Reduction of methicillin-resistant Staphylococcus aureus biofilm growth and development using arctic berry extracts.

Introduction: Surgical site infection remains a devastating and feared complication of surgery caused mainly by Staphylococcus aureus (S. aureus). More specifically, methicillin-resistant S. aureus (MRSA) infection poses a serious threat to global health. Therefore, developing new antibacterial agents to address drug resistance are urgently needed. Compounds derived from natural berries have shown a strong antimicrobial potential. Methods: This study aimed to evaluate the effect of various extracts from two arctic berries, cloudberry (Rubus chamaemorus) and raspberry (Rubus idaeus), on the development of an MRSA biofilm and as treatment on a mature MRSA biofilm. Furthermore, we evaluated the ability of two cloudberry seed-coat fractions, hydrothermal extract and ethanol extract, and the wet-milled hydrothermal extract of a raspberry press cake to inhibit and treat biofilm development in a wound-like medium. To do so, we used a model strain and two clinical strains isolated from infected patients. Results: All berry extracts prevented biofilm development of the three MRSA strains, except the raspberry press cake hydrothermal extract, which produced a diminished anti-staphylococcal effect. Discussion: The studied arctic berry extracts can be used as a treatment for a mature MRSA biofilm, however some limitations in their use exist.

Agrobacterium-Mediated Genetic Transformation of the Medicinal Plant Veratrum dahuricum.

Veratrum dahuricum L. (Liliaceae), a monocotyledonous species distributed throughout the Changbai mountains of Northeast China, is pharmaceutically important, due to the capacity to produce the anticancer drug cyclopamine. An efficient transformation system of Veratrum dahuricum mediated with Agrobacterium tumefaciens is presented. Murashige and Skoog (MS) medium containing 8 mg/L picloram was used to induce embryogenic calli from immature embryos with 56% efficiency. A. tumefaciens LBA4404 carrying the bar gene driven by the cauliflower mosaic virus 35S promoter was employed for embryogenic callus inoculation. A. tumefaciens cell density OD660 = 0.8 for inoculation, half an hour infection period, and three days of co-culture duration were found to be optimal for callus transformation. Phosphinothricin (PPT, 16 mg/L) was used as the selectable agent, and a transformation efficiency of 15% (transgenic plants/100 infected calli) was obtained. The transgenic nature of the regenerated plants was confirmed by PCR and Southern blot analysis, and expression of the bar gene was detected by RT-PCR and Quick PAT/bar strips. The steroid alkaloids cyclopamine, jervine, and veratramine were detected in transgenic plants, in non-transformed and control plants collected from natural sites. The transformation system constitutes a prerequisite for the production of the pharmaceutically important anticancer drug cyclopamine by metabolic engineering of Veratrum.

Methyljasmonate Elicitation Increases Terpenoid Indole Alkaloid Accumulation in Rhazya stricta Hairy Root Cultures.

Methyl jasmonate is capable of initiating or improving the biosynthesis of secondary metabolites in plants and therefore has opened up a concept for the biosynthesis of valuable constituents. In this study, the effect of different doses of methyl jasmonate (MeJA) elicitation on the accumulation of terpenoid indole alkaloids (TIAs) in the hairy root cultures of the medicinal plant, Rhazya stricta throughout a time course (one-seven days) was investigated. Gas chromatography-mass spectrometry (GC-MS) analyses were carried out for targeted ten major non-polar alkaloids. Furthermore, overall alterations in metabolite contents in elicited and control cultures were investigated applying proton nuclear magnetic resonance (1H NMR) spectroscopy. Methyl jasmonate caused dosage- and time course-dependent significant rise in the accumulation of TIAs as determined by GC-MS. The contents of seven alkaloids including eburenine, quebrachamine, fluorocarpamine, pleiocarpamine, tubotaiwine, tetrahydroalstonine, and ajmalicine increased compared to non-elicited cultures. However, MeJA-elicitation did not induce the accumulation of vincanine, yohimbine (isomer II), and vallesiachotamine. Furthermore, principal component analysis (PCA) of 1H NMR metabolic profiles revealed a discrimination between elicited hairy roots and control cultures with significant increase in total vindoline-type alkaloid content and elevated levels of organic and amino acids. In addition, elicited and control samples had different sugar and fatty acid profiles, suggesting that MeJA also influences the primary metabolism of R. stricta hairy roots. It is evident that methyl jasmonate is applicable for elevating alkaloid accumulation in "hairy root" organ cultures of R. strica.

Biotransformation of Cyclodextrine-Complexed Semisynthetic Betulin Derivatives by Plant Cells.

In this study, three semisynthetic betulonic acid-based compounds, 20(29)-dihydrolup-2-en[2,3-d]isoxazol-28-oic acid, 1-betulonoylpyrrolidine, and lupa-2,20(29)-dieno[2,3-b]pyrazin-28-oic acid, were studied in biotransformation experiments using Nicotiana tabacum and Catharanthus roseus cell suspension cultures. Biotransformation was performed using cyclodextrin to aid dissolving poorly water-soluble substrates. Several new derivatives were found, consisting of oxidized and glycosylated (pentose- and hexose-conjugated) products.

Genetically engineered hairy root cultures of Hyoscyamus senecionis and H. muticus: ploidy as a promising parameter in the metabolic engineering of tropane alkaloids.

KEY MESSAGE: Tetraploidy improves overexpression of h6h and scopolamine production of H. muticus, while in H. senecionis, pmt overexpression and elicitation can be used as effective methods for increasing tropane alkaloids. The effects of metabolic engineering in a polyploid context were studied by overexpression of h6h in the tetraploid hairy root cultures of H. muticus. Flow cytometry analysis indicated genetic stability in the majority of the clones, while only a few clones showed genetic instability. Among all the diploid and tetraploid clones, the highest level of h6h transgene expression and scopolamine accumulation was interestingly observed in the tetraploid clones of H. muticus. Therefore, metabolic engineering of the tropane biosynthetic pathway in polyploids is suggested as a potential system for increasing the production of tropane alkaloids. Transgenic hairy root cultures of Hyoscyamus senecionis were also established. While overexpression of pmt in H. senecionis was correlated with a sharp increase in hyoscyamine production, the h6h-overexpressing clones were not able to accumulate higher levels of scopolamine than the leaves of intact plants. Applying methyl jasmonate was followed by a sharp increase in the expression of pmt and a drop in the expression of tropinone reductase II (trII) which consequently resulted in the higher biosynthesis of hyoscyamine and total alkaloids in H. senecionis.

Biotechnology of the medicinal plant Rhazya stricta: a little investigated member of the Apocynaceae family.

Rhazya stricta Decne. (Apocynaceae) is an important medicinal plant that is widely distributed in the Middle East and Indian sub-continent. It produces a large number of terpenoid indole alkaloids (TIAs) some of which possess important pharmacological properties. However, the yields of these compounds are very low. Establishment of a reliable, reproducible and efficient transformation method and induction of hairy roots system is a vital prerequisite for application of biotechnology in order to improve secondary metabolite yields. In the present review, recent biotechnological attempts and advances in TIAs production through transformed hairy root cultures in R. stricta are reviewed to draw the attention to its metabolic engineering potential.

Fermentation and dry fractionation increase bioactivity of cloudberry (Rubus chamaemorus).

Phenolic composition and bioactivity of cloudberry was modified by bioprocessing, and highly bioactive fractions were produced by dry fractionation of the press cake. During fermentation polymeric ellagitannins were partly degraded into ellagic acid derivatives. Phenolic compounds were differentially distributed in seed coarse and fine fractions after dry fractionation process. Tannins concentrated in fine fraction, and flavonol derivatives were mainly found in coarse fraction. Ellagic acid derivatives were equally distributed between the dry fractions. Fermentation and dry fractionation increased statistically significantly anti-adhesion and anti-inflammatory activity of cloudberry. The seed fine fraction showed significant inhibition of P fimbria-mediated haemagglutination assay of uropathogenic Escherichia coli. The seed coarse fraction significantly reduced NO and IL-6 production and iNOS expression in activated macrophages. Fermentation did not affect antimicrobial activity, but slight increase in activity was detected in dry fractions. The results indicate the potential of cloudberry in pharma or health food applications.

Establishment of transgenic Rhazya stricta hairy roots to modulate terpenoid indole alkaloid production.

KEY MESSAGE: Transgenic hairy roots of R. stricta were developed for investigation of alkaloid accumulations. The contents of five identified alkaloids, including serpentine as a new compound, increased compared to non-transformed roots. Rhazya stricta Decne. is a rich source of pharmacologically active terpenoid indole alkaloids (TIAs). In order to study TIA production and enable metabolic engineering, we established hairy root cultures of R. stricta by co-cultivating cotyledon, hypocotyl, leaf, and shoot explants with wild-type Agrobacterium rhizogenes strain LBA 9402 and A. rhizogenes carrying the pK2WG7-gusA binary vector. Hairy roots initiated from the leaf explants 2 to 8 weeks. Transformation was confirmed by polymerase chain reaction and in case of GUS clones with GUS staining assay. Transformation efficiency was 74 and 83% for wild-type and GUS hairy root clones, respectively. Alkaloid accumulation was monitored by HPLC, and identification was achieved by UPLC-MS analysis. The influence of light (16 h photoperiod versus total darkness) and media composition (modified Gamborg B5 medium versus Woody Plant Medium) on the production of TIAs were investigated. Compared to non-transformed roots, wild-type hairy roots accumulated significantly higher amounts of five alkaloids. GUS hairy roots contained higher amounts two of alkaloids compared to non-transformed roots. Light conditions had a marked effect on the accumulation of five alkaloids whereas the composition of media only affected the accumulation of two alkaloids. By successfully establishing R. stricta hairy root clones, the potential of transgenic hairy root systems in modulating TIA production was confirmed.

Determination of terpenoid indole alkaloids in hairy roots of Rhazya stricta (Apocynaceae) by GC-MS.

INTRODUCTION: Rhazya stricta Decne. (Apocynaceae) is a medicinal plant rich in terpenoid indole alkaloids (TIAs), some of which possess important pharmacological properties. The study material including transgenic hairy root cultures have been developed and their potential for alkaloid production are being investigated. OBJECTIVE: In this study, a comprehensive GC-MS method for qualitative and quantitative analysis of alkaloids from Rhazya hairy roots was developed. METHODS: The composition of alkaloids was determined by using GC-MS. In quantification, the ratio between alkaloid and internal standard was based on extracted ion from total ion current (TIC) analyses. RESULTS: The developed method was validated. An acceptable precision with RSD ≤ 8% over a linear range of 1 to 100 µg/mL was achieved. The accuracy of the method was within 94-107%. Analysis of hairy root extracts indicated the occurrence of a total of 20 TIAs. Six of them, pleiocarpamine, fluorocarpamine, vincamine, ajmalicine and two yohimbine isomers are reported here for the first time in Rhazya. Trimethylsilyl (TMS) derivatisation of the extracts resulted in the separation of two isomers for yohimbine and also for vallesiachotamine. Clearly improved chromatographic profiles of TMS-derivatives were observed for vincanine and for minor compounds vincamine and rhazine. CONCLUSION: The results show that the present GC-MS method is reliable and well applicable for studying the variation of indole alkaloids in Rhazya samples.

Evaluation of tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots for the production of geraniol, the first committed step in terpenoid indole alkaloid pathway.

The terpenoid indole alkaloids are one of the major classes of plant-derived natural products and are well known for their many applications in the pharmaceutical, fragrance and cosmetics industries. Hairy root cultures are useful for the production of plant secondary metabolites because of their genetic and biochemical stability and their rapid growth in hormone-free media. Tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots, which do not produce geraniol naturally, were engineered to express a plastid-targeted geraniol synthase gene originally isolated from Valeriana officinalis L. (VoGES). A SPME-GC-MS screening tool was developed for the rapid evaluation of production clones. The GC-MS analysis revealed that the free geraniol content in 20 hairy root clones expressing VoGES was an average of 13.7 µg/g dry weight (DW) and a maximum of 31.3 µg/g DW. More detailed metabolic analysis revealed that geraniol derivatives were present in six major glycoside forms, namely the hexose and/or pentose conjugates of geraniol and hydroxygeraniol, resulting in total geraniol levels of up to 204.3 µg/g DW following deglycosylation. A benchtop-scale process was developed in a 20-L wave-mixed bioreactor eventually yielding hundreds of grams of biomass and milligram quantities of geraniol per cultivation bag.

CathaCyc, a metabolic pathway database built from Catharanthus roseus RNA-Seq data.

The medicinal plant Madagascar periwinkle (Catharanthus roseus) synthesizes numerous terpenoid indole alkaloids (TIAs), such as the anticancer drugs vinblastine and vincristine. The TIA pathway operates in a complex metabolic network that steers plant growth and survival. Pathway databases and metabolic networks reconstructed from 'omics' sequence data can help to discover missing enzymes, study metabolic pathway evolution and, ultimately, engineer metabolic pathways. To date, such databases have mainly been built for model plant species with sequenced genomes. Although genome sequence data are not available for most medicinal plant species, next-generation sequencing is now extensively employed to create comprehensive medicinal plant transcriptome sequence resources. Here we report on the construction of CathaCyc, a detailed metabolic pathway database, from C. roseus RNA-Seq data sets. CathaCyc (version 1.0) contains 390 pathways with 1,347 assigned enzymes and spans primary and secondary metabolism. Curation of the pathways linked with the synthesis of TIAs and triterpenoids, their primary metabolic precursors, and their elicitors, the jasmonate hormones, demonstrated that RNA-Seq resources are suitable for the construction of pathway databases. CathaCyc is accessible online (http://www.cathacyc.org) and offers a range of tools for the visualization and analysis of metabolic networks and 'omics' data. Overlay with expression data from publicly available RNA-Seq resources demonstrated that two well-characterized C. roseus terpenoid pathways, those of TIAs and triterpenoids, are subject to distinct regulation by both developmental and environmental cues. We anticipate that databases such as CathaCyc will become key to the study and exploitation of the metabolism of medicinal plants.

Metabolic engineering of plant secondary products: which way forward?

Secondary products are small molecular weight compounds produced by secondary metabolic pathways in plants. They are regarded as non-essential for normal growth and development but often confer benefits such as defense against pathogens, pests and herbivores or the attraction of pollinators. Many secondary products affect the survival and/or behavior of microbes, insects and mammals and they often have useful pharmacological effects in humans. Most secondary products can only be obtained as extracts from medicinal plants, many of which grow slowly and are difficult to cultivate. Chemical synthesis, although possible in principle, is often impractical or uneconomical due to the complexity of their molecular structures. The large scale production of secondary products by metabolic engineering has therefore been investigated in a number of heterologous systems including microbes, plant cell/organ cultures, and intact plants. In this critical review of production platforms for plant secondary products, we discuss the advantages and constraints of different approaches and the impact of post-genomics technologies on gene discovery and metabolite analysis. We highlight bottlenecks that remain to be overcome before the routine exploitation of secondary products can be achieved for the benefit of mankind.

Tropane and nicotine alkaloid biosynthesis-novel approaches towards biotechnological production of plant-derived pharmaceuticals.

Many plants belonging to the Solanaceae family have been used as a source of pharmaceuticals for centuries because of their active principles, tropane and nicotine alkaloids. Tropane alkaloids, atropine, hyoscyamine and scopolamine, are among the oldest drugs in medicine. On the other hand nicotine, the addictive agent in tobacco, has only recently gained attention as a backbone for novel potential alkaloids to be used for certain neurological diseases. The biotechnological production of alkaloids utilizing plant cells as hosts would be an attractive option. However, to date very little success in this field has been gained because of the lack of understanding how these compounds are synthesized in a plant cell. Metabolic engineering attempts have already shown that when the rate-limiting steps of the biosynthetic pathway are completely known and the respective genes cloned, the exact regulation towards desired medicinal products will be possible in the near future. The new functional genomics tools, which combine transcriptome and metabolome data, will create a platform to better understand a whole system and to engineer the complex plant biosynthetic pathways. With the help of this technology, it is not only possible to produce known plant metabolites more effectively but also to make arrays of new compounds in plants and cell cultures.

Biotransformation of hyoscyamine into scopolamine in transgenic tobacco cell cultures.

Hyoscyamine-6beta-hydroxylase (H6H) catalyses the conversion of hyoscyamine into its epoxide scopolamine, a compound with a higher added value in the pharmaceutical market than hyoscyamine. We report the establishment of tobacco cell cultures carrying the Hyoscyamus muticus h6h gene under the control of the promoter CAMV 35S. The cell cultures were derived from hairy roots obtained via genetically modified Agrobacterium rhizogenes carrying the pRi and pLAL21 plasmids. The cultures were fed with hyoscyamine, and 4 weeks later the amount of scopolamine produced was quantified by HPLC. The transgenic cell suspension cultures showed a considerable capacity for the bioconversion of hyoscyamine into scopolamine, and released it to the culture medium. Although the scale-up from shake-flask to bioreactor culture usually results in reduced productivities, our transgenic cells grown in a 5-L turbine stirred tank reactor in a batch mode significantly increased the scopolamine accumulation.

Development of in vitro techniques for the important medicinal plant Veratrum californicum.

VERATRUM CALIFORNICUM (Liliaceae) is an important monocotyledonous medicinal plant which is the only source of the anticancer compound cyclopamine. An IN VITRO culture system for somatic embryogenesis and green plant regeneration of VERATRUM CALIFORNICUM was developed. Embryogenic calli were induced from mature embryos on induction medium. Five basal media supplemented with different growth regulators were evaluated for embryogenic callus induction, modified MS medium with 4 mg/L picloram showing the best result for embryogenic callus production. Fine suspension cell lines were established by employing friable embryogenic calli as starting material and AA medium and L2 medium as culture media. The suspension cell lines cultured in AA medium with 4 mg/L NAA appeared to be fresh yellow and fast growing. The suspension cells were cryopreserved successfully and recovered at a high rate. Green plants were regenerated from embryogenic calli maintained on solid medium with 73 % regeneration ability (green plants/100 calli) in 27-month-old culture. The IN VITRO plantlets contained the steroid alkaloids cyclopamine and veratramine. This IN VITRO system will form the basis for metabolic engineering of VERATRUM cells in the context of biotechnological production of pharmaceutically important secondary metabolites. DMSO:dimethyl sulfoxide fw:fresh weight NAA:naphthaleneacetic acid 2,4-D:2,4-dichlorophenoxyacetic acid picloram:4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid dicamba:3,6-dichloro-2-methoxybenzoic acid.

Berry phenolics: antimicrobial properties and mechanisms of action against severe human pathogens.

Antimicrobial activity and mechanisms of phenolic extracts of 12 Nordic berries were studied against selected human pathogenic microbes. The most sensitive bacteria on berry phenolics were Helicobacter pylori and Bacillus cereus. Campylobacter jejuni and Candida albicans were inhibited only with phenolic extracts of cloudberry, raspberry, and strawberry, which all were rich in ellagitannins. Cloudberry extract gave strong microbicidic effects on the basis of plate count with all studied strains. However, fluorescence staining of liquid cultures of virulent Salmonella showed viable cells not detectable by plate count adhering to cloudberry extract, whereas Staphylococcus aureus cells adhered to berry extracts were dead on the basis of their fluorescence and plate count. Phenolic extracts of cloudberry and raspberry disintegrated the outer membrane of examined Salmonella strains as indicated by 1-N-phenylnaphthylamine (NPN) uptake increase and analysis of liberation of [14C]galactose- lipopolysaccharide. Gallic acid effectively permeabilized the tested Salmonella strains, and significant increase in the NPN uptake was recorded. The stability of berry phenolics and their antimicrobial activity in berries stored frozen for a year were examined using Escherichia coli and nonvirulent Salmonella enterica sv. Typhimurium. The amount of phenolic compounds decreased in all berries, but their antimicrobial activity was not influenced accordingly. Cloudberry, in particular, showed constantly strong antimicrobial activity during the storage.

Heterologous expression of Vitreoscilla hemoglobin (VHb) and cultivation conditions affect the alkaloid profile of Hyoscyamus muticus hairy roots.

Fast-growing hairy root cultures of Hyoscyamus muticus induced by Agrobacterium rhizogenes offer a potential production system for tropane alkaloids. Oxygen deficiency has been shown to limit growth and biomass accumulation of hairy roots, whereas little experimental data is available on the effect of oxygen on alkaloid production. We have investigated the effect of Vitreoscilla hemoglobin (VHb) expression and cultivation conditions on the complete alkaloid profile of H. muticus hairy roots in shake flasks and in a laboratory scale bioreactor. We optimized the growth medium composition and studied the effects of sucrose, ammonium, nitrate, and phosphate on growth and alkaloid production. Maximum biomass accumulation was achieved with the highest and maximum hyoscyamine content with the lowest sucrose concentration. The optimum nitrate concentration for growth was higher for the VHb line than the control. Neither VHb expression nor aeration improved the hyoscyamine content significantly, thus suggesting that hyoscyamine biosynthesis is not limited by oxygen availability. Interestingly, the effect of VHb expression on the alkaloid profile was slightly different from that of aeration. VHb expression did not affect the concentrations of cuscohygrine, which was increased by aeration. Therefore, the effect of VHb is probably not related only to its ability to increase the intracellular effective oxygen concentration.

Gene-to-metabolite networks for terpenoid indole alkaloid biosynthesis in Catharanthus roseus cells.

Rational engineering of complicated metabolic networks involved in the production of biologically active plant compounds has been greatly impeded by our poor understanding of the regulatory and metabolic pathways underlying the biosynthesis of these compounds. Whereas comprehensive genome-wide functional genomics approaches can be successfully applied to analyze a select number of model plants, these holistic approaches are not yet available for the study of nonmodel plants that include most, if not all, medicinal plants. We report here a comprehensive profiling analysis of the Madagascar periwinkle (Catharanthus roseus), a source of the anticancer drugs vinblastine and vincristine. Genome-wide transcript profiling by cDNA-amplified fragment-length polymorphism combined with metabolic profiling of elicited C. roseus cell cultures yielded a collection of known and previously undescribed transcript tags and metabolites associated with terpenoid indole alkaloids. Previously undescribed gene-to-gene and gene-to-metabolite networks were drawn up by searching for correlations between the expression profiles of 417 gene tags and the accumulation profiles of 178 metabolite peaks. These networks revealed that the different branches of terpenoid indole alkaloid biosynthesis and various other metabolic pathways are subject to differing hormonal regulation. These networks also served to identify a select number of genes and metabolites likely to be involved in the biosynthesis of terpenoid indole alkaloids. This study provides the basis for a better understanding of periwinkle secondary metabolism and increases the practical potential of metabolic engineering of this important medicinal plant.

Expression of Vitreoscilla hemoglobin enhances growth of Hyoscyamus muticus hairy root cultures.

The Vitreoscilla hemoglobin gene (vhb) was introduced into Hyoscyamus muticus with the aim of investigating its effect on growth and alkaloid production of Agrobacterium rhizogenes-induced hairy root cultures. We were able to generate several VHb-expressing hairy root lines with different integration patterns. Substantial somaclonal variation was observed in growth and hyoscyamine production amongst both VHb-expressing lines and controls. Despite this variation, the growth properties of single lines remained stable over time. Expression of VHb was found to improve growth of H. muticus hairy roots in shake-flask cultures. The dry weights of the root cultures expressing Vitreoscilla hemoglobin were on average 18 % higher than those of the controls. VHb expression also increased the volumetric hyoscyamine production, mainly due to the improved growth properties. However, this difference was not statistically significant due to the wide somaclonal variation and fluctuations over time in both VHb and control hairy root lines.

Bioactive berry compounds-novel tools against human pathogens.

Berry fruits are rich sources of bioactive compounds, such as phenolics and organic acids, which have antimicrobial activities against human pathogens. Among different berries and berry phenolics, cranberry, cloudberry, raspberry, strawberry and bilberry especially possess clear antimicrobial effects against, e.g. Salmonella and Staphylococcus. Complex phenolic polymers, like ellagitannins, are strong antibacterial agents present in cloudberry and raspberry. Several mechanisms of action in the growth inhibition of bacteria are involved, such as destabilisation of cytoplasmic membrane, permeabilisation of plasma membrane, inhibition of extracellular microbial enzymes, direct actions on microbial metabolism and deprivation of the substrates required for microbial growth. Antimicrobial activity of berries may also be related to antiadherence of bacteria to epithelial cells, which is a prerequisite for colonisation and infection of many pathogens. Antimicrobial berry compounds may have important applications in the future as natural antimicrobial agents for food industry as well as for medicine. Some of the novel approaches are discussed.