Production Of Valuable Secondary Metabolites Research Articles

Composition and antimicrobial activity of Rosmarinus officinalis L. and Artemisia monosperma L. leaf essential oils and methanolic extracts from plants grown in normal and saline habitats in Egypt

The present work aimed to investigate the effect of salinity in natural habitats in Egypt on the main secondary metabolites of Rosmarinus officinalis L. and Artemisia monosperma L. plants compared to plants grown at normal conditions. Plants grown under salinity were collected from Egyptian Western Coastal region habitats irrigated with underground water. Results showed that salinity increased the essential oil percentage of R. officinalis L. by 52.7% and A. monosperma L by 0.29% in addition to the total phenolics and flavonoids content in dry leaves compared to control plants. GC/MS analysis of rosemary essential oils revealed that salinity decreased the amount of some major oil monoterpenes component as verbenone, with a slight effect on 1,8 cineole and increased Camphor, endo- Boreneol, and linalool in addition to the appearance of new specific components such as Chrysanthenone monoterpene ketone and Caryophyllene sesquiterpene, while, in the case of Artemisia, the GC/MS showed that Artemisia ketone, Camphor, β -phellandrene monoterpenes andα-Bisabolol sesquiterpenewere the major oil components; salinity decreased Camphor and β -phellandrene content and increased artemisia ketone and α-Bisabolol oil content. About 11 new oil constituents were detected such as ( +)-2-Bornanone and Sesquisabinene hydrate. Mineral ions (N, K+, Ca+2, P, and Mg+2) uptake by R. officinalis and A. monosperma decreased in plants grown under salinity, while Na content increased compared to corresponding controls. Results demonstrated that both plants could tolerate the high salinity level in natural Western Coastal region soil which promoted more production of valuable secondary metabolites. The antimicrobial effect of R. officinalis L. and A. monosperma L. leaf methanolic extracts, results showed that R. officinalis extracts had an inhibitory response against all tested gram-positive and negative bacteria, in addition to the yeast (Candida albicans), whereas there was no any inhibitory effect concerning A. monosperma L extract on the tested species.

The Stilbene Synthase Family in Arachis: A Genome-Wide Study and Functional Characterization in Response to Stress.

Peanut (Arachis hypogaea) and its wild relatives are among the few species that naturally synthesize resveratrol, a well-known stilbenoid phytoalexin that plays a crucial role in plant defense against biotic and abiotic stresses. Resveratrol has received considerable attention due to its health benefits, such as preventing and treating various human diseases and disorders. Chalcone (CHS) and Stilbene (STS) Synthases are plant-specific type III Polyketide Synthases (PKSs) that share the same substrates and are key branch enzymes in the biosynthesis of flavonoids and stilbenoids, respectively. Although resveratrol accumulation in response to external stimulus has been described in peanut, there are no comprehensive studies of the CHS and STS gene families in the genus Arachis. In the present study, we identified and characterized 6 CHS and 46 STS genes in the tetraploid peanut and an average of 4 CHS and 22 STS genes in three diploid wild species (Arachis duranensis, Arachis ipaënsis and Arachis stenosperma). The CHS and STS gene and protein structures, chromosomal distributions, phylogenetic relationships, conserved amino acid domains, and cis-acting elements in the promoter regions were described for all Arachis species studied. Based on gene expression patterns of wild A. stenosperma STS genes in response to different biotic and abiotic stresses, we selected the candidate AsSTS4 gene, which is strongly induced by ultraviolet (UV) light exposure, for further functional investigation. The AsSTS4 overexpression in peanut hairy roots significantly reduced (47%) root-knot nematode infection, confirming that stilbene synthesis activation in transgenic plants can increase resistance to pathogens. These findings contribute to understanding the role of resveratrol in stress responses in Arachis species and provide the basis for genetic engineering for improved production of valuable secondary metabolites in plants.

Artificial Light Spectra and Its Impact on Plant Physiological Processes and Secondary Metabolism

Understanding the effects of artificial light spectra on plant physiological processes and secondary metabolism is critical for optimizing plant growth and productivity, particularly in controlled environment agriculture. This review synthesizes the current knowledge on this topic, exploring the fundamental principles of light spectra, their impacts on plant physiology, including photosynthesis and growth, as well as on secondary metabolism. Artificial light sources, such as light-emitting diodes (LEDs), have been shown to significantly influence these plant processes, owing to their controllable spectra and intensities. These artificial light spectra can enhance photosynthetic efficiency, manipulate growth and development, and stimulate the production of valuable secondary metabolites. The review further discusses the potential applications of this understanding in sectors like agriculture, pharmaceuticals, nutraceuticals, and even space agriculture. However, the interactions of artificial light spectra with other environmental factors, the development of custom light recipes for specific plant species or cultivars, and the need for long-term studies are identified as areas needing further research. This review contributes to the growing body of literature exploring the opportunities and challenges of utilizing artificial light spectra for improving plant performance and secondary metabolite production.

Effects of 2,4-Dichlorophenoxyacetic Acid on The Induction of Callus from Cotyledon and Hypocotyl Explants of Butterfly Pea (Clitoria ternatea)

Clitoria ternatea (Butterfly pea) is a tropical medicinal and fodder legume from the Fabaceae family possessing various beneficial phytochemical compounds linked to the mammalian neuroprotective mechanism. Callus and cell suspension cultures are excellent alternatives for harnessing secondary metabolites from medicinal plants. The current study aims to induce callus from cotyledon and hypocotyl explants of C. ternatea for the establishment of cell suspension cultures. Cotyledon and hypocotyl explants from two-weeks-old seedlings were subjected to half-strength MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at different concentrations (0.5 mg/L to 2.5 mg/L) and callus scoring and morphology were assessed at week 8 of culture. Results revealed that the treatment of 0.5 mg/L 2,4-D resulted in the highest percentage of callus induction (100%) and the highest callus scoring for both cotyledon and hypocotyl explants with friable callus morphology. Cotyledon explants exhibited a higher callus scoring with a relative value of 3.03 ± 0.20 compared to hypocotyl explants at 1.80 ± 0.12. This study thereby provides a basis for future studies on callus induction studies and the establishment of cell suspension cultures of C. ternatea for the production of valuable secondary metabolites linked to the memory enhancing properties of the plant.

Characterization of polyphenols by RP-HPLC in Basilicum polystachyon (L.) Moench with their antioxidant and antimicrobial properties

• Ultrasonic assisted leaf extract of Basilicum polystachyon revealed the phenolic compounds present in the free, esterified and glycosylated forms. • First comparative RP-HPLC analysis of different phenolic forms in vivo and in vitro B. polystachyon leaf. • Gallic acid, trans -cinnamic acid, ellagic acid, quercetin and vanillic acid in B. polystachyon for the first time along with caffeic acid, p- coumaric acid and rosmarinic acid were identified. • B. polystachyon could serve as a souce of antioxidants. • Phenolic compounds of in vivo plants exhibited the higher antimicrobial activities against B. subtilis, S. aureus, M. smegmatis, E. coli and C. albicans. Basilicum polystachyon (L.) Moench is a member of Lamiaceae family and important ethnomedicinal that is widely used in the treatment of several ailments and is reputed to possess an inhibitor of the dengue virus. In vitro propagation is an efficient strategy for large-scale production of plantlets. Such process is a good alternative method for the production of valuable secondary metabolites. Hence, the present study assesses the contents of phenolic compounds, radical scavenging activity and antimicrobial efficacy of in vivo grown compared with the in vitro grown B. polystachyon plants. Thus, the antioxidant capacity of leaf extracts was determined by scavengers like ABTS .+ , DPPH . , hydrogen peroxide, hydroxyl radical, nitric oxide along with lipid peroxidation inhibition, FRAP, reducing power and DNA damage protection assay. The antimicrobial activity was evaluated through disk diffusion assay and minimum inhibitory concentration assay. RP-HPLC was used to identify and quantify the phenolic compounds in the following forms: free, esterified and glycosylated. The leaf extract of in vivo plants exhibited stronger antioxidant activities. The FRAP assay revealed a maximum reducing power with leaf extract of in vivo grown plants (43.73±0.52 µM Fe(II) equivalent). Total phenolic content of in vivo plants presented a strong positive correlation to IC 50 values of ABTS, DPPH, and HPSA, whereas total flavonoid content of in vitro grown plant was strongly correlated to IC 50 values of DPPH and HPSA. In vivo plants showed the highest inhibition zone against Bacillus subtilis, Escherichia coli , and Staphylococcus aureus. RP-HPLC analysis showed the phenolic compounds present in all tested forms of B. polystachyon leaf. For the first time, gallic acid, trans -cinnamic acid, ellagic acid, quercetin and vanillic acid were identified in this extract. Also, caffeic acid, p -coumaric acid and rosmarinic acid were quantified. These findings strongly suggest that B. polystachyon may be exploited as a promising natural source of antioxidants along with efficient antimicrobial agents.

Nitric Oxide Signaling for Aerial Mycelium Formation in Streptomyces coelicolor A3(2) M145.

Nitric oxide (NO) is a well-known signaling molecule in various organisms. Streptomyces undergoes complex morphological differentiation, similar to that of fungi. A recent study revealed a nitrogen oxide metabolic cycle that forms NO in Streptomyces coelicolor A3(2) M145. Further, endogenously produced NO serves as a signaling molecule. Here, we report that endogenously produced NO regulates cyclic 3',5'-diguanylate (c-di-GMP) levels and controls aerial mycelium formation through the c-di-GMP-binding transcriptional regulator BldD in S. coelicolor A3(2) M145. These observations provide important insights into the mechanisms regulating morphological differentiation. This is the first study to demonstrate a link between NO and c-di-GMP in S. coelicolor A3(2) M145. Morphological differentiation is closely linked to the initiation of secondary metabolism in actinomycetes. Thus, the NO signaling-based regulation of aerial mycelium formation has potential applications in the fermentation industry employing useful actinomycetes. IMPORTANCE Eukaryotic and prokaryotic cells utilize nitric oxide (NO) to regulate physiological functions. Besides its role as a producer of different bioactive substances, Streptomyces is suggested to be involved in mycelial development regulated by endogenously produced NO. However, the regulatory mechanisms are unclear. In this study, we proposed that NO signaling is involved in aerial mycelium formation in S. coelicolor A3(2) M145. NO serves as a signaling molecule for the regulation of intracellular cyclic 3',5'-diguanylate (c-di-GMP) levels, resulting in aerial mycelium formation controlled by a c-di-GMP receptor, BldD. As the abundant production of valuable secondary metabolites is closely related to the initiation of morphological differentiation in Streptomyces, NO may provide value for application in industrial fermentation by serving as a tool for regulating secondary metabolism.

Biotechnological interventions of in vitro propagation and production ofvaluable secondary metabolites in Stevia rebaudiana.

Plant cell and tissue culture makes provision of a sustainable and nature-friendly strategy for the production of secondary metabolites, and modern progress in gene editing and genome engineering provides novel possibilities to improve both the qualitative and quantitative aspects of such phytochemicals. The ever-expanding quest for plant-based medicine to treat diabetes facilitates large-scale cultivation of Stevia rebaudiana to enhance the yield of its much-coveted low-calorie sweetener glycosides. The potential to process stevia as a "natural" product should enhance the acceptance of steviosides as a natural calorie-free sweetener especially suitable for use in diabetic and weight control drinks and foods. Besides sweetener agents, S. rebaudiana is a potent source of many antioxidant compounds and is used to cure immunodeficiencies, neurologic disorders, inflammation, diabetes mellitus, Parkinson's disease, and Alzheimer's disease. This comprehensive review presents the research outcomes of the many biotechnological interventions implicated to upscale the yield of steviol glycosides and its derivatives in in vitro cell, callus, tissue, and organ cultures with notes on the use of bioreactor and genetic engineering in relation to the production of these valuable compounds in S. rebaudiana. KEY POINTS: • Critical and updated assessment on sustainable production of steviol glycosides from Stevia rebaudiana. • In vitro propagation of S. rebaudiana and elicitation of steviol glycosides production. • Genetic fidelity and diversity assessment of S. rebaudiana using molecular markers.

Tissue culture of safflower and analysis of secondary metabolites in suspension cells

Safflower(Carthamus tinctorius), a valuable traditional Chinese medicinal plant, has attracted much attention in recent years. This study established a stable tissue culture system of safflower and analyzed the chromatogram of its secondary metabolites, providing high-quality experimental materials for further research on natural products in safflower. The calluses were established from the safflower seeds germinated in a sterile environment, and then they were differentiated into the aseptic seedlings, or cultured to obtain suspension cells in liquid medium. The ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry(UPLC-Q-TOF-MS), Progenesis QI, and principal component analysis(PCA) were used to detect and analyze the secondary metabolites in the suspension cells before and after induction with different elicitors(methyl jasmonate, silver nitrate, salicylic acid and yeast extract). A total of 23 secondary metabolites including flavonoids, phenylpropanoids, alkaloids, fatty acids and aromatic glycosides were detected in safflower suspension cells. In response to the four elicitors, 11 compounds showed increased or decreased relative content. The results indicate that different elicitors have various effects on the accumulation of secondary metabolites in safflower suspension cells, and yeast extract shows more obvious positive induction. Therefore, different elicitors may play a role in the expression of related genes in the biosynthetic pathway of specific secondary metabolites. The results facilitate the discovery of targeted elicitors and the large-scale production of valuable secondary metabolites in the future.

EFFECTS OF LIGHT ILLUMINATION AND SUBCULTURE FREQUENCY ON BIOMASS PRODUCTION IN CELL SUSPENSION CULTURES OF Clinacanthus nutans

Clinacanthus nutans (Burm. f.) Lindau is a medicinal plant rich in polyphenols and possesses multiple pharmacological properties. It is widely used as a side treatment for cancer and to prevent oxidative stress-related diseases. The present study aims to investigate the effects of light illumination and subculture frequency on the growth and proliferation of cells in the suspension culture of C. nutans. Friable callus induced from leaf explants C. nutans were cultured in liquid Murashige & Skoog medium supplemented with 0.25 mg/L 2, 4-dichlorophenoxyacetic acid, and 0.25 mg/L 6-benzylaminopurine to initiate the cell suspension culture. The suspension cultures were incubated under different conditions (16 hr photoperiod and continuous darkness). Subcultures were conducted every two weeks for ten subculture cycles. Results revealed that higher cell biomass was obtained under the exposure of 16 hr photoperiod with the highest biomass production at the fifth subculture cycle (growth index of 11.8) exhibiting stable growth from the eighth to the tenth subculture cycle. The suspension cells showed stable growth rates up to the 10th subculture cycle producing small aggregates and single cells. The present study indicated that light illumination significantly impacts the growth of suspension cultures for C. nutans in the production of valuable secondary metabolites.

Development of a Cell Suspension Culture System for Promoting Alkaloid and Vinca Alkaloid Biosynthesis Using Endophytic Fungi Isolated from Local Catharanthus roseus.

Cell and tissue cultures of Catharanthus roseus have been studied extensively as an alternative strategy to improve the production of valuable secondary metabolites. The purpose of this study was to produce C. roseus callus and suspension cell biomass of good quality and quantity to improve the total alkaloids and bis-indole alkaloids. The young stem derived-callus of C. roseus variety Quang Ninh (QN) was grown on MS medium supplemented with 1.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) plus 1.5 mg/L kinetin, and the growth rate increased by 67-fold after 20 days. The optimal conditions for maintaining the cell suspension culture were 150 mg/50 mL cell inoculum, a medium pH of 5.5 and a culture temperature of 25 °C. The low alkaloid content in the culture was compensated for by using endophytic fungi isolated from local C. roseus. Cell extracts of endophytic fungi—identified as Fusarium solani RN1 and Chaetomium funicola RN3—were found to significantly promote alkaloid accumulation. This elicitation also stimulated the accumulation of a tested bis-indole alkaloid, vinblastine. The findings are important for investigating the effects of fungal elicitors on the biosynthesis of vinblastine and vincristine, as well as other terpenoid indole alkaloids (TIAs), in C. roseus QN cell suspension cultures.

Responses of Medicinal and Aromatic Plants to Engineered Nanoparticles

Medicinal and aromatic plants have been used by mankind since ancient times. This is primarily due to their healing effects associated with their specific secondary metabolites (some of which are also used as drugs in modern medicine), or their structures, served as a basis for the development of new effective synthetic drugs. One way to increase the production of these secondary metabolites is to use nanoparticles that act as elicitors. However, depending on the specific particle size, composition, concentration, and route of application, nanoparticles may have several other benefits on medicinal and aromatic plants (e.g., increased plant growth, improved photosynthesis, and overall performance). On the other hand, particularly at applications of high concentrations, they are able to damage plants mechanically, adversely affect morphological and biochemical characteristics of plants, and show cytotoxic and genotoxic effects. This paper provides a comprehensive overview of the beneficial and adverse effects of metal-, metalloid-, and carbon-based nanoparticles on the germination, growth, and biochemical characteristics of a wide range of medicinal and aromatic plants, including the corresponding mechanisms of action. The positive impact of nanopriming and application of nanosized fertilizers on medicinal and aromatic plants is emphasized. Special attention is paid to the effects of various nanoparticles on the production of valuable secondary metabolites in these plants cultivated in hydroponic systems, soil, hairy root, or in vitro cultures. The beneficial impact of nanoparticles on the alleviation of abiotic stresses in medicinal and aromatic plants is also discussed.

Hybrid transcriptome sequencing approach improved assembly and gene annotation in Cynara cardunculus (L.)

BackgroundThe investigation of transcriptome profiles using short reads in non-model organisms, which lack of well-annotated genomes, is limited by partial gene reconstruction and isoform detection. In contrast, long-reads sequencing techniques revealed their potential to generate complete transcript assemblies even when a reference genome is lacking. Cynara cardunculus var. altilis (DC) (cultivated cardoon) is a perennial hardy crop adapted to dry environments with many industrial and nutraceutical applications due to the richness of secondary metabolites mostly produced in flower heads. The investigation of this species benefited from the recent release of a draft genome, but the transcriptome profile during the capitula formation still remains unexplored. In the present study we show a transcriptome analysis of vegetative and inflorescence organs of cultivated cardoon through a novel hybrid RNA-seq assembly approach utilizing both long and short RNA-seq reads.ResultsThe inclusion of a single Nanopore flow-cell output in a hybrid sequencing approach determined an increase of 15% complete assembled genes and 18% transcript isoforms respect to short reads alone. Among 25,463 assembled unigenes, we identified 578 new genes and updated 13,039 gene models, 11,169 of which were alternatively spliced isoforms. During capitulum development, 3424 genes were differentially expressed and approximately two-thirds were identified as transcription factors including bHLH, MYB, NAC, C2H2 and MADS-box which were highly expressed especially after capitulum opening. We also show the expression dynamics of key genes involved in the production of valuable secondary metabolites of which capitulum is rich such as phenylpropanoids, flavonoids and sesquiterpene lactones. Most of their biosynthetic genes were strongly transcribed in the flower heads with alternative isoforms exhibiting differentially expression levels across the tissues.ConclusionsThis novel hybrid sequencing approach allowed to improve the transcriptome assembly, to update more than half of annotated genes and to identify many novel genes and different alternatively spliced isoforms. This study provides new insights on the flowering cycle in an Asteraceae plant, a valuable resource for plant biology and breeding in Cynara and an effective method for improving gene annotation.

Heterologous Metabolic Pathways: Strategies for Optimal Expression in Eukaryotic Hosts

Heterologous pathways are linked series of biochemical reactions occurring in a host organism after the introduction of foreign genes. Incorporation of metabolic pathways into host organisms is a major strategy used to increase the production of valuable secondary metabolites. Unfortunately, simple introduction of the pathway genes into the heterologous host in most cases does not result in successful heterologous expression. Extensive modification of heterologous genes and the corresponding enzymes on many different levels is required to achieve high target metabolite production rates. This review summarizes the essential techniques used tocreate heterologous biochemical pathways, with a focus on the key challenges arising in the process and the major strategies for overcoming them.

The Effect of Non-biological Elicitors on Physiological and Biochemical Properties of Medicinal Plant Momordica charantia L.

Carla (Momordica charantia L.) is a medicinal plant of the Cucurbitaceae family, which has antibacterial, anticancer, anti-tumor, hypoglycemic, hypertensive, and cholesterol properties. In this study, the effects of zinc oxide nanoparticles (ZnO-NPs) (20, 60, and 100 ppm), jasmonate (100, 250, and 500 μM), and chitosan (10, 50, and 100 μM) were measured on the growth and some physiological and biochemical parameters of Carla plant. The results revealed the highest shoot weight of plants at 250 μM jasmonate and 10 μM chitosan, while ZnO-NPs had no significant effect on shoot weight. ZnO-NPs (20–60 ppm) and jasmonate (100 and 200 µM) significantly increased chlorophyll a content, but chitosan showed no effect on chlorophyll a content. Secondary metabolites such as phenols, flavonoids, and carotenoids as well as carbohydrate and proline content were significantly increased by all elicitors in a dose-dependent manner. Antioxidant enzyme activity showed varied responses to different concentrations of elicitors. Jasmonate increased catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidases (GPX) activity in a dose-dependent manner. Chitosan at all concentrations significantly increased CAT and APX enzymes activity, while at 100 μM concentration significantly increased GPX enzyme activity. ZnO-NPs did not affect GPX and AXP enzymes activity. Our findings confirmed for the first time that non-biological elicitors at specific levels have a significant growth promotion effect as well as increased production of valuable secondary metabolites in M. charantia.

Caffeoylquinic Acids with Potential Biological Activity from Plant In vitro Cultures as Alternative Sources of Valuable Natural Products.

For a long time, the researchers have been looking for new efficient methods to enhance production and obtain valuable plant secondary metabolites, which would contribute to the protection of the natural environment through the preservation of various plant species, often rare and endangered. These possibilities offer plant in vitro cultures which can be performed under strictly-controlled conditions, regardless of the season or climate and environmental factors. Biotechnological methods are promising strategies for obtaining the valuable plant secondary metabolites with various classes of chemical compounds including caffeoylquinic acids (CQAs) and their derivatives. CQAs have been found in many plant species which are components in the daily diet and exhibit a wide spectrum of biological activities, including antioxidant, immunomodulatory, antihypertensive, analgesic, anti-inflammatory, hepato- and neuroprotective, anti-hyperglycemic, anticancer, antiviral and antimicrobial activities. They have also been found to offer protection against Alzheimer's disease, and play a role in weight reduction and lipid metabolism control, as well as modulating the activity of glucose-6-phosphatase involved in glucose metabolism. This work presents the review of the recent advances in use in vitro cultures of various plant species for the alternative system to the production of CQAs and their derivatives. Production of the secondary metabolites in in vitro culture is usually performed with cell suspension or organ cultures, such as shoots and adventitious or transformed roots. To achieve high production of valuable secondary metabolites in in vitro cultures, the optimization of the culture condition is necessary with respect to both biomass accumulation and metabolite content. The optimization of the culture conditions can be achieved by choosing the type of medium, growth regulators or growth conditions, selection of high-productivity lines or culture period, supplementation of the culture medium with precursors or elicitor treatments. Cultivation for large-scale in bioreactors and genetic engineering: Agrobacterium rhizogenes transformation and expression improvement of transcriptional factor or genes involved in the secondary metabolite production pathway are also efficient strategies for enhancement of the valuable secondary metabolites. Many studies have been reported to obtain highly productive plant in vitro cultures with respect to CQAs. Among these valuable secondary metabolites, the most abundant compound accumulated in in vitro cultures was 5-CQA (chlorogenic acid). Highly productive cultures with respect to this phenolic acid were Leonurus sibiricus AtPAP1 transgenic roots, Lonicera macranthoides and Eucomia ulmoides cell suspension cultures which accumulated above 20 mg g-1 DW 5-CQA. It is known that di- and triCQAs are less common in plants than monoCQAs, but it was also possible to obtain them by biotechnological methods. The results indicate that the various in vitro cultures of different plant species can be a profitable approach for the production of CQAs. In particular, an efficient production of these valuable compounds is possible by Lonicera macranthoides and Eucomia ulmoides cell suspension cultures, Leonurus sibiricus transformed roots and AtPAP1 transgenic roots, Echinacea angustifolia adventitious shoots, Rhaponticum carthamoides transformed plants, Lavandula viridis shoots, Sausera involucrata cell suspension and Cichorium intybus transformed roots.

The Synergistic Effect of Co-Treatment of Methyl Jasmonate and Cyclodextrins on Pterocarpan Production in Sophora flavescens Cell Cultures.

Pterocarpans are derivatives of isoflavonoids, found in many species of the family Fabaceae. Sophora flavescens Aiton is a promising traditional Asian medicinal plant. Plant cell suspension cultures represent an excellent source for the production of valuable secondary metabolites. Herein, we found that methyl jasmonate (MJ) elicited the activation of pterocarpan biosynthetic genes in cell suspension cultures of S. flavescens and enhanced the accumulation of pterocarpans, producing mainly trifolirhizin, trifolirhizin malonate, and maackiain. MJ application stimulated the expression of structural genes (PAL, C4H, 4CL, CHS, CHR, CHI, IFS, I3’H, and IFR) of the pterocarpan biosynthetic pathway. In addition, the co-treatment of MJ and methyl-β-cyclodextrin (MeβCD) as a solubilizer exhibited a synergistic effect on the activation of the pterocarpan biosynthetic genes. The maximum level of total pterocarpan production (37.2 mg/g dry weight (DW)) was obtained on day 17 after the application of 50 μM MJ on cells. We also found that the combined treatment of cells for seven days with MJ and MeβCD synergistically induced the pterocarpan production (trifolirhizin, trifolirhizin malonate, and maackiain) in the cells (58 mg/g DW) and culture medium (222.7 mg/L). Noteworthy, the co-treatment only stimulated the elevated extracellular production of maackiain in the culture medium, indicating its extracellular secretion; however, its glycosides (trifolirhizin and trifolirhizin malonate) were not detected in any significant amounts in the culture medium. This work provides new strategies for the pterocarpan production in plant cell suspension cultures, and shows MeβCD to be an effective solubilizer for the extracellular production of maackiain in the cell cultures of S. flavescens.

Heterologous Metabolic Pathways: Strategies for Optimal Expression in Eukaryotic Hosts.

Heterologous pathways are linked series of biochemical reactions occurring in a host organism after the introduction of foreign genes. Incorporation of metabolic pathways into host organisms is a major strategy used to increase the production of valuable secondary metabolites. Unfortunately, simple introduction of the pathway genes into the heterologous host in most cases does not result in successful heterologous expression. Extensive modification of heterologous genes and the corresponding enzymes on many different levels is required to achieve high target metabolite production rates. This review summarizes the essential techniques used to create heterologous biochemical pathways, with a focus on the key challenges arising in the process and the major strategies for overcoming them.

In vitro plant regeneration and jasmonic acid induced bioactive chemical accumulations in two Hypericum species from Turkey

The aim of the study was to introduce two endangered species from Turkish flora, Hypericum aviculariifolium and H. pruinatum, into in vitro culture and to determine the elicitation effect of jasmonic acid (JA) on the accumulation of secondary metabolites in vitro regenerated flowering plants. A Murashige and Skoog (MS) medium supplemented with plant growth regulators were used for seed germination and seedling culture. Seedling shoot explants in MS medium supplemented with combination of 1 mg/l BA and IAA resulted in maximum and simultaneous induction of roots and shoots. The regenerants of both species survived (98%) in greenhouse cultivation and were assayed for bioactive compounds. The content of secondary metabolites of flowering plants, originating from in vitro culture and treated with different doses of JA was higher than that of intact, i.e. maternal plants providing seeds and control plants. However, the intact and control plants did not differ significantly in the amounts of the bioactive compounds tested. The chemical profile of both species was similar with quantitative differences of individual compounds. Successful establishment of a highly efficient in vitro multiplication protocol in the present study offers a crucial in vitro strategy for the further conservation of endemic and rare species and enhanced production of valuable secondary metabolites.

Use of the kojA promoter, involved in kojic acid biosynthesis, for polyketide production in Aspergillus oryzae: implications for long-term production

BackgroundAspergillus oryzae, a useful industrial filamentous fungus, produces limited varieties of secondary metabolites, such as kojic acid. Thus, for the production of valuable secondary metabolites by genetic engineering, the species is considered a clean host, enabling easy purification from cultured cells. A. oryzae has been evaluated for secondary metabolite production utilizing strong constitutive promoters of genes responsible for primary metabolism. However, secondary metabolites are typically produced by residual nutrition after microbial cells grow to the stationary phase and primary metabolism slows. We focused on a promoter of the secondary metabolism gene kojA, a component of the kojic acid biosynthetic gene cluster, for the production of other secondary metabolites by A. oryzae.ResultsA kojA disruptant that does not produce kojic acid was utilized as a host strain for production. Using this host strain, a mutant that expressed a polyketide synthase gene involved in polyketide secondary metabolite production under the kojA gene promoter was constructed. Then, polyketide production and polyketide synthase gene expression were observed every 24 h in liquid culture. From days 0 to 10 of culture, the polyketide was continuously produced, and the synthase gene expression was maintained. Therefore, the kojA promoter was activated, and it enabled the continuous production of polyketide for 10 days.ConclusionsThe combined use of the kojA gene promoter and a kojA disruptant proved useful for the continuous production of a polyketide secondary metabolite in A. oryzae. These findings suggest that this combination can be applied to other secondary metabolites for long-term production.

Effect of explant source, perlite nanoparticles and TiO2/perlite nanocomposites on phytochemical composition of metabolites in callus cultures of Hypericum perforatum

It appears that the biologically-synthesized nanoparticles (NPs) have potential to perform as effective elicitors for the production of valuable secondary metabolites in plants. Besides, it has been reported that the toxicity of the biologically-synthesized NP is not as much as that of the chemically-synthesized NPs. Therefore, it is necessary to test their advantages aspects. In this study, the physical synthesis of perlite NPs and biologically-synthesis of TiO2/perlite nanocomposites (NCs) were conducted. Subsequently, their effects and explant source influence on the growth characteristics and secondary metabolite profiles of Hypericum perforatum callus cultures were evaluated. According to the obtained results, morphology of the synthesized perlite NPs and TiO2/perlite NCs were mesoporous and spherical with sizes ranging about 14.51–23.34 and 15.50–24.61 nm, respectively. Addition of perlite NPs and TiO2/perlite NCs to the culture medium at the concentration range of 25–200 mg/L showed no adverse impacts on the growth characteristics of H. perforatum calli. According to the GC-MS analysis, the stress caused by perlite NPs and TiO2/perlite NCs led to an increase in the variety, amount and number of volatile compounds. The calli obtained from in vitro grown plants produced more volatile compounds relative to the calli obtained from field grown plants under the nanomaterial stress conditions. The production of hypericin and pseudohypericin were also determined in the callus cultures under desired nanomaterials elicitation. Accordingly, our results suggest that perlite NPs and TiO2/perlite NCs can possibly be considered as effective elicitors for the production of volatile compounds, hypericin, and pseudohypericin in callus cultures of H. perforatum.