O-methyltransferase Research Articles

Host-Induced Gene Silencing of the Aspergillus flavus O-Methyl Transferase Gene Enhanced Maize Aflatoxin Resistance

Maize is one of the major crops that are susceptible to Aspergillus flavus infection and subsequent aflatoxin contamination, which poses a serious health threat to humans and domestic animals. Here, an RNA interference (RNAi) approach called Host-Induced Gene Silencing (HIGS) was employed to suppress the O-methyl transferase gene (omtA, also called aflP), a key gene involved in aflatoxin biosynthesis. An RNAi vector carrying part of the omtA gene was introduced into the B104 maize line. Among the six transformation events that were positive for containing the omtA transgene, OmtA-6 and OmtA-10 were self-pollinated from T1 to T4, and OmtA-7 and OmtA-12 to the T6 generation. These four lines showed at least an 81.3% reduction in aflatoxin accumulation at the T3 generation under laboratory conditions. When screened under field conditions with artificial inoculation, OmtA-7 at T5 and T6 generations and OmtA-10 at T4 generation showed a reduction in aflatoxin contamination between 60% and 91% (p < 0.02 to p < 0.002). In order to develop commercial maize lines with enhanced aflatoxin resistance, the omtA transgene in OmtA-7 was introduced into three elite inbred lines through crossing, and the resulting crosses also exhibited significantly lower aflatoxin accumulation compared to crosses with non-transgenic controls (p < 0.04). In addition, high levels of omtA-specific small RNAs were only detected in the transgenic kernel and leaf tissues. These results demonstrate that suppression of omtA through HIGS can enhance maize resistance to aflatoxin contamination, and this resistance can be transferred to elite backgrounds, providing a viable and practical approach to reduce aflatoxin contamination in maize.

A chromosome level reference genome of Diviner’s sage (Salvia divinorum) provides insight into salvinorin A biosynthesis

BackgroundDiviner’s sage (Salvia divinorum; Lamiaceae) is the source of the powerful hallucinogen salvinorin A (SalA). This neoclerodane diterpenoid is an agonist of the human Κ-opioid receptor with potential medical applications in the treatment of chronic pain, addiction, and post-traumatic stress disorder. Only two steps of the approximately twelve step biosynthetic sequence leading to SalA have been resolved to date.ResultsTo facilitate pathway elucidation in this ethnomedicinal plant species, here we report a chromosome level genome assembly. A high-quality genome sequence was assembled with an N50 value of 41.4 Mb and a BUSCO completeness score of 98.4%. The diploid (2n = 22) genome of ~ 541 Mb is comparable in size and ploidy to most other members of this genus. Two diterpene biosynthetic gene clusters were identified and are highly enriched in previously unidentified cytochrome P450s as well as crotonolide G synthase, which forms the dihydrofuran ring early in the SalA pathway. Coding sequences for other enzyme classes with likely involvement in downstream steps of the SalA pathway (BAHD acyl transferases, alcohol dehydrogenases, and O-methyl transferases) were scattered throughout the genome with no clear indication of clustering. Differential gene expression analysis suggests that most of these genes are not inducible by methyl jasmonate treatment.ConclusionsThis genome sequence and associated gene annotation are among the highest resolution in Salvia, a genus well known for the medicinal properties of its members. Here we have identified the cohort of genes responsible for the remaining steps in the SalA pathway. This genome sequence and associated candidate genes will facilitate the elucidation of SalA biosynthesis and enable an exploration of its full clinical potential.

Genome-wide investigation of caffeic acid O-methyltransferases and expression analysis under different abiotic stresses and anthracnose infection in pepper (Capsicum annuum L.)

ABSTRACT The caffeic acid O-methyl transferases (COMTs) facilitate the biosynthesis of lignin and play important roles in many processes, including growth, development and response to stresses. Pepper (Capsicum annuum L.) is a major horticultural crop with significant nutritional and medicinal values. Nevertheless, a comprehensive assessment of the COMT genes in pepper and their involvement in drought, salt tolerance and anthracnose resistance remains unestablished. In the present study, 80 pepper COMT genes were identified and classified into four major groups based on domain structure and phylogenetics. Additionally, the sequence analysis, gene structures, and duplication events were analysed. The genes were unevenly distributed across 10 chromosomes, and 10 pairs of tandem and 13 pairs of segmental duplicated genes were detected in the COMT gene family. Examination of cis-acting regulatory elements detected the presence of phytohormone-specific, stress-receptive and light responsive elements in the pepper COMT promoters. Moreover, qRT-PCR analysis revealed significant upregulated expression of CanCOMT48 to drought stress, CanCOMT14 to salinity stress and CanCOMT18 to anthracnose infection by Colletotrichum truncatum, suggesting their important functions in different stresses. Our results provide significant insights into the expression and probable function of selective COMTs which could be used as potential candidate genes for pepper tolerance breeding.

Systematic Identification and Characterization of O-Methyltransferase Gene Family Members Involved in Flavonoid Biosynthesis in Chrysanthemum indicum L.

Chrysanthemum indicum L. capitulum is an enriched source of flavonoids with broad-ranging biological activities, mainly due to their anti-inflammatory, anti-cancer, immune regulation, anti-microbial activity, hepatoprotective, and neuroprotective effects. The O-methylation of various secondary metabolites has previously been demonstrated to be mainly catalyzed by S-adenosyl-L-methionine-dependent O-methyltransferase (OMT) proteins encoded by the OMT gene family. However, limited comprehensive study was published on the OMT gene family, especially the CCoAOMT subfamily, involved in the O-methylation of flavonoids in Chrysanthemum. Here, we analyzed the spatiotemporal expression patterns of C. indicum OMT genes in leaf and flower at different developmental stages. Transcriptome sequencing and qRT-PCR analysis showed that COMTs were mainly highly expressed in capitulum, especially in full bloom, while CCoAOMTs were mainly highly expressed in leaves. Correlation analysis of OMT gene expression and flavonoids accumulation revealed that four OMTs (CHR00029120, CHR00029783, CHR00077404, and CHR00078333) were putatively involved in most methylated flavonoids biosynthesis in the capitulum. Furthermore, we identified a true CCoAOMT enzyme, CiCCoAOMT1, and found that it catalyzed O-methylation of quercetin and luteolin at the 3'-OH position. In summary, this work provides an important theoretical basis for further research on the biological functions of OMTs in C. indicum.

Addressing the Ethnicity Gap in Catechol O-Methyl Transferase Inhibitor Trials in Parkinson's Disease: A Review of Available Global Data.

Catechol-O-methyltransferase inhibitors (COMT-Is) have significantly improved the quality of life and symptom management for those at advanced stages of Parkinson's Disease (PD). Given that PD is one of the fastest-growing neurodegenerative diseases worldwide, there is a need to establish a clear framework for the systematic distribution of COMT-Is, considering inter-individual and intra-individual variations in patient response. One major barrier to this is the underrepresentation of ethnic minority participants in clinical trials investigating COMT-Is. To investigate this, we performed a narrative review. We searched PubMed for clinical trials investigating COMT-Is in patients with PD and examined the ethnic diversity of cohorts. A total of 63 articles were identified, with 34 trials found to match our inclusion criteria. Among the 34 trials meeting our inclusion criteria, only 8 reported participants' ethnic backgrounds. Our findings reveal a consistent underrepresentation of ethnic minority groups in trials investigating COMT-Is in PD cohorts-a trend that reflects broader concerns across clinical research. In this review, we explore potential reasons for the underrepresentation of ethnic minorities in clinical trials and propose strategies to address this issue.

ScRNA-seq reveals the spatiotemporal distribution of camptothecin pathway and transposon activity in Camptotheca acuminata shoot apexes and leaves.

Camptotheca acuminata Decne., a significant natural source of the anticancer drug camptothecin (CPT), synthesizes CPT through the monoterpene indole alkaloid (MIA) pathway. In this study, we used single-cell RNA sequencing (scRNA-seq) to generate datasets encompassing over 60,000 cells from C. acuminata shoot apexes and leaves. After cell clustering and annotation, we identified five major cell types in shoot apexes and four in leaves. Analysis of MIA pathway gene expression revealed that most of them exhibited heightened expression in proliferating cells (PCs) and vascular cells (VCs). In contrast to MIA biosynthesis in Catharanthus roseus, CPT biosynthesis in C. acuminata did not exhibit multicellular compartmentalization. Some putative genes encoding enzymes and transcription factors (TFs) related to the biosynthesis of CPT and its derivatives were identified through co-expression analysis. These include 19 cytochrome P450 genes, 8 O-methyltransferase (OMT) genes, and 62 TFs. Additionally, these pathway genes exhibited dynamic expression patterns during VC and EC development. Furthermore, by integrating gene and transposable element (TE) expression data, we constructed novel single-cell transcriptome atlases for C. acuminata. This approach significantly facilitated the identification of rare cell types, including peripheral zone cells (PZs). Some TE families displayed cell type specific, tissue specific, or developmental stage-specific expression patterns, suggesting crucial roles for these TEs in cell differentiation and development. Overall, this study not only provides novel insights into CPT biosynthesis and spatial-temporal TE expression characteristics in C. acuminata, but also serves as a valuable resource for further comprehensive investigations into the development and physiology of this species.

The Clausena lansium genome provides new insights into alkaloid diversity and the evolution of the methyltransferase family

Wampee (Clausena lansium) is an important evergreen fruit tree native to southern China that has a long history of use for medicinal purposes. Here, a chromosome-level genome of C. lansium was constructed with a genome size of 282.9 Mb and scaffold N50 of 30.75 Mb. The assembled genome contains 48.70% repetitive elements and 24,381 protein-coding genes. Comparative genomic analysis showed that C. lansium diverged from Aurantioideae 15.91–24.95 million years ago. Additionally, some expansive and specific gene families related to methyltransferase activity and S-adenosylmethionine-dependent methyltransferase activity were also identified. Further analysis indicated that N-methyltransferase (NMT) is mainly involved in alkaloid biosynthesis and O-methyltransferase (OMT) participates in the regulation of coumarin accumulation in wampee. This suggested that wampee's richness in alkaloids and coumarins might be due to the gene expansions of NMT and OMT. The tandem repeat event was one of the major reasons for the NMT expansion. Hence, the reference genome of C. lansium will facilitate the identification of some useful medicinal compounds from wampee resources and reveal their biosynthetic pathways.

Functional identification of three regiospecific flavonoid O-methyltransferases in Rhododendron delavayi and their applications in the biotechnological production of methoxyflavonoids

Rhododendrons produce a variety of methoxyflavonoids, including rarely found 3-methoxyflavonoids and 5-methoxyflavonoids. It was thus suggested that they have a series of regiospecific flavonoid O-methyltransferases (FOMTs). The 18 Class II O-methyltransferase (OMT) genes were retrieved from the Rhododendron delavayi genome, designating them as RdOMTs. A comprehensive biochemical characterization of RdOMTs was performed to identify functional FOMTs. The FOMT activity of recombinant RdOMTs was assayed with flavonoid substrates of different subclasses. Among the examined RdOMTs, RdOMT3, RdOMT10, and RdOMT12 showed FOMT activity for diverse flavonoids. In particular, RdOMT3 consumed only flavonols as a substrate. Structural analyses of the methylated products demonstrated that RdOMT3, RdOMT10, and RdOMT12 catalyze regiospecific methylation of flavonoids at the 3'/5'-, 3-, and 4'-hydroxyl groups, respectively. Their broad substrate spectrum and different regiospecificity suggest that these RdOMTs contribute to the formation of complex methoxyflavonoids in R. delavayi. Bioconversion of flavonoids using E. coli harboring each RdOMT demonstrated that RdOMT3, RdOMT10, and RdOMT12 are useful tools for the biotechnological production of valuable methoxyflavonoids, including the rarely found 3-methoxyflavonoids.

An O-methyltransferase gene, RrCCoAOMT1, participates in the red flower color formation of Rosa rugosa

Rosa rugosa, a valuable ornamental plant species, has high aromatic, ornamental and medicinal values. Diversification of flower color is one of the main purposes for ornamental horticultural breeding, especially the red phenotype. Understanding the molecular mechanism of red color formation regulated by O-methyltransferase (OMT) during anthocyanin biosynthesis is necessary. In this study, 38 candidate members belonging to the CCoAOMT (11) and COMT (27) subfamilies were systematically identified with average lengths of 227 and 360 amino acids, respectively. Two subfamily members had different chromosome distribution characteristics, exon/intron structures, and conserved motifs, consistent with the phylogenetic analysis result of 257 OMT members from six species. Cis-elements in the promoter region of RrCCoAOMTs were mainly involved in hormone and stress response. RrCCoAOMT1 was mainly expressed in petals, and its expression level was significantly decreased in red flowers with high relative percentage of cyanidin. Moreover, RrCCoAOMT1 was separated from other RrCCoAOMTs based on the multiple sequence alignment analysis. Silent expression of RrCCoAOMT1 caused the formation of red/deep-pink phenotypes on purple- and pink-flowered germplasms with an increase in the ratio of Cy3G5G to Pn3G5G. Overall, RrCCoAOMT1 was involved in the modification of red petal color in R. rugosa via changing the ratio of Cy3G5G to Pn3G5G, and may be transcriptionally regulated by MYB transcription factors. These findings provided a theoretical foundation for further studies on the regulation mechanism of red phenotype formation in R. rugosa, and provided important gene resources for flower color improvement breeding.

Identification of O-Methyltransferases Potentially Contributing to the Structural Diversity of 2-(2-Phenylethyl)chromones in Agarwood.

2-(2-Phenylethyl)chromones (PECs) are the primary constituents responsible for the promising pharmacological activities and unique fragrance of agarwood. However, the O-methyltransferases (OMTs) involved in the formation of diverse methylated PECs have not been reported. In this study, we identified one Mg2+-dependent caffeoyl-CoA-OMT subfamily enzyme (AsOMT1) and three caffeic acid-OMT subfamily enzymes (AsOMT2-4) from NaCl-treated Aquilaria sinensis calli. AsOMT1 not only converts caffeoyl-CoA to feruloyl-CoA but also performs nonregioselective methylation at either the 6-OH or 7-OH position of 6,7-dihydroxy-PEC. On the other hand, AsOMT2-4 preferentially utilizes PECs as substrates to produce structurally diverse methylated PECs. Additionally, AsOMT2-4 also accepts nonPEC-type substrates such as caffeic acid and apigenin to generate methylated products. Protein structure prediction and site-directed mutagenesis revealed that residues of L313 and I318 in AsOMT3, as well as S292 and F313 in AsOMT4 determine the distinct regioselectivity of these two OMTs toward apigenin. These findings provide important biochemical evidence of the remarkable structural diversity of PECs in agarwood.

Identification and characterization of two O-methyltransferases involved in methylated 2-(2-phenethyl) chromones biosynthesis in agarwood.

The 2-(2-phenethyl)chromones (PECs) are the signature constituents responsible for the fragrance and pharmacological properties of agarwood. O-methyltransferases (OMTs) are necessary for the methylated PECs biosynthesis, however, there is little information known about OMTs in Aquilaria sinensis. In this study, we identified 29 OMT genes from the A. sinensis genome. Expression analysis showed they were differentially expressed in different tissues and responded to drill wounding. Comprehensive analysis of the gene expression and methylated PECs content revealed that the AsOMT2, AsOMT8, AsOMT11, AsOMT16, and AsOMT28 could potentially be involved in methylated PECs biosynthesis. The vitro enzyme assays and functional analysis in Nicotiana benthamiana demonstrated that AsOMT11 and AsOMT16 could methylate 6-hydroxy-2-(2-phenylethyl)chromone to form 6-methoxy-2-(2-phenylethyl) chromone. The transient overexpression experiment in Qi-Nan revealed that AsOMT11 and AsOMT16 could significantly promote the accumulation of three major methylated PECs. Our results provide candidate genes for the mass production of methylated PECs using synthetic biology.

Antibacterial insights into alternariol and its derivative alternariol monomethyl ether produced by a marine fungus.

Alternaria alternata FB1 is a marine fungus identified as a candidate for plastic degradation in our previous study. This fungus has been recently shown to produce secondary metabolites with significant antimicrobial activity against various pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and the notorious aquaculture pathogen Vibrio anguillarum. The antibacterial compounds were purified and identified as alternariol (AOH) and its derivative, alternariol monomethyl ether (AME). We found that AOH and AME primarily inhibited pathogenic bacteria (MRSA or V. anguillarum) by disordering cell division and some other key physiological and biochemical processes. We further demonstrated that AOH could effectively inhibit the unwinding activity of MRSA topoisomerases, which are closely related to cell division and are the potential action target of AOH. The antibacterial activities of AOH and AME were verified by using zebrafish as the in vivo model. Notably, AOH and AME did not significantly affect the viability of normal human liver cells at concentrations that effectively inhibited MRSA or V. anguillarum. Finally, we developed the genetic operation system of A. alternata FB1 and blocked the biosynthesis of AME by knocking out omtI (encoding an O-methyl transferase), which facilitated A. alternata FB1 to only produce AOH. The development of this system in the marine fungus will accelerate the discovery of novel natural products and further bioactivity study.IMPORTANCEMore and more scientific reports indicate that alternariol (AOH) and its derivative alternariol monomethyl ether (AME) exhibit antibacterial activities. However, limited exploration of their detailed antibacterial mechanisms has been performed. In the present study, the antibacterial mechanisms of AOH and AME produced by the marine fungus Alternaria alternata FB1 were disclosed in vitro and in vivo. Given their low toxicity on the normal human liver cell line under the concentrations exhibiting significant antibacterial activity against different pathogens, AOH and AME are proposed to be good candidates for developing promising antibiotics against methicillin-resistant Staphylococcus aureus and Vibrio anguillarum. We also succeeded in blocking the biosynthesis of AME, which facilitated us to easily obtain pure AOH. Moreover, based on our previous results, A. alternata FB1 was shown to enable polyethylene degradation.

Neofunctionalization of an OMT cluster dominates polymethoxyflavone biosynthesis associated with the domestication of citrus

Polymethoxyflavones (PMFs) are a class of abundant specialized metabolites with remarkable anticancer properties in citrus. Multiple methoxy groups in PMFs are derived from methylation modification catalyzed by a series of hydroxylases and O-methyltransferases (OMTs). However, the specific OMTs that catalyze the systematic O-methylation of hydroxyflavones remain largely unknown. Here, we report that PMFs are highly accumulated in wild mandarins and mandarin-derived accessions, while undetectable in early-diverging citrus species and related species. Our results demonstrated that three homologous genes, CreOMT3, CreOMT4, and CreOMT5, are crucial for PMF biosynthesis in citrus, and their encoded methyltransferases exhibit multisite O-methylation activities for hydroxyflavones, producing seven PMFs in vitro and in vivo. Comparative genomic and syntenic analyses indicated that the tandem CreOMT3, CreOMT4, and CreOMT5 may be duplicated from CreOMT6 and contributes to the genetic basis of PMF biosynthesis in the mandarin group through neofunctionalization. We also demonstrated that N17 in CreOMT4 is an essential amino acid residue for C3-, C5-, C6-, and C3'-O-methylation activity and provided a rationale for the functional deficiency of OMT6 to produce PMFs in early-diverging citrus and some domesticated citrus species. A 1,041-bp deletion in the CreOMT4 promoter, which is found in most modern cultivated mandarins, has reduced the PMF content relative to that in wild and early-admixture mandarins. This study provides a framework for reconstructing PMF biosynthetic pathways, which may facilitate the breeding of citrus fruits with enhanced health benefits.

A Novel O- and S-Methyltransferase from Pleurotus sapidus Is Involved in Flavor Formation.

Increasing consumer aversion to non-natural flavoring substances is prompting a heightened interest in enzymatic processes for flavor production. This includes methylation reactions, which are often performed by using hazardous chemicals. By correlation of aroma profile data and transcriptomic analysis, a novel O-methyltransferase (OMT) catalyzing a respective reaction within the formation of p-anisaldehyde was identified in the mushroom Pleurotus sapidus. Heterologous expression in E. coli followed by purification allowed for further characterization of the enzyme. Besides p-hydroxybenzaldehyde, the proposed precursor of p-anisaldehyde, the enzyme catalyzed the methylation of further hydroxylated aromatic compounds at the meta- and para-position. The Km values determined for p-hydroxybenzaldehyde and S-adenosyl-l-methionine were 80 and 107 μM, respectively. Surprisingly, the studied enzyme enabled the transmethylation of thiol-nucleophiles, as indicated by the formation of 2-methyl-3-(methylthio)furan from 2-methyl-3-furanthiol. Moreover, the enzyme was crystallized at a resolution of 2.0 Å, representing the first published crystal structure of a basidiomycetous OMT.

Genome-wide identification, expression profiling, and protein interaction analysis of the CCoAOMT gene family in the tea plant (Camellia sinensis)

BackgroundThe caffeoyl-CoA-O methyltransferase (CCoAOMT) family plays a crucial role in the oxidative methylation of phenolic substances and is involved in various plant processes, including growth, development, and stress response. However, there is a limited understanding of the interactions among CCoAOMT protein members in tea plants.ResultsIn this study, we identified 10 members of the CsCCoAOMT family in the genome of Camellia sinensis (cultivar ‘HuangDan’), characterized by conserved gene structures and motifs. These CsCCoAOMT members were located on six different chromosomes (1, 2, 3, 4, 6, and 14). Based on phylogenetic analysis, CsCCoAOMT can be divided into two groups: I and II. Notably, the CsCCoAOMT members of group Ia are likely to be candidate genes involved in lignin biosynthesis. Moreover, through the yeast two-hybrid (Y2H) assay, we established protein interaction networks for the CsCCoAOMT family, revealing 9 pairs of members with interaction relationships.ConclusionsWe identified the CCoAOMT gene family in Camellia sinensis and conducted a comprehensive analysis of their classifications, phylogenetic and synteny relationships, gene structures, protein interactions, tissue-specific expression patterns, and responses to various stresses. Our findings shed light on the evolution and composition of CsCCoAOMT. Notably, the observed interaction among CCoAOMT proteins suggests the potential formation of the O-methyltransferase (OMT) complex during the methylation modification process, expanding our understanding of the functional roles of this gene family in diverse biological processes.

Metabolome and Transcriptome Analysis Revealed the Basis of the Difference in Antioxidant Capacity in Different Tissues of Citrus reticulata 'Ponkan'.

Citrus is an important type of fruit, with antioxidant bioactivity. However, the variations in the antioxidant ability of different tissues in citrus and its metabolic and molecular basis remain unclear. Here, we assessed the antioxidant capacities of 12 tissues from Citrus reticulata 'Ponkan', finding that young leaves and root exhibited the strongest antioxidant capacity. Secondary metabolites accumulated differentially in parts of the citrus plant, of which flavonoids were enriched in stem, leaf, and flavedo; phenolic acids were enriched in the albedo, while coumarins were enriched in the root, potentially explaining the higher antioxidant capacities of these tissues. The spatially specific accumulation of metabolites was related to the expression levels of biosynthesis-related genes such as chalcone synthase (CHS), chalcone isomerase (CHI), flavone synthase (FNS), O-methyltransferase (OMT), flavonoid-3'-hydroxylase (F3'H), flavonoid-6/8-hydroxylase (F6/8H), p-coumaroyl CoA 2'-hydroxylase (C2'H), and prenyltransferase (PT), among others, in the phenylpropane pathway. Weighted gene co-expression network analysis (WGCNA) identified modules associated with flavonoids and coumarin content, among which we identified an OMT involved in coumarin O-methylation, and related transcription factors were predicted. Our study identifies key genes and metabolites influencing the antioxidant capacity of citrus, which could contribute to the enhanced understanding and utilization of bioactive citrus components.

Unraveling the Role of COMT Polymorphism in Dopamine-Mediated Vasopressor Effects: An Observational Cross-Sectional Study.

To evaluate the association between rs4680 polymorphism in the COMT gene and the vasoconstrictive effects of commonly used vasopressors. Dopamine is a medication that is given intravenously to critically ill patients to help increase blood pressure. Catechol O-Methyl Transferase (COMT) breaks down dopamine and other catecholamines. There is a genetic variation in the COMT gene called rs4680 that can affect how well the enzyme works. Studies have shown that people with this genetic variation may have different blood pressure levels. However, no one has looked at how this genetic variation affects the way dopamine works to increase blood pressure. To investigate the impact of the rs4680 polymorphism in the COMT gene on the pharmacodynamic response to dopamine. Critically ill patients administered dopamine were included following the consent of their legally acceptable representatives. Details on their demographic characteristics, diagnosis, drug-related details, changes in the heart rate, blood pressure, and urinary output were obtained. The presence of rs4680 polymorphism in the COMT gene was evaluated using a validated method. One hundred and seventeen patients were recruited, and we observed a prevalence of rs4680 polymorphism in 57.3% of our critically ill patients. Those with mutant genotypes were observed with an increase in the median rate of change in mean arterial pressure (mm Hg/hour) [wild: 8.9 (-22.6 to 49.1); heterozygous mutant: 5.9 (-34.1 to 61.6); and homozygous mutant: 19.5 (-2.5 to 129.2)] and lowered urine output (ml/day) [wild: 1080 (21.4 to 5900); heterozygous mutant: 380 (23.7 to 15800); and homozygous mutant: 316.7 (5.8 to 2308.3)]. V158M (rs4680) polymorphism is widely prevalent in the population and was significantly associated with altered effects as observed clinically. This finding suggests valuable insights into the molecular basis of COMT function and its potential impact on neurotransmitter metabolism and related disorders. Large-scale studies delineating the effect of these polymorphisms on various vasopressors are the need of the hour.

Biochemical characterization of a regiospecific flavonoid 3'-O-methyltransferase from orange

Citrus plants have diverse methoxyflavonoids including, chrysoeriol, isosakuranetin, and nobiletin. In plants, O-methyltransferases (OMTs) participate in the methylation of a vast array of secondary metabolites, including flavonoids, phenylpropanoids, and alkaloids. To identify functional OMTs involved in the formation of methoxyflavonoids, orange (Citrus sinensis) OMT (CsOMT) genes were retrieved from the Citrus Genome Database. The phylogenetic relationships with functional OMTs suggested that three CsOMTs, CsOMT15, CsOMT16, and CsOMT30, are possible candidates for flavonoid OMTs (FOMTs). These CsOMTs were heterologously expressed in Escherichia coli, and their OMT activity was examined with flavonoid substrates. Of the examined CsOMTs, CsOMT16 catalyzed the regiospecific 3'-O-methylation of flavonoids to the respective 3'-methoxyflavonoids. A kinetic study demonstrated that CsOMT16 accepts diverse flavonoids as a substrate with a comparable preference. The flavonoids eriodictyol, luteolin, and quercetin were efficiently converted to homoeriodictyol, chrysoeriol, and isorhamnetin by CsOMT16-transformed E. coli cells, respectively. These findings suggest that CsOMT16 contributes to the methoxyflavonoid formation in orange and is applicable to the biotechnological production of 3'-methoxyflavonoids.

Integrated untargeted metabolomic and transcriptomic analyses reveal OMT genes controlling polymethoxyflavonoid biosynthesis in the pericarp of Citrus reticulata ‘Chachi’

The dried pericarp of Citrus reticulata 'Chachi' (CRC) is commonly utilized in Traditional Chinese Medicine (TCM) and food consumption. Geqingpi (GQP), Sihuaqingpi (SHQP) and Guangchenpi (CRP) are TCM materials derived from different developmental stages of CRC pericarp, each possessing distinct medicinal properties. However, there is currently limited study on the active substances in these three stages. Metabolome data suggested that the discrepancies in flavonoid content, particularly polymethoxyflavonoids (PMFs), predominantly contribute to the variations across the three periods. Additionally, through multivariate statistical analysis, 7 potential biomarkers were identified and nobiletin (NOB) possessed the most significant contribution. O-methyltransferases (OMTs) play a crucial role to catalyze the formation of -OCH3 in PMFs. The analysis of transcriptome and genome data revealed the presence of 93 CrOMTs in CRC and 43 were expressed on pericarp. Further investigation identified that 28 of these genes were potentially responsible for catalyzing the biosynthesis of important PMFs such as NOB, sinensetin (SIN), and 3,3′,4′,5,6,7,8-heptamethoxyflavone (HEP). Our results may provide a new perspective for the study of GQP, SHQP and CRP, as well as the regulatory mechanism of PMFs in CRC.

Biosynthetic pathway of prescription bergenin from Bergenia purpurascens and Ardisia japonica.

Bergenin is a typical carbon glycoside and the primary active ingredient in antitussive drugs widely prescribed for central cough inhibition in China. The bergenin extraction industry relies on the medicinal plant species Bergenia purpurascens and Ardisia japonica as their resources. However, the bergenin biosynthetic pathway in plants remains elusive. In this study, we functionally characterized a shikimate dehydrogenase (SDH), two O-methyltransferases (OMTs), and a C-glycosyltransferase (CGT) involved in bergenin synthesis through bioinformatics analysis, heterologous expression, and enzymatic characterization. We found that BpSDH2 catalyzes the two-step dehydrogenation process of shikimic acid to form gallic acid (GA). BpOMT1 and AjOMT1 facilitate the methylation reaction at the 4-OH position of GA, resulting in the formation of 4-O-methyl gallic acid (4-O-Me-GA). AjCGT1 transfers a glucose moiety to C-2 to generate 2-Glucosyl-4-O-methyl gallic acid (2-Glucosyl-4-O-Me-GA). Bergenin production ultimately occurs in acidic conditions or via dehydration catalyzed by plant dehydratases following a ring-closure reaction. This study for the first time uncovered the biosynthetic pathway of bergenin, paving the way to rational production of bergenin in cell factories via synthetic biology strategies.