Molecular Cloning and Characterization of a DFR from Developing Seeds of Blue-grained Wheat in Anthocyanin Biosynthetic Pathway

Blue-grained wheat derived from the hybrid Triticum aestivum L. X Thinopyrum ponticum (Podp.) Barkworth et D. R. Dewey (Agropyron elongatum (Host) P. Beauv., 2n=70). The molecular biological mechanism of the biosynthetic pathway of blue pigments in the blue grain remains unclear yet. Dihydroflavonol 4-reductase (DFR) is one of the key enzymes controlling flavonoid synthesis in anthocyanin biosynthetic pathway, and may directly participate in the formation of blue pigment in the aleurone layer of blue-grained wheat. Here we cloned a DFR cDNA (TaDFR) from the developing seeds of blue-grained wheat, and four DFR genomic DNAs from Th. ponticum (ThpDFR.t), blue-grained wheat (TaDFR.bg), white-grained offspring of light blue-grained wheat (TaDFR.wg) and Chinese Spring (2n=42) (TaDFR.csg), respectively. TaDFR cDNA encodes a 354 amino-acids polypeptide with high identity to DFR from Hordeum vulgare L. (94%), Oryza sativa L. (83%), Zea mays L.(84%). The result of cluster analysis showed that TaDFR cDNA nucleotide sequence has 100% identity with that of TaDFR.csg. The four DFR genomic DNAs have extraordinary high homology and each has three introns. The differences of the four DFR genomic DNAs mainly exist in introns. Southern blotting analysis showed that there are at least 3-5 DFR copies in wheat, the copy numbers in different color grain wheats are not significantly different. The hybridization band patterns were the same, but different from that of Th. ponticum. DFR in blue-grained wheat belongs to a DFR superfamily. Northern blotting analysis indicated that the DFR expressed in the developing seeds of both blue- and white-grained wheat at 15 d after flowering (DAF), the mRNA levels of DFR reached the highest at 18 DAF, then declined quickly and disappeared at 33 DAF But the expression levels in blue-grained seeds were higher than that in white grain at the same seed developing stages. DFR transcripts accumulated in young leaves, and leaf sheaths of blue- and white-grained wheat and Th ponticum, but not detected in roots from different color wheats and developing seeds of Th. ponticum. Results indicated that there may exist some regulatory gene(s) which can increase the expression of DFR in the aleurone layer of blue-grained wheat, and thus resulting in the formation of blue pigments.

Comparative Study on the Expression of Genes Involved in Carotenoid and ABA Biosynthetic Pathway in Response to Salt Stress in Tomato

Carotenoid biosynthetic pathway produces not only pigments that protect photosynthetic system against photo-oxidative damage, but also precursors of abscisic acid, the major hormone regulates stress responses. To understand the response of carotenoid biosynthetic pathway to salt stress, the expression of the genes involved in carotenoid and ABA biosynthesis were compared in cultivated tomato Solanum lycopersicon cv. Moneymaker and its relative wild genotype S. pimpinellifolium （PI365967） together with the contents of carotenoids and ABA. The results showed that 11 of the 15 genes investigated were up-regulated and four unaltered in Moneymaker after 5 h of salt stress; whereas only four genes were up-regulated, four unaltered, and seven down-regulated in PI365967 after stress. Further comparison revealed that 11 salinity-induced genes were expressed significantly lower in Moneymaker than in PI365967 under normal condition, and 8 of them were induced to similar levels after salt stress. In consistence, ABA level was doubled in Moneymaker but kept consistent in PI365967 after salt stress, though the contents of neoxanthin, violaxanthin, [3-carotene, lutein, and total carotenoids were kept unchanged in both species. Since it is known that PI365967 is more tolerant to salt stress than Moneymaker, we proposed that the constitutive high level of carotenoid and ABA biosynthetic pathway under normal growth condition could be benefit to PI365967 for establishing the early response to salt stress. In addition, CrtR-bl and CrtR-b2 that encode [3-carotenoid hydroxylases were the only genes in carotenoid biosynthetic pathway that were up-regulated by salt stress in both species. The CrtR-b2 gene was cloned from both species and no essential difference was found in the encoded amino acid sequences. Transformation of CrtR-b2 to tobacco improved the seed germination under salt stress condition, indicating that the hydrolysis of β-carotenoid is the target of transcriptional regulation of the carotenoid biosynthesis in both tomato cultivar and wild relative.

Insights into the pyrimidine biosynthetic pathway of human malaria parasite Plasmodium falciparum as chemotherapeutic target

Malaria is a major cause of morbidity and mortality in humans. Artemisinins remain as the first-line treatment for Plasmodium falciparum(P. falciparum) malaria although drug resistance has already emerged and spread in Southeast Asia. Thus, to fight this disease, there is an urgent need to develop new antimalarial drugs for malaria chemotherapy. Unlike human host cells, P. falciparum cannot salvage preformed pyrimidine bases or nucleosides from the extracellular environment and relies solely on nucleotides synthesized through the de novo biosynthetic pathway. This review presents significant progress on understanding the de novo pyrimidine pathway and the functional enzymes in the human parasite P. falciparum. Current knowledge in genomics and metabolomics are described, particularly focusing on the parasite purine and pyrimidine nucleotide metabolism. These include gene annotation, characterization and molecular mechanism of the enzymes that are different from the human host pathway. Recent elucidation of the three-dimensional crystal structures and the catalytic reactions of three enzymes: dihydroorotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate decarboxylase, as well as their inhibitors are reviewed in the context of their therapeutic potential against malaria.

Clinical metabolic analysis combined with traceability of biosynthetic pathway:a new approach to quality marker of Chinese materia medica

Quality marker(Q-marker)of Chinese materia medica(CMM)plays an important role in quality control of CMM products.However,its research strategy and technique remain unclear.Based on the fact that quality standard of CMM should be associated with clinical efficacy,taking Jinqi Jiangtang tablet treating type 2 diabetes as an example,the Q-marker related to activity via the reverse analysis of drug metabolism in clinic and traceability of botanic biosynthetic pathways is discovered and validated.Therefore,we proposed a new research strategy of Q-marker of CMM with"Discovery of clinical active constituents as guidance,Reverse analysis of metabolic transformations as link,and Traceability of biosynthesis pathways as key",to improve quality control of CMM products.

Analyzing and engineering of the biosynthetic pathway of mollemycin A for enhancing its production

Mollemycin A(MOMA)is a unique glyco-hexadepsipeptide-polyketide that was isolated from a Streptomyces sp.derived from the Australian marine environment.MOMA exhibits remarkable inhibitory activity against both drug-sensitive and multidrug-resistant malaria parasites.Optimizing MOMA through structural modifications or product enhancements is necessary for the development of effective analogues.However,modifying MOMA using chemical approaches is challenging,and the production titer of MOMA in the wild-type strain is low.This study identified and characterized the biosynthetic gene cluster of MOMA for the first time,proposed its complex biosynthetic pathway,and achieved an effective two-pronged enhancement of MOMA production.The fermentation medium was optimized to increase the yield of MOMA from 0.9 mg L^(-1)to 1.3 mg L^(-1),a 44%boost.Additionally,a synergistic mutant strain was developed by deleting the momB3 gene and overexpressing momB2,resulting in a 2.6-fold increase from 1.3 mg L^(-1)to 3.4 mg L^(-1).These findings pave the way for investigating the biosynthetic mechanism of MOMA,creating opportunities to produce a wide range of MOMA analogues,and developing an efficient strain for the sustainable and economical production of MOMA and its analogues.

The biosynthetic pathway of the hallucinogen mescaline and its heterologous reconstruction

Mescaline,among the earliest identified natural hallucinogens,holds great potential in psychotherapy treatment.Nonetheless,despite the existence of a postulated biosynthetic pathway for more than half a century,the specific enzymes involved in this process are yet to be identified.In this study,we investigated the cactus Lophophora williamsii(Peyote),the largest known natural producer of the phenethylamine mescaline.We employed a multi-faceted approach,combining de novo whole-genome and transcriptome sequencing with comprehensive chemical profiling,enzymatic assays,molecular modeling,and pathway engineering for pathway elucidation.We identified four groups of enzymes responsible for the six catalytic steps in the mescaline biosynthetic pathway,and an N-methyltransferase enzyme that N-methylates all phenethylamine intermediates,likely modulating mescaline levels in Peyote.Finally,we reconstructed the mescaline biosynthetic pathway in both Nicotiana benthamiana plants and yeast cells,providing novel insights into several challenges hindering complete heterologous mescaline production.Taken together,our study opens up avenues for exploration of sustainable production approaches and responsible utilization of mescaline,safeguarding this valuable natural resource for future generations.

Functional characterization of CYP81C16 involved in the tanshinone biosynthetic pathway in Salvia miltiorrhiza

Danshen,the dried roots and rhizomes of Salvia miltiorrhiza Bunge(S.miltiorrhiza),is widely used in the treatment of cardiovascular and cerebrovascular diseases.Tanshinones,the bioactive compounds from Danshen,exhibit a wide spectrum of pharmacological properties,suggesting their potential for future therapeutic applications.Tanshinone biosynthesis is a complex process involving at least six P450 enzymes that have been identified and characterized,most of which belong to the CYP76 and CYP71 families.In this study,CYP81C16,a member of the CYP71 clan,was identified in S.miltiorrhiza.An in vitro assay revealed that it could catalyze the hydroxylation of four para-quinone-type tanshinones,namely neocryptotanshinone,deoxyneocryptotanshinone,and danshenxinkuns A and B.SmCYP81C16 emerged as a potential broad-spectrum oxidase targeting the C-18 position of para-quinone-type tanshinones with an impressive relative conversion rate exceeding 90%.Kinetic evaluations and in vivo assays underscored its highest affinity towards neocryptotanshinone among the tested substrates.The overexpression of SmCYP81C16 promoted the accumulation of(iso)tanshinone in hairy root lines.The characterization of SmCYP81C16 in this study accentuates its potential as a pivotal tool in the biotechnological production of tanshinones,either through microbial or plant metabolic engineering.

De novo nucleotide biosynthetic pathway and cancer

De novo nucleotide biosynthetic pathway is a highly conserved and essential biochemical pathway in almost all organisms.Both purine nucleotides and pyrimidine nucleotides are necessary for cell metabolism and proliferation.Thus,the dysregulation of the de novo nucleotide biosynthetic pathway contributes to the development of many human diseases,such as cancer.It has been shown that many enzymes in this pathway are overactivated in different cancers.In this review,we summarize and update the current knowledge on the de novo nucleotide biosynthetic pathway,regulatory mechanisms,its role in tumorigenesis,and potential targeting opportunities.

Different Expression Analysis in Fruit Softening and Ethylene Biosynthetic Pathways in Peaches of Different Flesh Textures

The aim of our study was to assess differences in the expression of genes involved in fruit softening and ethylene biosynthetic pathways under different temperature storage conditions. Different peach cultivars of ‘Xiacui' and ‘Yumyeong', which are stonyhard, ‘Yinhualu', which is softmelting, ‘Hujing Milu', which is hard-melting, and ‘Baby Gold 6', which is non-melting at 80% ripening, were collected as test materials. The results showed that only slight ethylene production was detected after harvesting of ‘Yumyeong' and ‘Xiacui' under either a room temperature(25 °C) or low temperature(4 °C). The fruit firmness of stonyhard cultivars was retained at a high level under room temperature over time, whereas a low temperature induced ‘Yumyeong' fruit to soften. Quantitative real-time PCR results indicated that the PpACS1 gene was highly expressed in soft-melting, hard-melting and non-melting cultivars; however, expression was extremely low in stonyhard peaches. PpACS2 or PpACS3, however,was not detected in all five cultivars. Interestingly, cold treatment significantly decreased firmness along with endo-PG expression obviously upregulated in ‘Yumyeong', but not in ‘Xiacui' peaches. In conclusion, this study revealed that fruit softening of peaches with different flesh textures was closely related to ethylene biosynthesis during the storage period, which was controlled via regulating relevant gene expression levels under different storage temperatures.

Multi-omics analysis reveals the evolutionary origin of diterpenoid alkaloid biosynthesis pathways in Aconitum

Diterpenoid alkaloids(DAs) have been often utilized in clinical practice due to their analgesic and anti-infammatory properties. Natural DAs are prevalent in the family Ranunculaceae, notably in the Aconitum genus. Nevertheless, the evolutionary origin of the biosynthesis pathway responsible for DA production remains unknown.In this study, we successfully assembled a highquality, pseudochromosome-level genome of the DA-rich species Aconitum vilmorinianum(A.vilmorinianum)(5.76 Gb). An A. vilmorinianumspecific whole-genome duplication event was discovered using comparative genomic analysis,which may aid in the evolution of the DA biosynthesis pathway. We identified several genes involved in DA biosynthesis via integrated genomic, transcriptomic, and metabolomic analyses. These genes included enzymes encoding target ent-kaurene oxidases and aminotransferases, which facilitated the activation of diterpenes and insertion of nitrogen atoms into diterpene skeletons, thereby mediating the transformation of diterpenes into DAs. The divergence periods of these genes in A. vilmorinianum were further assessed, and it was shown that two major types of genes were involved in the establishment of the DA biosynthesis pathway. Our integrated analysis offers fresh insights into the evolutionary origin of DAs in A.vilmorinianum as well as suggestions for engineering the biosynthetic pathways to obtain desired DAs.

A comprehensive review on the chemical constituents,sesquiterpenoid biosynthesis and biological activities of Sarcandra glabra

Sarcandra glabra(Thunb.)Nakai is a perennial evergreen herb categorised within the Sarcandra Gardner genus under the Chloranthaceae family.Indigenous to tropical and subtropical regions of East Asia and India,this species is extensively distributed across China,particularly in the southern regions(Sichuan,Yunnan,and Jiangxi).In addition to its high ornamental value,S.glabra has a rich history of use in traditional Chinese medicine,evident through its empirical prescriptions for various ailments like pneumonia,dysentery,fractures,bruises,numbness,amenorrhea,rheumatism,and other diseases.Besides,modern pharmacological studies have revealed various biological activities,such as antitumour,anti-bacterial,anti-viral anti-inflammatory and immunomodulatory effects.The diverse chemical constituents of S.glabra have fascinated natural product researchers since the 1900s.To date,over 400 compounds including terpenoids,coumarins,lignans,flavonoids,sterols,anthraquinones,organic acids,and organic esters have been isolated and characterised,some featuring unprecedented structures.This review comprehensively examines the current understanding of S.glabra’s phytochemistry and pharmacology,with emphasis on the chemistry and biosynthesis of its unique chemotaxonomic marker,the lindenane-type sesquiterpenoids.

Chromosome-level genome of Himalayan yew provides insights into the origin and evolution of the paclitaxel biosynthetic pathway

Taxus,commonly known as yew,is a well-known gymnosperm with great ornamental and medicinal value.In this study,by assembling a chromosome-level genome of the Himalayan yew(Taxus wallichiana)with 10.9 Gb in 12 chromosomes,we revealed that tandem duplication acts as the driving force of gene family evolution in the yew genome,resulting in the main genes for paclitaxel biosynthesis,i.e.those encoding the taxadiene synthase,P450s,and transferases,being clustered on the same chromosome.The tandem duplication may also provide genetic resources for the nature to sculpt the core structure of taxoids at different positions and subsequently establish the complex pathway of paclitaxel by neofunctionalization.Furthermore,we confirmed that there are two genes in the cluster encoding isoenzymes of a known enzyme in the paclitaxel biosynthetic pathway.The reference genome of the Himalayan yew will serve as a platform for decoding the complete biosynthetic pathway of paclitaxel and understanding the chemodi-versity of taxoids in gymnosperms.

Biosynthetic pathway of terpenoid indole alkaloids in Catharanthus roseus

Catharanthus roseus is one of the most extensively investigated medicinal plants, which can produce more than 130 alkaloids, including the powerful antitumor drugs vinblastine and vincristine. Here we review the recent advances in the biosynthetic pathway of terpenoid indole alkaloids （TIAs） in C. roseus, and the identification and characterization of the corresponding enzymes involved in this pathway. Strictosidine is the central intermediate in the biosynthesis of different TIAs, which is formed by the condensation of secologanin and tryptamine. Secologanin is derived from terpenoid （isoprenoid） biosynthetic pathway, while tryptamine is derived from indole biosynthetic pathway. Then various specific end products are produced by different routes during downstream process. Although many genes and corresponding enzymes have been characterized in this pathway, our knowledge on the whole TIA biosynthetic pathway still remains largely unknown up to date. Full elucidation of TIA biosynthetic pathway is an important prerequisite to understand the regulation of the TIA biosynthesis in the medicinal plant and to produce valuable TIAs by synthetic biological technology.

Genome sequencing provides potential strategies for drug discovery and synthesis

Medicinal plants are renowned for their abundant production of secondary metabolites,which exhibit notable pharmacological activities and great potential for drug development.The biosynthesis of secondary metabolites is highly intricate and influenced by various intrinsic and extrinsic factors,resulting in substantial species diversity and content variation.Consequently,precise regulation of secondary metabolite synthesis is of utmost importance.In recent years,genome sequencing has emerged as a valuable tool for investigating the synthesis and regulation of secondary metabolites in medicinal plants,facilitated by the widespread use of high-throughput sequencing technologies.This review highlights the latest advancements in genome sequencing within this field and presents several strategies for studying secondary metabolites.Specifically,the article elucidates how genome sequencing can unravel the pathways for secondary metabolite synthesis in medicinal plants,offering insights into the functions and regulatory mechanisms of participating enzymes.Comparative analyses of plant genomes allow identification of shared pathways of metabolite synthesis among species,thereby providing novel avenues for obtaining cost-effective biosynthetic intermediates.By examining individual genomic variations,genes or gene clusters associated with the synthesis of specific compounds can be discovered,indicating potential targets and directions for drug development and the exploration of alternative compound sources.Moreover,the advent of gene-editing technology has enabled the precise modifications of medicinal plant genomes.Optimization of specific secondary metabolite synthesis pathways becomes thus feasible,enabling the precise editing of target genes to regulate secondary metabolite production within cells.These findings serve as valuable references and lessons for future drug development endeavors,conservation of rare resources,and the exploration of new resources.

Analyzing the Prebiotic Potential of Glucosamine for Targeting the Gut Microbiome Health

Recognizing the composition and modulation of the microbiome, a viable therapeutic tool for multi-targeted therapy is a new strategy that has recently been explored. Glucosamine (GS) is being studied for its prebiotic potential in addition to being the most abundant and naturally occurring amino monosaccharide. The current study focuses on glucosamine’s prebiotic potential by assessing the stability of various GS concentrations (1% - 5%) in the gastrointestinal tract (GIT) and its ability to be fermented by the gut microbiota. The results showed that GS stimulated the most growth in L. acidophilus even after a longer incubation time than B. bifidum and L. acidophilus growth was concentration-dependent, with maximum growth at 3% with a simultaneous decrease in pH (5.6 - 1.7). The decrease in GS concentration with time also represented the growth of bacterial species, demonstrating the species’ utilization of GS. Furthermore, at 3%, GS also represented the prebiotic index of 1.9. In addition, the concentration of GS in various simulated GIT fluids was estimated in both fast and fed conditions to examine GS stability at various levels in the gut. The results showed that GS remained unaffected and non-digestible in all of the simulated GIT fluids (salivary, gastric, intestinal, and colonic), but there was a slight decrease in GS concentration (2.8%) in the fasted state of gastric fluid due to low pH levels (1.6). As a result, the findings are conclusive and suggest that GS possesses prebiotic properties.

Expression of. Arabidopsis tryptophan biosynthetic pathway genes: effect of the 5' coding region of phosphoribosylanthranilate isomerase gene

There are three non-allelic isogenes encoding phosphoribosylanthranilate isomerase (PAI) in Arabidopsis thaliana. The expression plasmids were constructed by fusion of the GUS reporter gene to the three PAI promoters with or without the 5' region encoding PAI N-terminal polypeptides and transferred into Arabidopsis plants by Agrobacterium tumefaciens. Analysis of GUS activity revealed that the PAI 5' coding region was necessary for high expression of GUS activity. GUS activity in transgenic plants transformed with the expression plasmids containing the 5' coding region of PAH or PAD was 60-100-fold higher than that without the corresponding 5' region. However, the effect of 5' coding region of PAI2 gene on the GUS activity was very small (only about 1 time difference) . The GUS histochemical staining showed a similar result as revealed by GUS activity assay. It was expressed in the mesophyll cells and guard cells, but not in the epidermic cells, indicating that the N-terminal polypeptides encoded by the 5' region of PAI genes have the function of PTP.

Expression of Recombinant Tryptophan Decarboxylase in Different Subcellular Compartments in Tobacco Plant

The gene encoded for tryptophan decarboxylase (TDC), which is the key enzyme in terpenoil indole alkaloids pathway, was targeted to different subcellular compartments and stably expressed in transgenic tobacco (Nicotiana tabacum L.) plants at the levels detected by Western blot and tryptamine accumulation analysis. It was shown that the TDC was located in subcellular compartments, the chloroplasts and cytosol. The recombinant TDC targeted to chloroplasts and cytosol in tobacco plants was effectively expressed as soluble protein by Western blot analysis and enzymatic assay. The level of tryptamine accumulation in chloroplast was higher than that in cytosol and very low in vacuole and endoplasmic reticulum (ER) to be hardly detected by Western blot analysis. It was indicated that the highest amount of tryptamine was in chloroplasts, lower in endoplasmic reticula and the lowest in vacuoles as compared to those in wild type plants. The TDC targeted to different subcellular compartments of tobacco plants and its expression level were studied by different nucleotide sequences coding signal peptides at 5'-end of tdc gene in order to know the effects of the TDC in compartmentation on its functionality.

Connexin:a potential novel target for protecting the central nervous system?

Connexin subunits are proteins that form gap junction channels, and play an important role in communication between adjacent cells. This review article discusses the function of connexins/hemichannels/gap junctions under physiological conditions, and summarizes the findings re-garding the role of connexins/hemichannels/gap junctions in the physiological and pathological mechanisms underlying central nervous system diseases such as brain ischemia, traumatic brain and spinal cord injury, epilepsy, brain and spinal cord tumor, migraine, neuroautoimmune disease, Alzheimer’s disease, Parkinson’s disease, X-linked Charcot-Marie-Tooth disease, Peli-zaeus-Merzbacher-like disease, spastic paraplegia and maxillofacial dysplasia. Connexins are considered to be a potential novel target for protecting the central nervous system.

Progress on the Chemical Constituents Derived from Glucosinolates in Maca(Lepidium meyenii)

Maca(Lepidium meyenii Walp.),a famous food supplement,has drawn an unprecedented international interest over the last two decades.It was assumed that glucosinolates,macamides,macaenes,and alkaloids are the main bioactive components of Maca before.Recently,a series of novel thiohydantoins which generally exhibit a variety of activities have been isolated from Maca.This review focuses on the progress on the main bioactive components of Maca and their biosynthetic pathway,which indicates that macamides,thiohydantoins,and some alkaloids may originate from glucosinolates.Interestingly,thiohydantoins from Maca are the first type of thiohydantoin derivatives to be found from a natural source and may contribute to some significant effects of Maca.

Three New Heptelidic Acid Derivatives from the Culture of Mushroom Lentinellus ursinus

Three new heptelidic acid derivatives(1-3)including two new dimeric esters and two known heptelidic acid analogues(4 and 5)were isolated from the solid culture of mushroom Lentinellus ursinus.The structures of new compounds were confirmed by the analysis of NMR and HRESIMS spectroscopic data.The biosynthetic origin of compounds 1-5 was postulated.Compounds 1-5 exhibited no antibacterial activity against Staphylococcus aureus and Escherichia coli at the dose of 100 μM.