential showed pathways related to isoprenoid precursor, haem and purine biosynthesis were significantly higher in abundance in calves that later tested positive for C. parvum (q ≤ 0.25). These pathways are either absent or streamlined in the C. parvum parasites. Though the de novo production of isoprenoid precursors, haem and purines are absent, C. parvum has been shown to encode enzymes that catalyse the downstream reactions of these pathway metabolites, indicating that C. parvum may scavenge those products from an external source.ConclusionsThe host has previously been put forward as the source of essential metabolites, but our study suggests that C. parvum may also be able to harness specific metabolic pathways of the microbiota in order to survive and replicate. This finding is important as components of these microbial pathways could be exploited as potential therapeutic targets for the prevention or mitigation of cryptosporidiosis in bovine neonates.