Plant in vitro culture for the production of antioxidants--a review

Plant in vitro cultures are able to produce and accumulate many medicinally valuable secondary metabolites. Antioxidants are an important group of medicinal preventive compounds as well as being food additives inhibiting detrimental changes of easily oxidizable nutrients. Many different in vitro approaches have been used for increased biosynthesis and the accumulation of antioxidant compounds in plant cells. In the present review some of the most active antioxidants derived from plant tissue cultures are described; they have been divided into the main chemical groups of polyphenols and isoprenoids, and several examples also from other chemical classes are presented. The strategies used for improving the antioxidants in vitro production efficiency are also highlighted, including media optimization, biotransformation, elicitation, Agrobacterium transformation and scale-up.     

Opportunistic emissions of volatile isoprenoids

Isoprene, monoterpenes and sesquiterpenes are synthesized and emitted by some plant species, but not all plant species have this ability. These volatile, nonessential isoprenoid compounds share the same biochemical precursors as larger essential isoprenoids such as gibberellic acids and carotenoids. They have many protective and ecological functions for the plant species that produce them, but plant species that do not produce these compounds also grow and reproduce successfully. Here, we develop an 'opportunist hypothesis' suggesting that (i) volatile isoprenoid production takes advantage of dimethylallyl diphosphate (DMAPP) and its isomer isopentenyl diphosphate (IPP), which are synthesized primarily to produce essential isoprenoids, and (ii) conditions affecting synthesis of the higher isoprenoids will affect the production and emission of volatile isoprenoids.     

AM fungi root colonization increases the production of essential isoprenoids vs. nonessential isoprenoids especially under drought stress conditions or after jasmonic acid application

Previous studies have shown that root colonization by arbuscular mycorrhiza (AM) fungi enhances plant resistance to abiotic and biotic stressors and finally plant growth. However, little is known about the effect of AM on isoprenoid foliar and root content. In this study we tested whether the AM symbiosis affects carbon resource allocation to different classes of isoprenoids such as the volatile nonessential isoprenoids (monoterpenes and sesquiterpenes) and the non-volatile essential isoprenoids (abscisic acid, chlorophylls and carotenoids). By subjecting the plants to stressors such as drought and to exogenous application of JA, we wanted to test their interaction with AM symbiosis in conditions where isoprenoids usually play a role in resistance to stress and in plant defence. Root colonization by AM fungi favoured the leaf production of essential isoprenoids rather than nonessential ones, especially under drought stress conditions or after JA application. The increased carbon demand brought on by AM fungi might thus influence not only the amount of carbon allocated to isoprenoids, but also the carbon partitioning between the different classes of isoprenoids, thus explaining the not previously shown decrease of root volatile isoprenoids in AM plants. We propose that since AM fungi are a nutrient source for the plant, other carbon sinks normally necessary to increase nutrient uptake can be avoided and therefore the plant can devote more resources to synthesize essential isoprenoids for plant growth.     

Gas composition strategies for the successful scale-up of Catharanthus roseus cell cultures for the production of ajmalicine

Ajmalicine, serpentine, catharanthine, and vindoline are monoterpenoid indole alkaloids (MIAs) of commercial interest which are produced by the Catharanthus roseus plant. Cultures of C. roseus have been investigated as a potential source of these pharmaceutically important compounds since the early 1960s. In addition, their production from C. roseus cultures has served as a model system for investigating secondary metabolism and for evaluating production-enhancing strategies. Initially, this review will survey (1) the MIAs of interest for large-scale production from plant cell cultures and (2) the volumetric productivities of a specific MIA, ajmalicine, achieved and projected using plant cell cultures. To meet the need for these valuable compounds, the production of these MIAs from plant cell cultures must be successfully reproduced in large-scale aerated and agitated reactors. While the large-scale cultivation of plant cell cultures is currently feasible, initial attempts at scale-up may yield results that differ from that optimized in flasks. To bridge the jump between production in flasks and production in large-scale bioreactors, changes introduced with scale-up such as gas composition must be identified and rationally manipulated to reproduce or even improve growth and secondary metabolite production. Hence, this review will (1) identify the effects of gas composition (i.e., O₂, CO₂, ethylene, or other endogenous volatile compounds) on growth and secondary metabolism and (2) draw operating strategies for optimizing the gas composition for growth of C. roseus cultures and the production of ajmalicine.     

Roles of glycine betaine and proline in improving plant abiotic stress resistance

Glycine betaine (GB) and proline are two major organic osmolytes that accumulate in a variety of plant species in response to environmental stresses such as drought, salinity, extreme temperatures, UV radiation and heavy metals. Although their actual roles in plant osmotolerance remain controversial, both compounds are thought to have positive effects on enzyme and membrane integrity along with adaptive roles in mediating osmotic adjustment in plants grown under stress conditions. While many studies have indicated a positive relationship between accumulation of GB and proline and plant stress tolerance, some have argued that the increase in their concentrations under stress is a product of, and not an adaptive response to stress. In this article, we review and discuss the evidence supporting each of these arguments. As not all plant species are capable of natural production or accumulation of these compounds in response to stress, extensive research has been conducted examining various approaches to introduce them into plants. Genetically-engineered plants containing transgenes for production of GB or proline have thus far faced with the limitation of being unable to produce sufficient amounts of these compounds to ameliorate stress effects. An alternative “shot-gun” approach of exogenous application of GB or proline to plants under stress conditions, however, has gained some attention. A review of the literature indicates that in many, but not all, plant species such applications lead to significant increases in growth and final crop yield under environmental stresses. In this review article, numerous examples of successful application of these compounds to improve plant stress tolerance are presented. However, to streamline useful and economic applications of these compounds, further investigations are needed to determine the most effective concentrations and number of applications as well as the most responsive growth stage(s) of the plant. All these factors may vary from species to species. Furthermore, a better understanding of the mechanisms of action of exogenously applied GB and proline is expected to aid their effective utilization in crop production in stress environments.     

Improvement of hairy root cultures and plants by changing biosynthetic pathways leading to pharmaceutical metabolites: Strategies and applications

A plethora of bioactive plant metabolites has been explored for pharmaceutical, food chemistry and agricultural applications. The chemical synthesis of these structures is often difficult, so plants are favorably used as producers. While whole plants can serve as a source for secondary metabolites and can be also improved by metabolic engineering, more often cell or organ cultures of relevant plant species are of interest. It should be noted that only in few cases the production for commercial application in such cultures has been achieved. Their genetic manipulation is sometimes faster and the production of a specific metabolite is more reliable, because of less environmental influences. In addition, upscaling in bioreactors is nowadays possible for many of these cultures, so some are already used in industry. There are approaches to alter the profile of metabolites not only by using plant genes, but also by using bacterial genes encoding modifying enzymes. Also, strategies to cope with unwanted or even toxic compounds are available. The need for metabolic engineering of plant secondary metabolite pathways is increasing with the rising demand for (novel) compounds with new bioactive properties. Here, we give some examples of recent developments for the metabolic engineering of plants and organ cultures, which can be used in the production of metabolites with interesting properties.     

Terpenoid biomaterials

Terpenoids (isoprenoids) encompass more than 40 000 structures and form the largest class of all known plant metabolites. Some terpenoids have well-characterized physiological functions that are common to most plant species. In addition, many of the structurally diverse plant terpenoids may function in taxonomically more discrete, specialized interactions with other organisms. Historically, specialized terpenoids, together with alkaloids and many of the phenolics, have been referred to as secondary metabolites. More recently, these compounds have become widely recognized, conceptually and/or empirically, for their essential ecological functions in plant biology. Owing to their diverse biological activities and their diverse physical and chemical properties, terpenoid plant chemicals have been exploited by humans as traditional biomaterials in the form of complex mixtures or in the form of more or less pure compounds since ancient times. Plant terpenoids are widely used as industrially relevant chemicals, including many pharmaceuticals, flavours, fragrances, pesticides and disinfectants, and as large-volume feedstocks for chemical industries. Recently, there has been a renaissance of awareness of plant terpenoids as a valuable biological resource for societies that will have to become less reliant on petrochemicals. Harnessing the powers of plant and microbial systems for production of economically valuable plant terpenoids requires interdisciplinary and often expensive research into their chemistry, biosynthesis and genomics, as well as metabolic and biochemical engineering. This paper provides an overview of the formation of hemi-, mono-, sesqui- and diterpenoids in plants, and highlights some well-established examples for these classes of terpenoids in the context of biomaterials and biofuels.     

Biotechnology of flavonoids and other phenylpropanoid-derived natural products. Part I: Chemical diversity, impacts on plant biology and human health

Plant natural products derived from phenylalanine and the phenylpropanoid pathway are impressive in their chemical diversity and are the result of plant evolution, which has selected for the acquisition of large repertoires of pigments, structural and defensive compounds, all derived from a phenylpropanoid backbone via the plant-specific phenylpropanoid pathway. These compounds are important in plant growth, development and responses to environmental stresses and thus can have large impacts on agricultural productivity. While plant-based medicines containing phenylpropanoid-derived active components have long been used by humans, the benefits of specific flavonoids and other phenylpropanoid-derived compounds to human health and their potential for long-term health benefits have been only recognized more recently. In this part of the review, we discuss the diversity and biosynthetic origins of phenylpropanoids and particularly of the flavonoid and stilbenoid natural products. We then review data pertaining to the modes of action and biological properties of these compounds, referring on their effects on human health and physiology and their roles as plant defense and antimicrobial compounds. This review continues in Part II discussing the use of biotechnological tools targeting the rational reconstruction of multienzyme pathways in order to modify the production of such compounds in plants and model microbial systems for the benefit of agriculture and forestry.     

Biosynthesis of isoprenoids, polyunsaturated fatty acids and flavonoids in Saccharomyces cerevisiae

Industrial biotechnology employs the controlled use of microorganisms for the production of synthetic chemicals or simple biomass that can further be used in a diverse array of applications that span the pharmaceutical, chemical and nutraceutical industries. Recent advances in metagenomics and in the incorporation of entire biosynthetic pathways into Saccharomyces cerevisiae have greatly expanded both the fitness and the repertoire of biochemicals that can be synthesized from this popular microorganism. Further, the availability of the S. cerevisiae entire genome sequence allows the application of systems biology approaches for improving its enormous biosynthetic potential. In this review, we will describe some of the efforts on using S. cerevisiae as a cell factory for the biosynthesis of high-value natural products that belong to the families of isoprenoids, flavonoids and long chain polyunsaturated fatty acids. As natural products are increasingly becoming the center of attention of the pharmaceutical and nutraceutical industries, the use of S. cerevisiae for their production is only expected to expand in the future, further allowing the biosynthesis of novel molecular structures with unique properties.     

Isoprenoid generating systems in plants — A handy toolbox how to assess contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthetic process

Isoprenoids comprise an astonishingly diverse group of metabolites with numerous potential and actual applications in medicine, agriculture and the chemical industry. Generation of efficient platforms producing isoprenoids is a target of numerous laboratories. Such efforts are generally enhanced if the native biosynthetic routes can be identified, and if the regulatory mechanisms responsible for the biosynthesis of the compound(s) of interest can be determined.In this review a critical summary of the techniques applied to establish the contribution of the two alternative routes of isoprenoid production operating in plant cells, the mevalonate and methylerythritol pathways, with a focus on their co-operation (cross-talk) is presented. Special attention has been paid to methodological aspects of the referred studies, in order to give the reader a deeper understanding for the nuances of these powerful techniques. This review has been designed as an organized toolbox, which might offer the researchers comments useful both for project design and for interpretation of results obtained.     

Strategies of isoprenoids production in engineered bacteria

Isoprenoids are the largest family of natural products, over 40 000 compounds have been described, which have been widely used in various fields. Currently, the isoprenoid products are mainly produced by natural extraction or chemical synthesis, however, limited yield and high cost is far behind the increasing need. Most bacteria synthesize the precursors of isoprenoids through the methylerythritol 4‐phosphate pathway, microbial synthesis of isoprenoids by fermentation becomes more attractive mainly in terms of environmental concern and renewable resources. In this review, the strategies of isoprenoid production in bacteria by synthetic biology are discussed. Introducing foreign genes associated with desired products made it possible to produce isoprenoids in bacteria. Furthermore, the yield of isoprenoids is increased by the strategies of overexpression of native or foreign genes, introducing heterologous mevalonate pathway, balancing of the precursors and inactivating the competing pathway, these methods were used separately or simultaneously.     

Actinomycins inhibit the production of the siderophore pyoverdines in the plant pathogen Pseudomonas cichorii SPC9018

ABSTRACT

Pyoverdines, a group of peptide siderophores produced by Pseudomonas species, function not only in iron acquisition, but also in their virulence in hosts. Thus, chemical inhibition of pyoverdine production may be an effective strategy to control Pseudomonas virulence. In the plant pathogen Pseudomonas cichorii SPC9018 (SPC9018), pyoverdine production is required for virulence on eggplant. We screened microbial culture extracts in a pyoverdine-production inhibition assay of SPC9018 and found Streptomyces sp. RM-32 as a candidate-producer. We isolated two active compounds from RM-32 cultures, and elucidated their structures to be actinomycins X₂ and D. Actinomycins X₂ and D inhibited pyoverdine production by SPC9018 with IC₅₀ values of 17.6 and 29.6 μM, respectively. Furthermore, pyoverdine production in other Pseudomonas bacteria, such as the mushroom pathogen P. tolaasii, was inhibited by the actinomycins. Therefore, these actinomycins may be useful as chemical tools to examine pyoverdine functions and as seed compounds for anti-Pseudomonas virulence agents.     

Microbial biosurfactants as additives for food industries

Microbial biosurfactants with high ability to reduce surface and interfacial surface tension and conferring important properties such as emulsification, detergency, solubilization, lubrication and phase dispersion have a wide range of potential applications in many industries. Significant interest in these compounds has been demonstrated by environmental, bioremediation, oil, petroleum, food, beverage, cosmetic and pharmaceutical industries attracted by their low toxicity, biodegradability and sustainable production technologies. Despite having significant potentials associated with emulsion formation, stabilization, antiadhesive and antimicrobial activities, significantly less output and applications have been reported in food industry. This has been exacerbated by uneconomical or uncompetitive costing issues for their production when compared to plant or chemical counterparts. In this review, biosurfactants properties, present uses and potential future applications as food additives acting as thickening, emulsifying, dispersing or stabilising agents in addition to the use of sustainable economic processes utilising agro‐industrial wastes as alternative substrates for their production are discussed. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1097–1108, 2013     

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

Isoprenoids are the most numerous and structurally diverse family of natural products. Terpenoids, a class of isoprenoids often isolated from plants, are used as commercial flavor and fragrance compounds and antimalarial or anticancer drugs. Because plant tissue extractions typically yield low terpenoid concentrations, we sought an alternative method to produce high-value terpenoid compounds, such as the antimalarial drug artemisinin, in a microbial host. We engineered the expression of a synthetic amorpha-4,11-diene synthase gene and the mevalonate isoprenoid pathway from Saccharomyces cerevisiae in Escherichia coli. Concentrations of amorphadiene, the sesquiterpene olefin precursor to artemisinin, reached 24 microg caryophyllene equivalent/ml. Because isopentenyl and dimethylallyl pyrophosphates are the universal precursors to all isoprenoids, the strains developed in this study can serve as platform hosts for the production of any terpenoid compound for which a terpene synthase gene is available.     

Overexpressing 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase (HMGR) in the Lactococcal Mevalonate Pathway for Heterologous Plant Sesquiterpene Production

Isoprenoids are a large and diverse group of metabolites with interesting properties such as flavour, fragrance and therapeutic properties. They are produced via two pathways, the mevalonate pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. While plants are the richest source of isoprenoids, they are not the most efficient producers. Escherichia coli and yeasts have been extensively studied as heterologous hosts for plant isoprenoids production. In the current study, we describe the usage of the food grade Lactococcus lactis as a potential heterologous host for the production of sesquiterpenes from a local herbaceous Malaysian plant, Persicaria minor (synonym Polygonum minus). A sesquiterpene synthase gene from P. minor was successfully cloned and expressed in L. lactis. The expressed protein was identified to be a β-sesquiphellandrene synthase as it was demonstrated to be functional in producing β-sesquiphellandrene at 85.4% of the total sesquiterpenes produced based on in vitro enzymatic assays. The recombinant L. lactis strain developed in this study was also capable of producing β-sesquiphellandrene in vivo without exogenous substrates supplementation. In addition, overexpression of the strain’s endogenous 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR), an established rate-limiting enzyme in the eukaryotic mevalonate pathway, increased the production level of β-sesquiphellandrene by 1.25–1.60 fold. The highest amount achieved was 33 nM at 2 h post-induction.     

Antioxidant and Antimelanogenic Activities of Compounds Isolated from the Aerial Parts of Achillea alpina L.

Achillea alpina is widely distributed in Korea and is often used as a folk medicine for stomach disorders. Although a previous study isolated antioxidant compounds (flavonoid O‐glucoside, sesquiterpene) from this plant, no systematic study of its chemical constituents had been reported. The present study aimed to identify the phytochemicals present in a methanol extract of A. alpina, assess their potential antioxidant activities in vitro, and determine their effects on melanogenesis in B16F10 melanoma cells. Column chromatographic separation of aqueous fractions of A. alpina led to the isolation of 17 compounds. The chemical structures of these compounds were determined using spectroscopic data from electrospray ionization‐mass spectrometry and nuclear magnetic resonance. To the best of our knowledge, the present study is the first to identify compounds 2 – 10 and 12 – 17 in A. alpina. Furthermore, compound 6 possessed powerful antioxidant activity, while compound 15 suppressed intracellular tyrosinase activity and thus reduced melanogenesis in B16F10 cells. Therefore, our research suggested that these naturally occurring compounds have the potential to reduce oxidative stress and promote skin whitening. Further investigations will be required to elucidate the mechanisms underlying the antioxidant and antityrosinase activities of these compounds.     

Male pheromone composition depends on larval but not adult diet in Heliconius melpomene

1. Condition‐dependent traits can act as honest signals of mate quality, with fitter individuals being able to display preferred phenotypes. Nutrition is known to be an important determinant of individual condition, with diet known to affect many secondary sexual traits. 2. In Heliconius butterflies, male chemical signalling plays an important role in female mate choice. Potential male sex pheromone components have been identified previously, although it is unclear what information they convey to the female. 3. In the present study, the effect of diet on androconial and genital compound production is tested in male Heliconius melpomene rosina. To manipulate larval diet, larvae are reared on three different Passiflora host plants: Passiflora menispermifolia, the preferred host plant, Passiflora vitifolia and Passiflora platyloba. To manipulate adult diet, adult butterflies are reared with and without access to pollen, a key component of their diet. 4. No evidence is found to suggest that adult pollen consumption affects compound production in the first 10 days after eclosion. There is also a strong overlap in the chemical profiles of individuals reared on different larval host plants. The most abundant compounds produced by the butterflies do not differ between host plant groups. However, some compounds found in small amounts differ both qualitatively and quantitatively. Some of these compounds are predicted to be of plant origin and the others synthesised by the butterfly. Further electrophysiological and behavioural experiments will be needed to determine the biological significance of these differences.     

Isoprenoid biosynthesis is not compromised in a Zellweger syndrome mouse model.

Because several studies indicated that peroxisomes are important for the biosynthesis of isoprenoids, we wanted to investigate whether a reduced availability of isoprenoids could be one of the pathogenic factors contributing to the severe phenotype of the Pex5(-/-) mouse, a model for Zellweger syndrome. Total cholesterol was determined in plasma, brain and liver of newborn mice. In none of these tissues a significant difference was observed between Pex5(-/-) and wild type or heterozygous mice. The hepatic ubiquinone content was found to be even higher in Pex5(-/-) mice as compared to wild type or heterozygous littermates. To investigate whether the Pex5(-/-) fetuses are able to synthesise their own isoprenoids, fibroblasts derived from these mice were incubated with radiolabeled mevalonolactone as a substrate for isoprenoid synthesis. No significant difference was observed between the cholesterol production rates of Pex5(-/-) and normal fibroblasts. Our results show that there is no deficiency of isoprenoids in newborn Pex5(-/-) mice, excluding the possibility that a lack of these compounds is a determinant factor in the development of the disease state before birth.     

L-DOPA functions as a plant pheromone for belowground anti-herbivory communication

While mechanisms of plant–plant communication for alerting neighbouring plants of an imminent insect herbivore attack have been described aboveground via the production of volatile organic compounds (VOCs), we are yet to decipher the specific components of plant–plant signalling belowground. Using bioassay-guided fractionation, we isolated and identified the non-protein amino acid l -DOPA, released from roots of Acyrtosiphon pisum aphid-infested Vicia faba plants, as an active compound in triggering the production of VOCs released aboveground in uninfested plants. In behavioural assays, we show that after contact with l -DOPA, healthy plants become highly attractive to the aphid parasitoid (Aphidius ervi), as if they were infested by aphids. We conclude that l -DOPA, originally described as a brain neurotransmitter precursor, can also enhance immunity in plants.     

Medicinal biotechnology in the genus <Emphasis Type="Italic">scutellaria</Emphasis>

Plant-based medicines have an important role in the lives of millions of people. The ancient knowledge of the use of plants as medicines has led to the discovery of many important western pharmaceuticals, and the popularity of whole plant preparations for a range of therapeutic applications is growing rapidly. However, there are many challenges in the production of plant-based medicines, many of which put both the consumer and the plant populations at risk. Modern biotechnology can be optimized to mass-produce plants of specific chemical composition for use as particular treatments and applications. In this review, we have used one of the most important medicinal plant genera, Scutellaria, as a model to assess the potential of applications of biotechnology for the improvement of medicinal plants.