The parallel biosynthesis routes of hyperoside from naringenin in Hypericum monogynum
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PROVIDER: S-EPMC10506691 | biostudies-literature | 2023 Aug
REPOSITORIES: biostudies-literature
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Project description:Flavonoids that exhibit various biological activities such as antioxidant, antitumor, antiviral, antibacterial and anti-inflammatory properties are found in a wide range of medicinal plants. Among the flavonoid-producing plants identified so far, the genus Epimedium is recognised as a group of prolific prenyl-flavonoid glycoside producers with high economic value in the global dietary supplement market. To date, the biosynthetic genes for prenyl-flavonoid glycosides still remain elusive in Epimedium. Here, we identified five genes in Epimedium wushanense responsible for the biosynthesis of naringenin, the common precursor for flavonoid natural products. We successfully set up the biosynthetic pathway of naringenin using l-tyrosine as the precursor through enzymatic assays of these genes' encoding products, including phenylalanine ammonia-lyase (EwPAL), 4-coumarate-CoA ligase (Ew4CL1), chalcone synthase (EwCHS1), chalcone isomerase (EwCHI1) and CHI-like protein (EwCHIL3). Intriguingly, in vitro characterisation of the above catalytic enzymes' substrate specificity indicated a route parallel to naringenin biosynthesis, which starts from l-phenylalanine and ends in pinocembrin. The fact that there is no pinocembrin or pinocembrin-derived flavonoid accumulated in E. wushanense prompted us to propose that pinocembrin is likely converted into naringenin in vivo, constituting two parallel biosynthetic pathways for naringenin. Therefore, our study provides a basis for the full elucidation of the biosynthetic logic of prenyl-flavonoid glycoside in Epimedium, paving the way for future metabolite engineering and molecular breeding of E. wushanense to acquire a higher titre of desired, bioactive flavonoid compounds.
| S-EPMC8697707 | biostudies-literature
Project description:Hyperoside (quercetin 3-O-galactoside) exhibits many biological functions, along with higher bioactivities than quercetin. In this study, three UDP-dependent glycosyltransferases (UGTs) were screened for efficient hyperoside synthesis from quercetin. The highest hyperoside production of 58.5 mg·L-1 was obtained in a recombinant Escherichia coli co-expressing UGT from Petunia hybrida (PhUGT) and UDP-glucose epimerase (GalE, a key enzyme catalyzing the conversion of UDP-glucose to UDP-galactose) from E. coli. When additional enzymes (phosphoglucomutase (Pgm) and UDP-glucose pyrophosphorylase (GalU)) were introduced into the recombinant E. coli, the increased flux toward UDP-glucose synthesis led to enhanced UDP-galactose-derived hyperoside synthesis. The efficiency of the recombinant strain was further improved by increasing the copy number of the PhUGT, which is a limiting step in the bioconversion. Through the optimization of the fermentation conditions, the production of hyperoside increased from 245.6 to 411.2 mg·L-1. The production was also conducted using a substrate-fed batch fermentation, and the maximal hyperoside production was 831.6 mg·L-1, with a molar conversion ratio of 90.2% and a specific productivity of 27.7 mg·L-1·h-1 after 30 h of fermentation. The efficient hyperoside synthesis pathway described here can be used widely for the glycosylation of other flavonoids and bioactive substances.
| S-EPMC8949062 | biostudies-literature
Project description:To explore the accumulation of rutin, hyperoside and quercetin in Hypericum attenuatum Choisy under treatment with different plant growth regulators, 100 mg/L, 200 mg/L and 300 mg/L cycocel, 100 mg/L, 200 mg/L and 300 mg/L mepiquat chloride and 1 mg/L, 2 mg/L and 3 mg/L naphthalene acetic acid were foliage sprayed on Hypericum attenuatum Choisy plants at the early growth stage. We sampled and determined the important flavonoid contents at the flowering stage. The results showed that the three plant growth regulators had different effects on the accumulation of rutin, hyperoside and quercetin in the leaves, stems and flowers of Hypericum attenuatum Choisy at the flowering stage. After spraying 1 mg/L naphthalene acetic acid at the early growth stage, the rutin contents in the leaves, stems and flowers increased by approximately 60.33%, 223.85% and 192.02%, respectively (P < 0.05). Spraying 100 mg/L mepiquat chloride increased the hyperoside contents in the leaves and flowers by approximately 7.77% and 12.87%, respectively (P < 0.05). Spraying 2 mg/L naphthalene acetic acid significantly increased the quercetin contents in the flowers and leaves by approximately 95.62% and 47.85%, respectively (P < 0.05). Therefore, at the early growth stage, spraying 1 mg/L naphthalene acetic acid significantly increased rutin content, spraying 100 mg/L mepiquat chloride significantly increased hyperoside content, and spraying 2 mg/L naphthalene acetic acid significantly increased quercetin content in Hypericum attenuatum Choisy. In conclusion, the accumulation of flavonoids in Hypericum attenuatum Choisy was regulated by plant growth regulators.
| S-EPMC10156007 | biostudies-literature
Project description:Species of the genus Hypericum contain a rich array of unusual polyketides, however, only a small proportion of the over 450 Hypericum species, other than the popular medicinal supplement St. John's Wort (Hypericum perforatum), have even been chemically characterized. Hypericum gentianoides, a small annual used medicinally by Cherokee Americans, contains bioactive acylphloroglucinols. Here, we identify acylphloroglucinol constituents of H. gentianoides and determine a potential pathway to their synthesis. Liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) and HPLC-UV indicate that the level of accumulation and profile of acylphloroglucinols in H. gentianoides vary little seasonally when grown in a greenhouse, but do vary with development and are highly dependent on the accession, highlighting the importance of the selection of plant material for study. We identify the chemical structures of the nine prevalent polyketides, based on LC/ESI-MS and hybrid quadrupole orthogonal time-of-flight (Q-TOF) mass spectrometry; these metabolites include one monomeric phlorisobutyrophenone (PIB) derivative and eight dimeric acylphloroglucinols. Q-TOF spectrometry was used to identify eight additional PIB derivatives that were not detected by LC/ESI-MS. These data lead us to propose that diacylphloroglucinols are synthesized via modification of PIB to yield diverse phloroglucinol and filicinic acids moieties, followed by dimerization of a phloroglucinol and a filicinic acid monomer to yield the observed complement of diacylphloroglucinols. The metabolomics data from H. gentianoides are accessible in plant metabolomics resource (PMR) (http://www.metnetdb.org/pmr), a public metabolomics database with analysis software for plants and microbial organisms.
| S-EPMC3687794 | biostudies-literature
Project description:Co-culture engineering is an emerging approach for microbial biosynthesis of a variety of biochemicals. In this study, E. coli-E. coli co-cultures were developed for heterologous biosynthesis of the natural product naringenin. The co-cultures were composed of two independent E. coli strains dedicated to functional expression of different portions of the biosynthetic pathway, respectively. The co-culture biosynthesis was optimized by investigating the effect of carbon source, E. coli strain selection, timing of IPTG induction and the inoculation ratio between the co-culture strains. Compared with the mono-culture strategy, the utilization of the designed co-cultures significantly improved the naringenin production, largely due to the reduction of metabolic stress, employment of proper hosts for improving pathway enzyme activities, and flexible adjustment of the relative biosynthetic strength between the co-culture strains. The findings of this study extend the applicability of co-culture engineering in complex natural product biosynthesis.
| S-EPMC5655346 | biostudies-literature
Project description:Next generation sequencing technology rapidly developed research applications in the field of plant functional genomics. Several Hypericum spp. with an aim to generate and enhance gene annotations especially for genes coding the enzymes supposedly included in biosynthesis of valuable bioactive compounds were analyzed. The first de novo transcriptome profiling of Hypericum annulatum Moris, H. tomentosum L., H. kalmianum L., and H. androsaemum L. leaves cultivated in vitro was accomplished. All four species with only limited genomic information were selected on the basis of differences in ability to synthesize hypericins and presence of dark nodules accumulating these metabolites with purpose to enrich genomic background of Hypericum spp. H. annulatum was chosen because of high number of the dark nodules and high content of hypericin. H. tomentosum leaves are typical for the presence of only 1-2 dark nodules localized in the apical part. Both H. kalmianum and H. androsaemum lack hypericin and have no dark nodules. Four separated datasets of the pair-end reads were gathered and used for de novo assembly by Trinity program. Assembled transcriptomes were annotated to the public databases Swiss-Prot and non-redundant protein database (NCBI-nr). Gene ontology analysis was performed. Differences of expression levels in the marginal tissues with dark nodules and inner part of leaves lacking these nodules indicate a potential genetic background for hypericin formation as the presumed site of hypericin biosynthesis is in the cells adjacent to these structures. Altogether 165 contigs in H. annulatum and 100 contigs in H. tomentosum were detected as significantly differentially expressed (P < 0.05) and upregulated in the leaf rim tissues containing the dark nodules. The new sequences homologous to octaketide synthase and enzymes catalyzing phenolic oxidative coupling reactions indispensable for hypericin biosynthesis were discovered. The presented transcriptomic sequence data will improve current knowledge about the selected Hypericum spp. with proposed relation to hypericin biosynthesis and will provide a useful resource of genomic information for consequential studies in the field of functional genomics, proteomics and metabolomics.
| S-EPMC4942478 | biostudies-literature
Project description:BackgroundThe rapidly expanding synthetic biology toolbox allows engineers to develop smarter strategies to tackle the optimization of complex biosynthetic pathways. In such a strategy, multi-gene pathways are subdivided in several modules which are each dynamically controlled to fine-tune their expression in response to a changing cellular environment. To fine-tune separate modules without interference between modules or from the host regulatory machinery, a sigma factor (σ) toolbox was developed in previous work for tunable orthogonal gene expression. Here, this toolbox is implemented in E. coli to orthogonally express and fine-tune a pathway for the heterologous biosynthesis of the industrially relevant plant metabolite, naringenin. To optimize the production of this pathway, a practical workflow is still imperative to balance all steps of the pathway. This is tackled here by the biosensor-driven screening, subsequent genotyping of combinatorially engineered libraries and finally the training of three different computer models to predict the optimal pathway configuration.ResultsThe efficiency and knowledge gained through this workflow is demonstrated here by improving the naringenin production titer by 32% with respect to a random pathway library screen. Our best strain was cultured in a batch bioreactor experiment and was able to produce 286 mg/L naringenin from glycerol in approximately 26 h. This is the highest reported naringenin production titer in E. coli without the supplementation of pathway precursors to the medium or any precursor pathway engineering. In addition, valuable pathway configuration preferences were identified in the statistical learning process, such as specific enzyme variant preferences and significant correlations between promoter strength at specific steps in the pathway and titer.ConclusionsAn efficient strategy, powered by orthogonal expression, was applied to successfully optimize a biosynthetic pathway for microbial production of flavonoids in E. coli up to high, competitive levels. Within this strategy, statistical learning techniques were combined with combinatorial pathway optimization techniques and an in vivo high-throughput screening method to efficiently determine the optimal operon configuration of the pathway. This "pathway architecture designer" workflow can be applied for the fast and efficient development of new microbial cell factories for different types of molecules of interest while also providing additional insights into the underlying pathway characteristics.
| S-EPMC8962593 | biostudies-literature
Project description:Dihydrochalcones (DHCs) are flavonoids produced as a side branch of the phenylpropanoid pathway. DHCs are found at high concentrations in apples (Malus spp.) but not in pears (Pyrus spp.) or other members of the Rosaceae. Biosynthesis of DHCs in apple has been hypothesized to occur via reduction of p-coumaroyl CoA by a Malus × domestica hydroxycinnamoyl CoA double-bond reductase (MdHCDBR) followed by the action chalcone synthase to produce phloretin or via direct reduction of naringenin chalcone to phloretin via an unknown enzyme. In this study, we report that genetic downregulation of MdHCDBR does not reduce DHC concentrations in apple leaves. We used comparative transcriptome analysis to identify candidate naringenin chalcone reductases (NCRs), designated MdNCR1a-c, expressed in apple leaves but not fruit. These MdNCR1 genes form an expanded gene cluster found exclusively in apple. Transient expression of MdNCR1 genes in Nicotiana benthamiana leaves indicated they produced DHCs at high concentrations in planta. Recombinant MdNCR1 utilized naringenin chalcone to produce phloretin at high efficiency. Downregulation of NCR genes in transgenic apple reduced foliar DHC levels by 85% to 95%. Reducing DHC production redirected flux to the production of flavonol glycosides. In situ localization indicated that NCR proteins were likely found in the vacuolar membrane. Active site analysis of AlphaFold models indicated that MdNCR1a-c share identical substrate binding pockets, but the pockets differ substantially in related weakly active/inactive NCR proteins. Identifying the missing enzyme required for DHC production provides opportunities to manipulate DHC content in apple and other fruits and has other applications, e.g. in biofermentation and biopharming.
| S-EPMC11638483 | biostudies-literature
Project description:The cytosolic mevalonate (MVA) pathway in Hevea brasiliensis latex is the conventionally accepted pathway which provides isopentenyl diphosphate (IPP) for cis-polyisoprene (rubber) biosynthesis. However, the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway may be an alternative source of IPP since its more recent discovery in plants. Quantitative RT-PCR (qRT-PCR) expression profiles of genes from both pathways in latex showed that subcellular compartmentalization of IPP for cis-polyisoprene synthesis is related to the degree of plastidic carotenoid synthesis. From this, the occurrence of two schemes of IPP partitioning and utilization within one species is proposed whereby the supply of IPP for cis-polyisoprene from the MEP pathway is related to carotenoid production in latex. Subsequently, a set of latex unique gene transcripts was sequenced and assembled and they were then mapped to IPP-requiring pathways. Up to eight such pathways, including cis-polyisoprene biosynthesis, were identified. Our findings on pre- and post-IPP metabolic routes form an important aspect of a pathway knowledge-driven approach to enhancing cis-polyisoprene biosynthesis in transgenic rubber trees.
| S-EPMC3295384 | biostudies-literature
Project description:Understanding how organisms adapt to the environment is a major goal of modern biology. Parallel evolution-the independent evolution of similar phenotypes in different populations-provides a powerful framework to investigate the evolutionary potential of populations, the constraints of evolution, its repeatability and therefore its predictability. Here, we quantified the degree of gene expression and functional parallelism across replicated ecotype formation in Heliosperma pusillum (Caryophyllaceae), and gained insights into the architecture of adaptive traits. Population structure analyses and demographic modelling support a previously formulated hypothesis of parallel polytopic divergence of montane and alpine ecotypes. We detect a large proportion of differentially expressed genes (DEGs) underlying divergence within each replicate ecotype pair, with a strikingly low number of shared DEGs across pairs. Functional enrichment of DEGs reveals that the traits affected by significant expression divergence are largely consistent across ecotype pairs, in strong contrast to the nonshared genetic basis. The remarkable redundancy of differential gene expression indicates a polygenic architecture for the diverged adaptive traits. We conclude that polygenic traits appear key to opening multiple routes for adaptation, widening the adaptive potential of organisms.
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