Geranylgeranyl Diphosphate Synthase Research Articles

Periodontal ligament fibroblasts utilize isoprenoid intermediate farnesyl diphosphate for maintaining osteo/cementogenic differentiation abilities

Background/purposePeroxisome proliferator-activated receptor γ (PPARγ) is a major transcription factor of energy metabolism-associated genes, and three PPARγ isoforms have been identified in periodontal tissues and cells. When energy metabolism homeostasis is affected by PPARγ downregulation in periodontal ligament fibroblasts (PDLFs), osteo/cementogenic abilities are markedly lost. Herein, we investigated whether PPARγ agonists promote periodontal tissue regeneration, and which PPARγ isoforms and metabolic pathways are indispensable for osteo/cementogenic abilities. Materials and methodsA PPARγ agonist was locally administered to regenerate murine periodontal tissue. The distinct functions of the PPARγ isoforms in PDLFs were assessed using an overexpression strategy. Candidate metabolic processes were searched using gene ontology analysis of PPARγ-knockdown PDLFs. In vitro differentiation assays were performed to evaluate the effects of farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), two major isoprenoid intermediates. ResultsPPARγ agonists accelerated periodontal tissue regeneration. Full-length PPARγ overexpression specifically enhanced the osteo/cementogenic differentiation of PPARγ agonist–induced PDLFs. The isoprenoid metabolic process was the top-ranked downregulated metabolism-associated pathway following PPARγ knockdown; FPP and GGPP enhanced and suppressed PDLFs' differentiation, respectively. Gene expression analysis of human clinical periodontal tissues revealed that osteocalcin correlated with farnesyl pyrophosphate synthetase (FDPS), which catalyzes FPP production, but not with two FPP conversion enzymes: geranylgeranyl diphosphate synthase 1 (GGPS1) or farnesyl diphosphate farnesyltransferase 1 (FDFT1). ConclusionPreferable PPARγ agonistic actions depend on the full-length PPARγ isoform. FPP increased PDLFs' osteo/cementogenic abilities. Therefore, administering FPP and precisely controlling FDPS, GGPS1, and FDFT1 activities could be a novel strategy for accelerating periodontal tissue regeneration.

Short-term salicylic acid application induces the expression of GPPS and NEPS1 in catnip

AbstractThe catnip plant, Nepeta cataria L., produces bioactive iridoid monoterpenes known as nepetalactones. These monoterpenes follow a noncanonical biosynthetic route starting as isoprene monomers in the methylerythritol (MEP) pathway after condensation via geranyl pyrophosphate synthase (GPPS) and later cyclization through the iridoid pathway via nepetalactol-related short-chain dehydrogenase enzymes (NEPS). Nepetalactones exhibit insect repellant properties against disease-carrying vectors such as mosquitoes, bed bugs and ticks. Chemical elicitors such as salicylic acid have been used to induce biochemical changes in medicinal and aromatic plant species including catnip that can temporarily increase the yield and quality of secondary metabolites. This is the first study to correlate the effects of short-term salicylic acid application on the genes in the nepetalactone biosynthetic pathway. Both 0.5 mM SA and 1.0 mM SA resulted in upregulation of GPPS and NEPS1. However, within the 24-h time course of the study significant changes in nepetalactone yield were not observed. Correlation analysis revealed a significant relationship between the mRNA transcript abundance for GPPS and NEPS1 demonstrating a low positive co-expression strength for the two genes.

Integrative metabolomic and transcriptomic analyses reveals the accumulation patterns of key metabolites associated with flavonoids and terpenoids of Gynostemma pentaphyllum (Thunb.) Makino.

Gynostemma pentaphyllum (Thunb.) Makino (G. pentaphyllum) is a medicinal and edible plant with multiple functions of liver protection, anti-tumor, anti-inflammation, balancing blood sugar and blood lipids. The nutritional value of the G. pentaphyllum plant is mainly due to its rich variety of biologically active substances, such as flavonoids, terpenes and polysaccharides. In this study, we performed a comprehensive analysis combining metabolomics and root, stem and leaf transcriptomic data of G. pentaphyllum. We used transcriptomics and metabolomics data to construct a dynamic regulatory network diagram of G. pentaphyllum flavonoids and terpenoids, and screened the transcription factors involved in flavonoids and terpenoids, including basic helix-loop-helix (bHLH), myb-related, WRKY, AP2/ERF. Transcriptome analysis results showed that among the DEGs related to the synthesis of flavonoids and terpenoids, dihydroflavonol 4-reductase (DFR) and geranylgeranyl diphosphate synthases (GGPPS) were core genes. This study presents a dynamic image of gene expression in different tissues of G. pentaphyllum, elucidating the key genes and metabolites of flavonoids and terpenoids. This study is beneficial to a deeper understanding of the medicinal plants of G. pentaphyllum, and also provides a scientific basis for further regulatory mechanisms of plant natural product synthesis pathways and drug development.

Isoprenyl diphosphate synthases of terpenoid biosynthesis in rose-scented geranium (Pelargonium graveolens)

The essential oil of Pelargonium graveolens (rose-scented geranium), an important aromatic plant, comprising mainly mono- and sesqui-terpenes, has applications in food and cosmetic industries. This study reports the characterization of isoprenyl disphosphate synthases (IDSs) involved in P. graveolens terpene biosynthesis. The six identified PgIDSs belonged to different classes of IDSs, comprising homomeric geranyl diphosphate synthases (GPPSs; PgGPPS1 and PgGPPS2), the large subunit of heteromeric GPPS or geranylgeranyl diphosphate synthases (GGPPSs; PgGGPPS), the small subunit of heteromeric GPPS (PgGPPS.SSUI and PgGPPS.SSUII), and farnesyl diphosphate synthases (FPPS; PgFPPS).All IDSs exhibited maximal expression in glandular trichomes (GTs), the site of aroma formation, and their expression except PgGPPS.SSUII was induced upon treatment with MeJA. Functional characterization of recombinant proteins revealed that PgGPPS1, PgGGPPS and PgFPPS were active enzymes producing GPP, GGPP/GPP, and FPP respectively, whereas both PgGPPS.SSUs and PgGPPS2 were inactive. Co-expression of PgGGPPS (that exhibited bifunctional G(G)PPS activity) with PgGPPS.SSUs in bacterial expression system showed lack of interaction between the two proteins, however, PgGGPPS interacted with a phylogenetically distant Antirrhinum majus GPPS.SSU. Further, transient expression of AmGPPS.SSU in P. graveolens leaf led to a significant increase in monoterpene levels. These findings provide insight into the types of IDSs and their role in providing precursors for different terpenoid components of P. graveolens essential oil.

The Biosynthesis of the Monoterpene Tricyclene in E. coli through the Appropriate Truncation of Plant Transit Peptides

Tricyclene, a tricyclic monoterpene naturally occurring in plant essential oils, holds potential for the development of medicinal and fuel applications. In this study, we successfully synthesized tricyclene in E. coli by introducing the heterologous mevalonate (MVA) pathway along with Abies grandis geranyl diphosphate synthase (GPPS) and Nicotiana sylvestris tricyclene synthase (TS) XP_009791411. Initially, the shake-flask fermentation at 30 ◦C yielded a tricyclene titer of 0.060 mg/L. By increasing the copy number of the TS-coding gene, we achieved a titer of 0.103 mg/L. To further enhance tricyclene production, optimal truncation in the N-terminal region of TS XP_009791411 resulted in an impressive highest titer of 47.671 mg/L, approximately a 794.5-fold improvement compared to its wild-type counterpart. To the best of our knowledge, this is the highest titer of the heterologous synthesis of tricyclene in E. coli. The SDS-PAGE analysis revealed that lowering induction temperature and truncating the random coil N-terminal region effectively improved TS solubility, which was closely associated with tricyclene production levels. Furthermore, by truncating other TSs, the titers of tricyclene were improved to different degrees.

The Ginkgo biloba microRNA160-ERF4 module participates in terpene trilactone biosynthesis.

Terpene trilactones (TTLs) are important secondary metabolites in ginkgo (Ginkgo biloba); however, their biosynthesis gene regulatory network remains unclear. Here, we isolated a G. biloba ethylene response factors 4 (GbERF4) involved in TTL synthesis. Overexpression of GbERF4 in tobacco (Nicotiana tabacum) significantly increased terpenoid content and upregulated the expression of key enzyme genes (3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), 1-deoxy-D-xylulose-5-phosphate synthase (DXS), acetyl-CoA C-acetyltransferase (AACT), and geranylgeranyl diphosphate synthase (GGPPS)) in the terpenoid pathway in tobacco, suggesting that GbERF4 functions in regulating the synthesis of terpenoids. The expression pattern analysis and previous microRNA (miRNA) sequencing showed that gb-miR160 negatively regulates the biosynthesis of TTLs. Transgenic experiments showed that overexpression of gb-miR160 could significantly inhibit the accumulation of terpenoids in tobacco. Targeted inhibition and dual-luciferase reporter assays confirmed that gb-miR160 targets and negatively regulates GbERF4. Transient overexpression of GbERF4 increased TTL content in G. biloba, and further transcriptome analysis revealed that DXS, HMGS, CYPs, and transcription factor genes were upregulated. In addition, yeast one-hybrid and dual-luciferase reporter assays showed that GbERF4 could bind to the promoters of the HMGS1, AACT1, DXS1, levopimaradiene synthase (LPS2), and GGPPS2 genes in the TTL biosynthesis pathway and activate their expression. In summary, this study investigated the molecular mechanism of the gb-miR160-GbERF4 regulatory module in regulating the synthesis of TTLs. It provides information for enriching the understanding of the regulatory network of TTL biosynthesis and offers important gene resources for the genetic improvement of G. biloba with high contents of TTLs.

α-Amino bisphosphonate triazoles serve as GGDPS inhibitors

Depletion of cellular levels of geranylgeranyl diphosphate by inhibition of the enzyme geranylgeranyl diphosphate synthase (GGDPS) is a potential strategy for disruption of protein transport by limiting the geranylgeranylation of the Rab proteins that regulate intracellular trafficking. As such, there is interest in the development of GGDPS inhibitors for the treatment of malignancies characterized by abnormal protein production, including multiple myeloma. Our previous work has explored the structure-function relationship of a series of isoprenoid triazole bisphosphonate-based GGDPS inhibitors, with modifications having impact on enzymatic, cellular and in vivo activities. We have synthesized a new series of α-amino bisphosphonates to understand the impact of modifying the alpha position with a moiety that is potentially linkable to other agents. Bioassays evaluating the enzymatic and cellular activities of these compounds demonstrate that incorporation of the α-amino group affords compounds with GGDPS inhibitory activity which is modulated by isoprenoid tail chain length and olefin stereochemistry. These studies provide further insight into the complexity of the structure-function relationship and will enable future efforts focused on tumor-specific drug delivery.

Transcriptome and volatile compounds analyses of floral development provide insight into floral scent formation in Paeonia lactiflora 'Wu Hua Long Yu'.

Herbaceous peony (Paeonia lactiflora) is a well-known ornamental plant in China, celebrated for its beautiful flowers that can emit fragrances. However, exact molecular mechanisms governing synthesis of floral volatiles within herbaceous peony remain unclear. To address this gap in knowledge, our study focused on analyzing the transcriptome and the levels of floral volatile compounds in P. lactiflora 'Wu Hua Long Yu' at different stages of flower development. Using gas chromatography-mass spectrometry (GC-MS), we obtained eighteen major volatile compounds, with monoterpenes being the dominant components among them. Our transcriptome analysis, based on pooled sequencing data, revealed the most differentially expressed genes (DEGs) existed between stages S1 and S3 of flower development. Among these DEGs, we identified 89 functional genes associated with the synthesis of volatile monoterpenes, with 28 of these genes showing a positive correlation with the release of monoterpenes. Specifically, key regulators of monoterpene synthesis in herbaceous peony appear to be 1-deoxy-D-xylulose 5-phosphate synthase (DXS), geranyl pyrophosphate synthase (GPPS), and terpene synthase (TPS). Additionally, our study identified some transcription factors (TFs) that may be involved in the biosynthesis of monoterpenes. These discoveries offer invaluable illumination into the intricate molecular underpinnings orchestrating the generation of floral fragrances in herbaceous peonies, and they offer a foundation for further research to identify and utilize candidate gene resources for this purpose.

Cloning and Expression Analysis of Key Enzyme Gene CoGPPS Involved in Iridoid Glycoside Synthesis in Cornus officinalis.

Cornus iridoid glycosides (CIGs), including loganin and morroniside, are the main active components of Cornus officinalis. As one of the key enzymes in the biosynthesis of CIGs, geranyl pyrophosphate synthase (GPPS) catalyzes the formation of geranyl pyrophosphate, which is the direct precursor of CIGs. In this study, the C. officinalis geranyl pyrophosphate synthase (CoGPPS) sequence was cloned from C. officinalis and analyzed. The cDNA sequence of the CoGPPS gene was 915 bp (GenBank No. OR725699). Phylogenetic analysis showed that CoGPPS was closely related to the GPPS sequence of Actinidia chinensis and Camellia sinensis, but relatively distantly related to Paeonia lactiflora and Tripterygium wilfordii. Results from the quantitative real-time PCR showed the spatiotemporal expression pattern of CoGPPS; that is, CoGPPS was specifically expressed in the fruits. Subcellular localization assay proved that CoGPPS was specifically found in chloroplasts. Loganin and morroniside contents in the tissues were detected by high-performance liquid chromatography, and both compounds were found to be at higher levels in the fruits than in leaves. Thus, this study laid the foundation for further studies on the synthetic pathway of CIGs.

Functional characterization of a geranylgeranyl diphosphate synthase in the leaf beetle Monolepta hieroglyphica.

Geranylgeranyl diphosphate synthase (GGPPS) as the short-chain prenyltransferases for catalyzing the formation of the acyclic precursor (E)-GGPP has been extensively investigated in mammals, plants, and microbes, but its functional plasticity is poorly understood in insect species. Here, a single GGPPS in leaf beetle Monolepta hieroglyphica, MhieGGPPS, was functionally investigated. Phylogenetic analysis showed that MhieGGPPS was clustered in one clade with homologs and had six conserved motifs. Molecular docking results indicated that binding sites of dimethylallyl diphosphate (DMAPP), (E)-geranyl pyrophosphate (GPP), and (E)-farnesyl pyrophosphate (FPP) were in the chain-length determination region of MhieGGPPS, respectively. In vitro, recombiant MhieGGPPS could catalyze the formation of (E)-geranylgeraniol against different combinations of substrates including isopentenyl pyrophosphate (IPP)/DMAPP, IPP/(E)-GPP, and IPP/(E)-FPP, suggesting that MhieGGPPS could not only use (E)-FPP but also (E)-GPP and DMAPP as the allylic cosubstrates. In kinetic analysis, the (E)-FPP was most tightly bound to MhieGGPPS than that of others. It was proposed that MhieGGPPS as a multifunctional enzyme is differentiated from the other GGPPSs in the animals and plants, which only accepted (E)-FPP as the allylic cosubstrate. These findings provide valuable insights into understanding the functional plasticity of GGPPS in M. hieroglyphica and the novel biosynthesis mechanism in the isoprenoid pathway.

Nudix hydrolase 23 post-translationally regulates carotenoid biosynthesis in plants.

Carotenoids are essential for photosynthesis and photoprotection. Plants must evolve multifaceted regulatory mechanisms to control carotenoid biosynthesis. However, the regulatory mechanisms and the regulators conserved among plant species remain elusive. Phytoene synthase (PSY) catalyzes the highly regulated step of carotenogenesis and geranylgeranyl diphosphate synthase (GGPPS) acts as a hub to interact with GGPP-utilizing enzymes for the synthesis of specific downstream isoprenoids. Here, we report a function of Nudix hydrolase 23 (NUDX23), a Nudix domain-containing protein, in post-translational regulation of PSY and GGPPS for carotenoid biosynthesis. NUDX23 expresses highly in Arabidopsis (Arabidopsis thaliana) leaves. Overexpression of NUDX23 significantly increases PSY and GGPPS protein levels and carotenoid production, whereas knockout of NUDX23 dramatically reduces their abundances and carotenoid accumulation in Arabidopsis. NUDX23 regulates carotenoid biosynthesis via direct interactions with PSY and GGPPS in chloroplasts, which enhances PSY and GGPPS protein stability in a large PSY-GGPPS enzyme complex. NUDX23 was found to co-migrate with PSY and GGPPS proteins and to be required for the enzyme complex assembly. Our findings uncover a regulatory mechanism underlying carotenoid biosynthesis in plants and offer promising genetic tools for developing carotenoid-enriched food crops.

The potential of R. toruloides mevalonate pathway genes in increasing isoprenoid yields in S. cerevisiae: Evaluation of GGPPS and HMG-CoA reductase

The enzymes of the mevalonate pathway need to be improved to achieve high yields of isoprenoids in the yeast Saccharomyces cerevisiae. The red yeast Rhodosporidium toruloides produces high levels of carotenoids and may have evolved to carry a naturally high flux of isoprenoids. Enzymes from such yeasts are likely to be promising candidates for improvement. Towards this end, we have systematically investigated the various enzymes of the mevalonate pathway of R. toruloides and custom synthesized, expressed, and evaluated six key enzymes in S. cerevisiae. The two nodal enzymes geranyl pyrophosphate synthase (RtGGPPS) and truncated HMG-CoA reductase (RttHMG) of R. toruloides showed a significant advantage to the cells for isoprenoid production as seen by a visual carotenoid screen. These two were analyzed further, and attempts were also made at further improvement. RtGGPPS was confirmed to be superior to the S. cerevisiae enzyme, as seen from in vitro activity determinations and in vivo production of the heterologous diterpenoid sclareol. Four mutants were created through rational mutagenesis but were unable to improve the activity further. In the case of RttHMG, functional evaluation of the enzyme revealed that it was very unstable despite functioning very well in S. cerevisiae. We succeeded in stabilizing the enzyme through mutation of a conserved serine in the catalytic region, which did not alter the enzyme activity per se. In vivo evaluation of the mutant revealed that it could enable better sclareol yields. Therefore, these two enzymes from the red yeast are excellent candidates for heterologous isoprenoid production.

Engineering Nannochloropsis oceanica for the production of diterpenoid compounds

AbstractPhotosynthetic microalgae like Nannochloropsis hold enormous potential as sustainable, light‐driven biofactories for the production of high‐value natural products such as terpenoids. Nannochloropsis oceanica is distinguished as a particularly robust host with extensive genomic and transgenic resources available. Its capacity to grow in wastewater, brackish, and sea waters, coupled with advances in microalgal metabolic engineering, genome editing, and synthetic biology, provides an excellent opportunity. In the present work, we demonstrate how N. oceanica can be engineered to produce the diterpene casbene—an important intermediate in the biosynthesis of pharmacologically relevant macrocyclic diterpenoids. Casbene accumulated after stably expressing and targeting the casbene synthase from Daphne genkwa (DgTPS1) to the algal chloroplast. The engineered strains yielded production titers of up to 0.12 mg g−1 total dry cell weight (DCW) casbene. Heterologous overexpression and chloroplast targeting of two upstream rate‐limiting enzymes in the 2‐C‐methyl‐ d‐erythritol 4‐phosphate pathway, Coleus forskohlii 1‐deoxy‐ d‐xylulose‐5‐phosphate synthase and geranylgeranyl diphosphate synthase genes, further enhanced the yield of casbene to a titer up to 1.80 mg g−1 DCW. The results presented here form a basis for further development and production of complex plant diterpenoids in microalgae.

Geranyl diphosphate synthase large subunits OfLSU1/2 interact with small subunit OfSSUII and are involved in aromatic monoterpenes production in Osmanthus fragrans

Osmanthus fragrans is a famous ornamental tree species for its pleasing floral fragrance. Monoterpenoids are the core floral volatiles of O. fragrans flowers, which have tremendous commercial value. Geranyl diphosphate synthase (GPPS) is a key enzyme that catalyzes the formation of GPP, the precursor of monoterpenoids. However, there are no reports of GPPSs in O. fragrans. Here, we performed RNA sequencing on the O. fragrans flowers and identified three GPPSs. Phylogenetic tree analysis showed that OfLSU1/2 belonged to the GPPS.LSU branch, while the OfSSUII belonged to the GPPS.SSU branch. OfLSU1, OfLSU2 and OfSSUII were all localized in chloroplasts. Y2H and pull-down assays showed that OfLSU1 or OfLSU2 interacted with OfSSUII to form heteromeric GPPSs. Site mutation experiments revealed that the conserved CXXXC motifs of OfLSU1/2 and OfSSUII were essential for the interaction between OfLSU1/2 and OfSSUII. Transient expression experiments showed that OfLSU1, OfLSU2 and OfSSUII co-expressed with monoterpene synthase genes OfTPS1 or OfTPS2 improved the biosynthesis of monoterpenoids (E)-β-ocimene and linalool. The heteromeric GPPSs formed by OfLSU1/2 interacting with OfSSUII further improves the biosynthesis of monoterpenoids. Overall, these preliminary results suggested that the GPPSs play a key role in regulating the production of aromatic monoterpenes in O. fragrans.

ERF5.1 modulates carotenoid accumulation by interacting with CCD4.1 in Lycium.

Carotenoids are important natural pigments and have medical and health functions for humans. Carotenoid cleavage dioxygenase 4 (CCD4) and ethylene responsive factor (ERF) participate in carotenoid metabolism, but their roles in Lycium have not been discovered. Here, we annotated LbCCDs from the Lycium reference genome and found that LbCCD4.1 expression was significantly correlated with the carotenoid metabolites during Lycium five fruit developmental stages. Over-expression of LbCCD4.1 in NQ's leaves resulted in a series of significantly lower contents of carotenoid metabolites, including β-carotene and β-cryptoxanthin. Moreover, LbERF5.1, a transcription factor belonging to the ERF family that was located in the nucleus, was isolated. Significant reductions in the carotenoids, especially lutein, violaxanthin and their derivatives, were observed in over-expressing ERF5.1 transgenic NQ's leaves. Over-expression or virus-induced gene silencing of LbERF5.1 in NQ's leaves induced a consistent up- or down-expression, respectively, of LbCCD4.1. Furthermore, yeast one-hybrid and dual-luciferase reporter assays showed that ERF5.1 interacted with the promoter of CCD4.1 to increase its expression, and LbERF5.1 could bind to any one of the three predicted binding sites in the promoter of LbCCD4.1. A transcriptome analysis of LbERF5.1 and LbCCD4.1 over-expressed lines showed similar global transcript expression, and geranylgeranyl diphosphate synthase, phytoene synthase, lycopene δ-cyclase cytochrome, cytochrome P450-type monooxygenase 97A, cytochrome P450-type monooxygenase 97C, and zeaxanthin epoxidase in the carotenoid biosynthesis pathway were differentially expressed. In summary, we uncovered a novel molecular mechanism of carotenoid accumulation that involved an interaction between ERF5.1 and CCD4.1, which may be used to enhance carotenoid in Lycium.

Characterisation of lepidopteran geranylgeranyl diphosphate synthase as a putative pesticide target.

Geranylgeranyl pyrophosphate (diphosphate) synthase (GGPPS) plays an important role in various physiological processes in insects, such as isoprenoid biosynthesis and protein prenylation. Here, we functionally characterised the GGPPS from the major agricultural lepidopteran pests Spodoptera frugiperda and Helicoverpa armigera. Partial disruption of GGPPS by CRISPR in S. frugiperda decreased embryo hatching rate and larval survival, suggesting that this gene is essential. Functional expression in vitro of Helicoverpa armigera GGPPS in Escherichia coli revealed a catalytically active enzyme. Next, we developed and optimised an enzyme assay to screen for potential inhibitors, such as the zoledronate and the minodronate, which showed a dose-dependent inhibition. Phylogenetic analysis of GGPPS across insects showed that GGPPS is highly conserved but also revealed several residues likely to be involved in substrate binding, which were substantially different in bee pollinator and human GGPPS. Considering the essentiality of GGPPS and its putative binding residue variability qualifies a GGPPS as a novel pesticide target. The developed assay may contribute to the identification of novel insecticide leads.

DNA methylation regulates biosynthesis of tanshinones and phenolic acids during growth of Salvia miltiorrhiza.

DNA methylation plays a crucial role in the regulation of plant growth and the biosynthesis of secondary metabolites. Danshen (Salvia miltiorrhiza) is a valuable Chinese herbal medicine commonly used to treat cardiovascular diseases; its active ingredients are tanshinones and phenolic acids, which primarily accumulate in roots. Here, we conducted a targeted metabolic analysis of S. miltiorrhiza roots at 3 distinct growth stages: 40 d old (r40), 60 d old (r60), and 90 d old (r90). The contents of tanshinones (cryptotanshinone, tanshinone I, tanshinone IIA, and rosmariquinone) and phenolic acids (rosmarinic acid and salvianolic acid B) gradually increased during plant development. Whole-genome bisulfite sequencing and transcriptome sequencing of roots at the 3 growth stages revealed an increased level of DNA methylation in the CHH context (H represents A, T, or C) context at r90 compared with r40 and r60. Increased DNA methylation levels were associated with elevated expression of various genes linked to epigenetic regulations, including CHROMOMETHYLASE2 (SmCMT2), Decrease in DNA Methylation 1 (SmDDM1), Argonaute 4 (SmAGO4), and DOMAINS REARRANGED METHYLTRANSFERASE 1 (SmDRM1). Moreover, expression levels of many genes involved in tanshinone and salvianolic acid biosynthesis, such as copalyldiphosphate synthase 5 (SmCPS5), cytochrome P450-related enzyme (SmCYP71D464), geranylgeranyl diphosphate synthase (SmGGPPS1), geranyl diphosphate synthase (SmGPPS), hydroxyphenylpyruvate reductase (SmHPPR), and hydroxyphenylpyruvate dioxygenase (SmHPPD), were altered owing to hyper-methylation, indicating that DNA methylation plays an important role in regulating tanshinone and phenolic acid accumulation. Our data shed light on the epigenetic regulation of root growth and the biosynthesis of active ingredients in S. miltiorrhiza, providing crucial clues for further improvement of active compound production via molecular breeding in S. miltiorrhiza.

Boosting production of cembratriene-ol in Saccharomyces cerevisiae via systematic optimization.

Cembratriene-ol is a good biodegradable biopesticide ingredient with future potential applications in the field of sustainable agriculture. Cembratriene-ol is a monocyclic diterpenoid compound that is synthesized only in the trichome gland of Nicotiana plants. In this study, geranylgeranyl diphosphate synthase gene ggpps from Taxus canadensis and cbts*Δp were heterologously expressed in Saccharomyces cerevisiae W303-1A to successfully synthesize cembratriene-ol. The titer of cembratriene-ol was increased by 1.84-fold compared to the control by overexpressing the S. cerevisiae bifunctional (2E,6E)-farnesyl diphosphate synthase genes ERG20 and cbts*Δp under one promoter PGAP . The titer of cembratriene-ol in the engineered S. cerevisiae BY4741 was increased by 1.39-fold compared to the engineered S. cerevisiae W303-1A. The titer of cembratriene-ol in the engineered S. cerevisiae BY4741 was increased by 2.22-fold compared to the control by overexpressing ERG20 and cbts*Δp, respectively, using two promoters PGAP . Cembratriene-ol was found to be successfully synthesized via the integrated expression of cbts*Δp, ggpps and ERG20 on the genome of S. cerevisiae BY4741. The titer of cembratriene-ol in S. cerevisiae S25 was further increased by 1.80-fold compared to the control via dynamic control of the squalene synthase gene ERG9. Overexpression of the genes cbts*Δp and ggpps using pY26-GPD-TEF in S. cerevisiae S25 with their integration expression increased the titer of cembratriene-ol by 26.1-fold compared to S. cerevisiae S25. The titer of cembratriene-ol was significantly enhanced by mitochondrial compartmentalized expression of cbts*Δp and ggpps, which was 76.3-fold higher than that of the initial strain constructed. It was indicated that the systematic optimization has great potential in facilitating high-level production of cembratriene-ol production in S. cerevisiae.

CircUBR1 knockdown relieves ventilator-induced lung injury through regulating miR-20a-5p/GGPPS1 pathway

ObjectiveTo assess the influences and underlying mechanism of circular RNA UBR1 (circUBR1) in ventilator-induced lung injury (VILI). MethodsIn mice and mouse alveolar epithelial cells, VILI model was established. CircUBR1 and miR-20a-5p expression was assessed via quantitative real time polymerase chain reaction. Western blot and immunohistochemistry were applied to assess geranylgeranyl diphosphate synthase 1 (GGPPS1) protein expression. In lung tissues, the histopathological changes were utilized using hematoxylin and eosin staining. Cell counting kit-8 assay and flow cytometer were applied to detect cell proliferation and apoptosis. The levels of inflammatory cytokines [interleukin (IL)-1β, IL-18, IL-6, and tumor necrosis factor (TNF)-α] were measured by western blot and enzyme-linked immunosorbent assay. ResultsIn lung tissues of VILI mice, circUBR1 and GGPPS1 expression were upregulated, while miR-20a-5p expression was downregulated. In vivo, circUBR1 knockdown alleviated lung injury, inhibited cell apoptosis, and decreased the levels of inflammatory cytokines. In cells treated with cyclic stretch (CS), circUBR1 knockdown promoted cell viability, inhibited cell apoptosis, and reduced inflammatory cytokines. CircUBR1 could sponge miR-20a-5p, and GGPPS1 was the target gene of miR-20a-5p. In addition, in cells treated with CS, downregulation of miR-20a-5p or the overexpression of GGPPS1 reversed the promotive effect of circUBR1 knockdown on cell viability and the inhibitive effect of circUBR1 knockdown on cell apoptosis and inflammation production. ConclusionsIn VILI, knockdown of circUBR1 attenuated lung injury and inflammation via regulating the miR-20a-5p/GGPPS1 pathway. Our study may provide a potential therapeutic target for treatment of VILI.

Drought stress-mediated alterations in secondary metabolites and biosynthetic gene expression in cumin plants: Insights from gene-specific and metabolite-level analyses

In this research, we investigated the impact of drought stress on secondary metabolite production in cumin plants and the associated transcriptional changes in biosynthetic genes. Drought stress induced a significant increase in the levels of beta-carotene, lycopene, terpenes, anthocyanin, phenolic compounds, flavonoids, and alkaloids, indicating an adaptive metabolic shift in response to adverse conditions. Concurrently, there was an upregulation in the expression of key biosynthetic genes, including Phenylalanine ammonia-lyase (PAL), 3-deoxy-7-phosphoheptulonate synthase (DAHP synthase), Deoxy xylose phosphate reductase (DXS), 3‑hydroxy-3-methylglutaryl-CoA reductase (HMGR), and Geranyl diphosphate synthase (GPPS), which corroborated the metabolite findings. Notably, while most secondary metabolites exhibited an increasing trend under drought stress, flavonoids displayed a disproportionate elevation, even as there was an overall decrease in phenolic compounds and other metabolites. This observation suggests the potential activation of alternative flavonoid synthesis pathways or the conversion of phenolic compounds to flavonoids during stress. Strong correlations were identified between the expression of biosynthetic genes and their respective metabolite concentrations, underscoring the central role of transcriptional regulation in secondary metabolite synthesis during drought stress. Collectively, our results provide a comprehensive understanding of the transcriptional and metabolic adaptations of cumin plants when exposed to drought stress. Such insights can be pivotal in designing drought-resilient crops, with significant implications for food security amidst escalating climatic challenges.