Enzyme In Biosynthesis Pathway Research Articles

ADP-glucose pyrophosphorylase gene family in soybean and implications in drought stress tolerance.

ADP-glucose pyrophosphorylase (AGPase) is the key rate-limiting enzyme in starch biosynthesis pathway, and has been identified as a potential target for manipulation strategies aimed at improving crop yield and quality. To identify the AGPase gene family members in soybean, and explore the potential implications of GmAGPS2 in drought stress tolerance. Thegenome-wide identification and sequence analysis of soybean AGPase gene family was carried out by bioinformatics methods. The GmAGP gene expression was analyzed using transcriptome data and quantitative real-time PCR (qRT-PCR). Furthermore, transgenic yeast strains overexpressing GmAGPS2 were generated, and their growth was observed under drought stress. In this study, we searched for AGPase genes (GmAGP) in the soybean genome and identified a total of 14 GmAGP genes. The GmAGP proteins had a unique conserved NTP_transferase domain and were mainly located in the chloroplast and cytosol. Evolutionarily, theGmAGP proteins can be clustered into two distinct subgroups; within the same subgroup, they displayed a similar distribution pattern of conserved motifs. The GmAGP genes exhibited an uneven distribution on 10 chromosomes, and segmental duplication contributed to AGPase gene family expansion in soybean. The GmAGP genes presented different tissue expression pattern, in which GmAGPL6, GmAGPL9, and GmAGPL10 mainly exhibited tissue-specific expression pattern. The promoter of GmAGP genes had multiple cis-acting elements related to phytohormones and stress responses, and 8 GmAGP genes contained drought-responsive cis-acting elements. qRT‒PCR analysis demonstrated a significant upregulation expression of GmAGPL6, GmAGPL10, and GmAGPS2 in response to drought stress. Further functional analysis indicated that GmAGPS2 gene could improve yeast growth under drought stress conditions and enhance the drought tolerance of yeast. These results will contribute to further elucidation of the function of GmAGP genes, and offer important candidate genes for the genetic improvement of starch and yield-related traits and the breeding of high drought stress tolerance varieties in soybean.

Short communication: An alternative pathway for melatonin synthesis in the skin of European flounder (Platichthys flesus)

The classic melatonin biosynthesis pathway (Mel; N-acetyl-5-methoxytryptamine) involves two consecutive enzymatic steps that are decisive in hormone production: conversion of serotonin (5-hydroxytryptamine; 5-HT) to N-acetylserotonin (NAS) and the methylation of the last compound to Mel. This pathway requires the activity of the enzymes: the first is of the category of N-acetyltransferases (AANAT, SNAT, or NAT) and the second is N-acetylserotonin O-methyltransferase (ASMT; also known as HIOMT). However, quite recently, new information has been provided on the possibility of an alternative Mel synthesis pathway; it would include a two-step action by these enzymes, but in reverse order, where ASMT (or ASMTL, the enzyme related to ASMT) methylates 5-HT to 5-methoxytryptamine (5-MT), and then the last compound is acetylated by an enzyme of the category of N-acetyltransferases to Mel. In our study on the activity of enzymes in the Mel biosynthesis pathway in flounder skin, we have found an increase in 5-MT level, as a result of the increase in 5-HT concentration, which is followed by a growing concentration of Mel. However, we have not found any increase in Mel concentration, despite an increase in NAS in the samples. Our data strongly suggest an alternative way of Mel production in flounder skin in which 5-HT is first methylated to 5-MT, which is then acetylated to Mel.

Regulation of cholesterol biosynthesis by CTCF and H3K27 methylation is critical for cell migration

CTCF is a key factor in three-dimensional chromatin folding and transcriptional control that was found to affect cancer cell migration by a mechanism that is still poorly understood. To identify this mechanism, we used mouse melanoma cells with a partial loss of function (pLoF) of CTCF. We found that CTCF pLoF inhibits cell migration rate while leading to an increase in the expression of multiple enzymes in the cholesterol biosynthesis pathway along with an elevation in the cellular cholesterol levels. In agreement with the cholesterol changes we detected altered membrane dynamics in CTCF pLoF cells as measured by reduced formation of migrasomes, extracellular vesicles formed at the rear side of migrating cells. Inhibition of cholesterol synthesis in CTCF pLoF cells restored the cellular migration rate and migrasome formation, suggesting that CTCF supports cell migration by suppressing cholesterol synthesis. Detailed analysis of the promoter of Hmgcs1, an early enzyme in the cholesterol synthesis pathway, revealed that CTCF prevents formation of a loop between that promoter and another promoter 200kb away. CTCF also supports PRC2 recruitment to the promoter and deposition of H3K27me3. H3K27me3 at the promoter of Hmgcs1 prevents SREBP2 binding and activation of transcription. By this mechanism, CTCF fine-tunes cholesterol levels to support cell migration. Notably, genome wide association studies suggest a link between CTCF and cholesterol-associated diseases, thus CTCF emerges as a new regulator of cholesterol biosynthesis.

Identification of genus Deinococcus strains by PCR detection using the gyrB gene and its extension to Bacteria domain

In radiation-resistant bacteria belonging to the genus Deinococcus, transposition events of insertion sequences (IS elements) leading to phenotypic changes from a reddish color to white were detected following exposure to gamma irradiation and hydrogen peroxide treatment. This change resulted from the integration of IS elements into the phytoene desaturase gene, a key enzyme in the carotenoid biosynthesis pathway. To facilitate species identification and distinguish among Deinococcus strains, the gyrB gene encoding the B subunit of DNA gyrase was utilized. The s gnificance of the gyrB gene is well recognized not only in genome replication through the regulation of supercoiling but also in phylogenetic analysis providing support for 16S rRNA-based identification. Its mutation rate surpasses that of the 16S rRNA gene, offering greater resolution between closely related species, particularly those exhibiting >99% similarity. In this study, phylogenetic analysis was conducted comparing the 16S rRNA and gyrB gene sequences of Deinococcus species. Species-specific and genus-specific primers targeting Deinococcus species were designed and experimentally validated for selective amplification and rapid identification of the targeted species. This approach allows for the omission of 16S rRNA sequencing in the targeted Deinococcus species. Therefore, the gyrB gene is useful for identifying bacterial species and genus-level detection from individual microbes or microbial consortia using specialized primer sets for PCR amplification.

Berberine Degradation Characteristics and its Degradation Pathway by a Newly Isolated Berberine-Utilizing Agrobacterium.

Berberine (BBR) is widely used as a botanical pesticide due to its broad-spectrum antibacterial and antifungal activities. However, BBR degradation pathway in soil microorganisms, which determines its impact on soil environment, remains poorly understood. Herein, a novel BBR-degrading bacterium Agrobacterium sp. V1 was isolated and characterized. Agrobacterium sp. V1 was able to utilize BBR as the sole carbon source for cell growth, and 50μg/mL of BBR was completely degraded within 48h. To reveal the possible BBR degradation pathway, whole genome sequencing of Agrobacterium sp. V1 was conducted, and proteins in Agrobacterium sp. V1 were aligned with enzymes involved in BBR biosynthesis in Rhizoma Coptidis. The results indicated that more than 60% of enzymes in BBR biosynthesis pathway had orthologs in Agrobacterium sp. V1. Combined with the primary mass spectra of BBR metabolites, a novel BBR degradation pathway in this bacterium was proposed. In summary, the proposed BBR degradation pathway offered new insights into the impact of BBR to the environment and also provided a reference for studying BBR metabolism in microorganisms.

GFPT2 expression is induced by gemcitabine administration and enhances invasion by activating the hexosamine biosynthetic pathway in pancreatic cancer.

Our previous studies revealed a novel link between gemcitabine (GEM) chemotherapy and elevated glutamine-fructose-6-phosphate transaminase 2 (GFPT2) expression in pancreatic cancer (PaCa) cells. GFPT2 is a rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP). HBP can enhance metastatic potential by regulating epithelial-mesenchymal transition (EMT). The aim of this study was to further evaluate the effect of chemotherapy-induced GFPT2 expression on metastatic potential. GFPT2 expression was evaluated in a mouse xenograft model following GEM exposure and in clinical specimens of patients after chemotherapy using immunohistochemical analysis. The roles of GFPT2 in HBP activation, downstream pathways, and cellular functions in PaCa cells with regulated GFPT2 expression were investigated. GEM exposure increased GFPT2 expression in tumors resected from a mouse xenograft model and in patients treated with neoadjuvant chemotherapy (NAC). GFPT2 expression was correlated with post-operative liver metastasis after NAC. Its expression activated the HBP, promoting migration and invasion. Treatment with HBP inhibitors reversed these effects. Additionally, GFPT2 upregulated ZEB1 and vimentin expression and downregulated E-cadherin expression. GEM induction upregulated GFPT2 expression. Elevated GFPT2 levels promoted invasion by activating the HBP, suggesting the potential role of this mechanism in promoting chemotherapy-induced metastasis.

Structures of trehalose-6-phosphate synthase, Tps1, from the fungal pathogen Cryptococcus neoformans : a target for novel antifungals.

Fungal infections are responsible for over a million deaths worldwide each year. Biosynthesis of a disaccharide, trehalose, is required for multiple pathogenic fungi to transition from the environment to the human host. Enzymes in the trehalose biosynthesis pathway are absent in humans and, therefore, are potentially significant targets for novel antifungal therapeutics. One enzyme in the trehalose biosynthesis is trehalose-6-phosphate synthase (Tps1). Here, we describe the cryo-electron microscopy structures of the CnTps1 homo-tetramer in the unliganded form and in complex with a substrate and a product. These structures and subsequent biochemical analysis reveal key details of substrate-binding residues and substrate specificity. These structures should facilitate structure-guided design of inhibitors against CnTps1.

The trichothecene mycotoxin deoxynivalenol facilitates cell-to-cell invasion during wheat-tissue colonization by Fusarium graminearum.

Fusarium head blight disease on small-grain cereals is primarily caused by the ascomycete fungal pathogen Fusarium graminearum. Infection of floral spike tissues is characterized by the biosynthesis and secretion of potent trichothecene mycotoxins, of which deoxynivalenol (DON) is widely reported due to its negative impacts on grain quality and consumer safety. The TRI5 gene encodes an essential enzyme in the DON biosynthesis pathway and the single gene deletion mutant, ΔTri5, is widely reported to restrict disease progression to the inoculated spikelet. In this study, we present novel bioimaging evidence revealing that DON facilitates the traversal of the cell wall through plasmodesmata, a process essential for successful colonization of host tissue. Chemical complementation of ΔTri5 did not restore macro- or microscopic phenotypes, indicating that DON secretion is tightly regulated both spatially and temporally. A comparative qualitative and quantitative morphological cellular analysis revealed infections had no impact on plant cell wall thickness. Immunolabelling of callose at plasmodesmata during infection indicates that DON can increase deposits when applied exogenously but is reduced when F. graminearum hyphae are present. This study highlights the complexity of the interconnected roles of mycotoxin production, cell wall architecture and plasmodesmata in this highly specialized interaction.

Effects of UVA on Flavonol Accumulation in Ginkgo biloba

Ginkgo is an economic tree species with high medicinal value, and flavonols are its main medicinal components. This research was conducted to investigate the molecular mechanism underlying the influence of Ultraviolet A (UVA) treatment on the synthesis of ginkgo flavonols with the aim of increasing their content. Ginkgo full-sib hybrid offspring were used as test materials. The phenylalanine ammonialyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate: CoA ligase (4CL) enzyme activities, as well as flavonol contents, were measured under the same intensity of white light (300 μmol·m−2·s−1) with the addition of 20, 40, and 60 μmol·m−2·s−1 UVA separately after 20 days of treatment. The control check (CK) and treatment with the highest flavonol content were chosen for transcriptome sequencing analysis. The results showed that the PAL, C4H, and 4CL enzyme activities, as well as the flavonol and totalflavonol glycoside contents, of ginkgo hybrid progeny differed significantly under different UVA treatments. They showed a tendency to increase and then decrease, reaching a maximum value under UVA-4 (40 μmol·m−2·s−1 ultraviolet UVA light intensity) treatment. Ribonucleic acid (RNA) sequencing revealed the presence of 4165 genes with differential expression, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the metabolic pathways commonly enriched across all four comparison groups included ‘phenylpropanoid biosynthesis’, while the pathways commonly enriched in green-leaf ginkgo UVA-4 treatment (TL), yellow-leaf ginkgo mutant CK treatment (CKY), and green-leaf ginkgo CK treatment (CKL) were related to ‘flavonoid biosynthesis’. Treatment with UVA light led to the increased expression of PAL and 4CL enzymes in the phenylpropanoid biosynthesis pathway, as well as increased expression of chalcone synthase (CHS), Flavanone 3-hydroxylase (F3H), and flavonol synthase (FLS) enzymes in the flavonoid biosynthesis pathway, thereby promoting the synthesis of ginkgo flavonols. In summary, the use of 40 μmol·m−2·s−1 UVA treatment for 20 days significantly increased the flavonol content and the expression of related enzyme genes in ginkgo hybrid offspring, enhancing ginkgo flavonoids and increasing the medicinal value of ginkgo.

Effects of MhMYB1 and MhMYB2 transcription factors on the monoterpenoid biosynthesis pathway in l-menthol chemotype of Mentha haplocalyx Briq.

Transcription factors MhMYB1 and MhMYB2 correlate with monoterpenoid biosynthesis pathway in l-menthol chemotype of Mentha haplocalyx Briq, which could affect the contents of ( -)-menthol and ( -)-menthone. Mentha haplocalyx Briq., a plant with traditional medicinal and edible uses, is renowned for its rich essential oil content. The distinct functional activities and aromatic flavors of mint essential oils arise from various chemotypes. While the biosynthetic pathways of the main monoterpenes in mint are well understood, the regulatory mechanisms governing different chemotypes remain inadequately explored. In this investigation, we identified and cloned two transcription factor genes from the M. haplocalyx MYB family, namely MhMYB1 (PP236792) and MhMYB2 (PP236793), previously identified by our research group. Bioinformatics analysis revealed that MhMYB1 possesses two conserved MYB domains, while MhMYB2 contains a conserved SANT domain. Yeast one-hybrid (Y1H) analysis results demonstrated that both MhMYB1 and MhMYB2 interacted with the promoter regions of MhMD and MhPR, critical enzymes in the monoterpenoid biosynthesis pathway of M. haplocalyx. Subsequent virus-induced gene silencing (VIGS) of MhMYB1 and MhMYB2 led to a significant reduction (P < 0.01) in the relative expression levels of MhMD and MhPR genes in the VIGS groups of M. haplocalyx. In addition, there was a noteworthy decrease (P < 0.05) in the contents of ( -)-menthol and ( -)-menthone in the essential oil of M. haplocalyx. These findings suggest that MhMYB1 and MhMYB2 transcription factors play a positive regulatory role in ( -)-menthol biosynthesis, consequently influencing the essential oil composition in the l-menthol chemotype of M. haplocalyx. This study serves as a pivotal foundation for unraveling the regulatory mechanisms governing monoterpenoid biosynthesis in different chemotypes of M. haplocalyx.

Rice myo-inositol-3-phosphate synthase 2 (RINO2) alleviates heat injury-induced impairment in pollen germination and tube growth by modulating Ca2+ signaling and actin filament cytoskeleton.

Low phytic acid (lpa) crop is considered as an effective strategy to improve crop nutritional quality, but a substantial decrease in phytic acid (PA) usually has negative effect on agronomic performance and its response to environment adversities. Myo-inositol-3-phosphate synthase (MIPS) is the rate-limiting enzyme in PA biosynthesis pathway, and regarded as the prime target for engineering lpa crop. In this paper, the rice MIPS gene (RINO2) knockout mutants and its wild type were performed to investigate the genotype-dependent alteration in the heat injury-induced spikelet fertility and its underlying mechanism for rice plants being imposed to heat stress at anthesis. Results indicated that RINO2 knockout significantly enhanced the susceptibility of rice spikelet fertility to heat injury, due to the severely exacerbated obstacles in pollen germination and pollen tube growth in pistil for RINO2 knockout under high temperature (HT) at anthesis. The loss of RINO2 function caused a marked reduction in inositol and phosphatidylinositol derivative concentrations in the HT-stressed pollen grains, which resulted in the strikingly lower content of phosphatidylinositol 4,5-diphosphate (PI (4,5) P2) in germinating pollen grain and pollen tube. The insufficient supply of PI (4,5) P2 in the HT-stressed pollen grains disrupted normal Ca2+ gradient in the apical region of pollen tubes and actin filament cytoskeleton in growing pollen tubes. The severely repressed biosynthesis of PI (4,5) P2 was among the regulatory switch steps leading to the impaired pollen germination and deformed pollen tube growth for the HT-stressed pollens of RINO2 knockout mutants.

Cloning, Expression Characteristics of Farnesyl Pyrophosphate Synthase Gene from Platycodon grandiflorus and Functional Identification in Triterpenoid Synthesis.

Platycodon grandiflorus is a medicinal plant whose main component is platycodins, which have a variety of pharmacological effects and nutritional values. The farnesyl pyrophosphate synthase (FPS) is a key enzyme in the isoprenoid biosynthesis pathway, which catalyzes the synthesis of farnesyl diphosphate (FPP). In this study, we cloned the FPS gene from P. grandiflorus (PgFPS) with an ORF of 1260 bp, encoding 419 amino acids with a deduced molecular weight and theoretical pI of 46,200.98 Da and 6.52, respectively. The squalene content of overexpressed PgFPS in tobacco leaves and yeast cells extract was 1.88-fold and 1.21-fold higher than that of the control group, respectively, and the total saponin content was also increased by 1.15 times in yeast cells extract, which verified the biological function of PgFPS in terpenoid synthesis. After 48 h of MeJA treatment and 6 h of ethephon treatment, the expression of the PgFPS gene in roots and stems reached its peak, showing a 3.125-fold and 3.236-fold increase compared to the untreated group, respectively. Interestingly, the expression of the PgFPS gene in leaves showed a decreasing trend after exogenous elicitors treatment. The discovery of this enzyme will provide a novel perspective for enhancing the efficient synthesis of platycodins.

A multilevel investigation to reveal the regulatory mechanism of lignin accumulation in juice sac granulation of pomelo

Granulation of juice sacs is a physiological disorder, which affects pomelo fruit quality. Here, the transcriptome and ubiquitinome of the granulated juice sacs were analyzed in Guanxi pomelo. We found that lignin accumulation in the granulated juice sacs was regulated at transcription and protein modification levels. In transcriptome data, we found that the genes in lignin biosynthesis pathway and antioxidant enzyme system of the granulated juice sacs were significantly upregulated. However, in ubiquitinome data, we found that ubiquitinated antioxidant enzymes increased in abundance but the enzyme activities decreased after the modification, which gave rise to reactive oxygen species (ROS) contents in granulated juice sacs. This finding suggests that ubiquitination level of the antioxidant enzymes is negatively correlated with the enzyme activities. Increased H2O2 is considered to be a signaling molecule to activate the key gene expressions in lignin biosynthesis pathway, which leads to the lignification in granulated juice sacs of pomelo. This regulatory mechanism in juice sac granulation of pomelo was further confirmed through the verification experiment using tissue culture by adding H2O2 or dimethylthiourea (DMTU). Our findings suggest that scavenging H2O2 and other ROS are important for reducing lignin accumulation, alleviating juice sac granulation and improving pomelo fruit quality.

Congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) is a rare autosomal recessive disease due to the deficient, but not absent, activity of uroporphyrinogen III synthase (UROS), the fourth enzyme in the heme biosynthesis pathway. Biallelic variants in the UROS gene result in decreased UROS enzymatic activity and the accumulation of non-physiologic Type I porphyrins in cells and fluids. Overproduced uroporphyrins in haematopoietic cells are released into the circulation and distributed to tissues, inducing primarily hematologic and dermatologic symptoms. The clinical manifestations vary in severity ranging from non-immune hydrops fetalis in utero to mild dermatologic manifestations in adults. Here, the biochemical, molecular and clinical features of CEP as well as current and new treatment options, including the rescue of UROS enzyme activity by chaperones, are presented.

In silico analysis and characterization of potential inhibitors of MmaA3, a methoxy mycolic acid synthase from Mycobacterium tuberculosis

The emergence of the multi-and extensively drug-resistant (MDR and XDR) strains of Mycobacterium tuberculosis (M.tb), necessitates paradigm-shifting therapeutic approaches. The impermeable waxy lipid layer, primarily composed of mycolic acids, is a key factor in conferring resistance to conventional drugs. This study introduces a novel strategy to combat drug resistance by targeting Methoxy mycolic acid synthase 3 (MmaA3), a critical enzyme in the mycolic acid biosynthesis pathway. MmaA3 is responsible for the O-methylation of hydroxymycolate precursors and emerges as a promising therapeutic target. Through homology-based modeling, we generated a three-dimensional structure of MmaA3, providing crucial insights into its structural characteristics. High throughput virtual screening was performed against the MmaA3 model, using diverse sources: knowledge-based, FDA-approved Drugbank, and Asinex-Elite libraries. Through rigorous computational analyses, including binding affinity assessments, molecular interactions analysis, and binding free energy calculations, potential inhibitors of MmaA3 have been identified. Subsequent validation studies evaluated the stability of top protein-ligand complexes, and free energy calculations using molecular dynamics simulations. The stability of complexes within the catalytic site was confirmed through RMSD and RMSF profile analyses. Furthermore, binding free energy calculations using the MM-GBSA approach revealed significant binding affinity of identified ligands for MmaA3 target protein, comparable to its substrate/cofactors. These findings underscore the potential of the proposed molecules as candidates for further experimental exploration, offering promising avenues for the development of effective inhibitors against M.tb. Overall, our research contributes to significantly advancing the formulation of progressive therapeutic strategies in combating drug-resistant tuberculosis. Communicated by Ramaswamy H. Sarma

Molecular and cellular consequences of mevalonate kinase deficiency

Mevalonate kinase deficiency (MKD) is an autosomal recessive metabolic disorder associated with recurrent autoinflammatory episodes. The disorder is caused by bi-allelic loss-of-function variants in the MVK gene, which encodes mevalonate kinase (MK), an early enzyme in the isoprenoid biosynthesis pathway. To identify molecular and cellular consequences of MKD, we studied primary fibroblasts from severely affected patients with mevalonic aciduria (MKD-MA) and more mildly affected patients with hyper IgD and periodic fever syndrome (MKD-HIDS). As previous findings indicated that the deficient MK activity in MKD impacts protein prenylation in a temperature-sensitive manner, we compared the subcellular localization and activation of the small Rho GTPases RhoA, Rac1 and Cdc42 in control, MKD-HIDS and MKD-MA fibroblasts cultured at physiological and elevated temperatures. This revealed a temperature-induced altered subcellular localization and activation in the MKD cells. To study if and how the temperature-induced ectopic activation of these signalling proteins affects cellular processes, we performed comparative transcriptome analysis of control and MKD-MA fibroblasts cultured at 37 °C or 40 °C. This identified cell cycle and actin cytoskeleton organization as respectively most down- and upregulated gene clusters. Further studies confirmed that these processes were affected in fibroblasts from both patients with MKD-MA and MKD-HIDS. Finally, we found that, similar to immune cells, the MK deficiency causes metabolic reprogramming in MKD fibroblasts resulting in increased expression of genes involved in glycolysis and the PI3K/Akt/mTOR pathway. We postulate that the ectopic activation of small GTPases causes inappropriate signalling contributing to the molecular and cellular aberrations observed in MKD.

Transcription factor PgNAC72 activates DAMMARENEDIOL SYNTHASE expression to promote ginseng saponin biosynthesis.

Ginsenosides, the primary bioactive constituents in ginseng (Panax ginseng), possess substantial pharmacological potential and are in high demand in the market. The plant hormone methyl jasmonate (MeJA) effectively elicits ginsenoside biosynthesis in P. ginseng, though the regulatory mechanism remains largely unexplored. NAC transcription factors are critical in intricate plant regulatory networks and participate in numerous plant physiological activities. In this study, we identified a MeJA-responsive NAC transcription factor gene, PgNAC72, from a transcriptome library produced from MeJA-treated P. ginseng callus. Predominantly expressed in P. ginseng flowers, PgNAC72 localizes to the nucleus. Overexpressing PgNAC72 (OE-PgNAC72) in P. ginseng callus notably elevated total saponin levels, particularly dammarane-type ginsenosides, by upregulating dammarenediol synthase (PgDDS), encoding a key enzyme in the ginsenoside biosynthesis pathway. Electrophoretic mobility shift assays and dual-luciferase assays confirmed that PgNAC72 binds to the NAC-binding elements in the PgDDS promoter, thereby activating its transcription. Further RNA-seq and terpenoid metabolomic data in the OE-PgNAC72 line confirmed that PgNAC72 enhances ginsenoside biosynthesis. These findings uncover a regulatory role of PgNAC72 in MeJA-mediated ginsenoside biosynthesis, providing insights into the ginsenoside regulatory network and presenting a valuable target gene for metabolic engineering.

The GFPT2-O-GlcNAcylation-YBX1 axis promotes IL-18 secretion to regulate the tumor immune microenvironment in pancreatic cancer

The immunosuppressive microenvironment caused by several intrinsic and extrinsic mechanism has brought great challenges to the immunotherapy of pancreatic cancer. We identified GFPT2, the key enzyme in hexosamine biosynthesis pathway (HBP), as an immune-related prognostic gene in pancreatic cancer using transcriptome sequencing and further confirmed that GFPT2 promoted macrophage M2 polarization and malignant phenotype of pancreatic cancer. HBP is a glucose metabolism pathway leading to the generation of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), which is further utilized for protein O-GlcNAcylation. We confirmed GFPT2-mediated O-GlcNAcylation played an important role in regulating immune microenvironment. Through cellular proteomics, we identified IL-18 as a key downstream of GFPT2 in regulating the immune microenvironment. Through CO-IP and protein mass spectrum, we confirmed that YBX1 was O-GlcNAcylated and nuclear translocated by GFPT2-mediated O-GlcNAcylation. Then, YBX1 functioned as a transcription factor to promote IL-18 transcription. Our study elucidated the relationship between the metabolic pathway of HBP in cancer cells and the immune microenvironment, which might provide some insights into the combination therapy of HBP vulnerability and immunotherapy in pancreatic cancer.

Genome-wide identification and characterization of flavonol synthase (FLS) gene family in Brassica vegetables and their roles in response to biotic and abiotic stresses

Flavonol synthase (FLS) serves as a pivotal enzyme in the flavonol biosynthesis pathway, contributing to the abundance of flavonol compounds found in Brassica vegetables. Nevertheless, there are limited investigations on the identification and analysis of FLS gene family and their precise roles in responding to biotic and abiotic stresses remain elusive in Brassica vegetables. Herein, a total of 6 (Brassica rapa), 9 (Brassica oleracea), and 14 (Brassica napus) FLS genes were identified and distributed on 4, 5, and 8 chromosomes, respectively. Notably, while FLS genes exhibited considerable variation in sequence, exon-intron structure, and molecular weight, they displayed highly conserved domain and motif compositions. Of the six identified BrFLSs, namely BrFLS1, BrFLS2, BrFLS3.1, BrFLS3.2, BrFLS3.3, and BrFLS4, all were located in the nucleus and cell membrane except for BrFLS4 exclusively in the nucleus. Specifically, BrFLS1, BrFLS3.2, and BrFLS3.3 showed up-regulation after 6-BA, ABA, BR, and GA3 treatments, while BrFLS2 exhibited down-regulation. Intriguingly, BrFLS members displayed distinct expression patterns under diverse abiotic and biotic stresses. Employing the virus-induced gene silencing (VIGS) technology, we validated BrFLS1/BrFLS3.2, BrFLS1/BrFLS3.3, BrFLS2, BrFLS1, and BrFLS1/BrFLS2/BrFLS3.1/BrFLS3.3 as vital candidate genes, contributing to the flavonol biosynthesis pathway under drought, salt, high temperature, low temperature, and anthracnose stresses in Brassica vegetables, respectively, and they enhanced their resistance to stresses by bolstering antioxidant capacity. Overall, our findings shed light on the biological roles of FLS gene family in the flavonoid biosynthesis pathway, offering valuable genetic resources for crop enhancement.

Arginine inhibits the arginine biosynthesis rate-limiting enzyme and leads to the accumulation of intracellular aspartate in Synechocystis sp. PCC 6803

Cyanobacteria are oxygen-evolving photosynthetic prokaryotes that affect the global carbon and nitrogen turnover. Synechocystis sp. PCC 6803 (Synechocystis 6803) is a model cyanobacterium that has been widely studied and can utilize and uptake various nitrogen sources and amino acids from the outer environment and media. l-arginine is a nitrogen-rich amino acid used as a nitrogen reservoir in Synechocystis 6803, and its biosynthesis is strictly regulated by feedback inhibition. Argininosuccinate synthetase (ArgG; EC 6.3.4.5) is the rate-limiting enzyme in arginine biosynthesis and catalyzes the condensation of citrulline and aspartate using ATP to produce argininosuccinate, which is converted to l-arginine and fumarate through argininosuccinate lyase (ArgH). We performed a biochemical analysis of Synechocystis 6803 ArgG (SyArgG) and obtained a Synechocystis 6803 mutant overexpressing SyArgG and ArgH of Synechocystis 6803 (SyArgH). The specific activity of SyArgG was lower than that of other arginine biosynthesis enzymes and SyArgG was inhibited by arginine, especially among amino acids and organic acids. Both arginine biosynthesis enzyme-overexpressing strains grew faster than the wild-type Synechocystis 6803. Based on previous reports and our results, we suggest that SyArgG is the rate-limiting enzyme in the arginine biosynthesis pathway in cyanobacteria and that arginine biosynthesis enzymes are similarly regulated by arginine in this cyanobacterium. Our results contribute to elucidating the regulation of arginine biosynthesis during nitrogen metabolism.