The production of plant secondary metabolites by means of large-scale culture of plant cells in bioreactors is technically feasible. The economy of such a production is the major bottleneck. For some ...costly products it is feasible, but unfortunately some of the most interesting products are only in very small amounts or not all produced in plant cell cultures. Screening, selection and medium optimization may lead to 20- to 30-fold increase in case one has producing cultures. In case of phytoalexins, elicitation will lead to high production. But for many of the compounds of interest the production is not inducible by elicitors. The culture of differentiated cells, such as (hairy) root or shoot cultures, is an alternative, but is hampered by problems in scaling up of such cultures. Metabolic engineering offers new perspectives for improving the production of compounds of interest. This approach can be used to improve production in the cell culture, in the plant itself or even production in other plant species or organisms. Studies on the production of terpenoid indole alkaloids have shown that the overexpression of single genes of the pathway may lead for some enzymes to an increased production of the direct product, but not necessarily to an increased alkaloid production. On the other hand feeding of such transgenic cultures with early precursors showed an enormous capacity for producing alkaloids, which is not utilized without feeding precursors. Overexpression of regulatory genes results in the upregulation of a series of enzymes in the alkaloid pathway, but not to an improved flux through the pathway, but feeding loganin does result in increased alkaloid production if compared with wild-type cells. Indole alkaloids could be produced in hairy root cultures of Weigelia by overexpression of tryptophan decarboxylase and strictosidine synthase. Alkaloids could be produced in transgenic yeast overexpressing strictosidine synthase and strictosidine glucosidase growing on medium made out the juice of Symphoricarpus albus berries to which tryptamine is added. Metabolic engineering thus seems a promising approach to improve the production of a cell factory.
Recent achievements have been made in the metabolic engineering of plant secondary metabolism. Various pathways have been altered using genes encoding biosynthetic enzymes or genes encoding ...regulatory proteins. In addition, antisense genes have been used to block competitive pathways, thereby increasing the flux towards the desired secondary metabolites.
Unravelling plant secondary metabolism is the way to successful applications in molecular farming, health food, functional food, and plant resistance. Various pathways have been altered using genes encoding biosynthetic enzymes or regulatory proteins and show enormous potential for the genetic engineering of plant secondary metabolism.
The geraniol-derived (seco)iridoid skeleton is a precursor for a large group of bioactive compounds with diverse therapeutic applications, including the widely used anticancer molecule vinblastine. ...Despite of this economic prospect, the pathway leading to iridoid biosynthesis from geraniol is still unclear. The first geraniol hydroxylation step has been reported to be catalyzed by cytochrome P450 enzymes such as CYP76B6 from Catharanthus roseus and CYP76C1 from Arabidopsis thaliana. In the present study, an extended functional analysis of CYP76 family members was carried-out to identify the most effective enzyme to be used for pathway reconstruction. This disproved CYP76C1 activity and led to the characterization of CYP76C4 from A. thaliana as a geraniol 9- or 8-hydroxylase. CYP76B6 emerged as a highly specialized multifunctional enzyme catalyzing two sequential oxidation steps leading to the formation of 8-oxogeraniol from geraniol. This dual function was confirmed in planta using a leaf-disc assay. The first step, geraniol hydroxylation, was very efficient and fast enough to outcompete geraniol conjugation in plant tissues. When the enzyme was expressed in leaf tissues, 8-oxogeraniol was converted into further oxidized and/or reduced compounds in the absence of the next enzyme of the iridoid pathway.
•Arabidopsis CYP76C4 converts geraniol into 8-hydroxy- or 9-geraniol.•C. roseus CYP76B6 catalyzes successive oxidations of geraniol into 8-oxogeraniol.•Homology modeling provides an explanation for CYP76s regioselectivities.•Enzyme activities can be tested in planta using a transfected leaf-disc assay.•CYP76B6 activity outcompetes geraniol glycosidases in plant tissues.
Plants produce alkaloids, among others, to protect themselves against microbial infection, herbivore attack or ultraviolet irradiation. For man, alkaloid metabolism is the source of many natural ...products with useful applications, including pharmaceuticals. A major mechanism regulating alkaloid production in plant cells is the control of the transcription of the biosynthetic genes. Several transcription factors involved in the regulation of alkaloid biosynthesis genes have been isolated and studied. There are indications that the abundance and activities of transcription factors themselves are regulated by external signals. The aim of this review is to give an update on the transcriptional regulation of terpenoid indole metabolism in Catharanthus roseus.
The AP2/ERF-domain transcription factor ORCA3 is a master regulator of primary and secondary metabolism in Catharanthus roseus (periwinkle). Here we demonstrate that ORCA3 specifically binds to and ...activates gene expression via a previously characterized jasmonate- and elicitor-responsive element (JERE) in the promoter of the terpenoid indole alkaloid biosynthetic gene Strictosidine synthase (Str). Functional characterization of different domains in the ORCA3 protein in yeast and plant cells revealed the presence of an N-terminal acidic activation domain and a serine-rich C-terminal domain with a negative regulatory function. Orca3 mRNA accumulation was rapidly induced by the plant stress hormone methyljasmonate with biphasic kinetics. A precursor and an intermediate of the jasmonate biosynthetic pathway also induced Orca3 gene expression, further substantiating the role for ORCA3 in jasmonate signaling. The protein synthesis inhibitor cycloheximide did not inhibit jasmonate-responsive expression of Orca3, nor of its target genes Str and Tryptophan decarboxylase (Tdc). In conclusion, ORCA3 regulates jasmonate-responsive expression of the Str gene via direct interaction with the JERE. The activating activities of ORCA proteins do not seem to depend on jasmonate-induced de novo protein synthesis, but presumably occur via modification of pre-existing ORCA protein.
Plant secondary metabolism is very important for traits such as flower color, flavor of food, and resistance against pests and diseases. Moreover, it is the source of many fine chemicals such as ...drugs, dyes, flavors, and fragrances. It is thus of interest to be able to engineer the secondary metabolite production of the plant cell factory, e.g. to produce more of a fine chemical, to produce less of a toxic compound, or even to make new compounds, Engineering of plant secondary metabolism is feasible nowadays, but it requires knowledge of the biosynthetic pathways involved. To increase secondary metabolite production different strategies can be followed, such as overcoming rate limiting steps, reducing flux through competitive pathways, reducing catabolism and overexpression of regulatory genes. For this purpose genes of plant origin can be overexpressed, but also microbial genes have been used successfully. Overexpression of plant genes in microorganisms is another approach, which might be of interest for bioconversion of readily available precursors into valuable fine chemicals. Several examples will be given to illustrate these various approaches. The constraints of metabolic engineering of the plant cell factory will also be discussed. Our limited knowledge of secondary metabolite pathways and the genes involved is one of the main bottlenecks.
The technology of large-scale plant cell culture is feasible for the industrial production of plant-derived fine chemicals. Due to low or no productivity of the desired compounds the economy is only ...in a few cases favorable. Various approaches are studied to increase yields, these encompass screening and selection of high producing cell lines, media optimization, elicitation, culturing of differentiated cells (organ cultures), immobilization. In recent years metabolic engineering has opened a new promising perspectives for improved production in a plant or plant cell culture.
The analysis of antibiotics in animal faeces is important to obtain more insight in the possible formation of bacterial resistance in the animals׳ gut, to learn about the dissemination of antibiotics ...to the environment, to monitor trends in antibiotic usage and to detect the illegal and off-label use of antibiotics. To facilitate these studies a comprehensive method for the analysis of trace levels of 44 antibiotic compounds including tetracyclines, quinolones, macrolides and sulfonamides in animal faeces by liquid chromatography in combination with tandem mass spectrometric (LC–MS/MS) detection is reported. The method is fully validated according to European regulation and showed satisfactory quantitative performance according to the stringent criteria adopted, with the exception of some of the macrolide compounds, which can be analysed with somewhat high measurement uncertainty. A large survey was carried out monitoring swine and cattle faeces and the outcomes were striking. In 55% of the swines, originating from 80% of the swine farms and in 75% of the calves, originating from 95% of the cattle farms, antibiotics were detected. Oxytetracycline, doxycycline and sulfadiazine were the most detected antibiotics, followed by tetracycline, flumequine, lincomycin and tylosin. Over 34% of the faeces samples contained two or more different antibiotics with a maximum of eight. Possible explanations for these findings are given and the effects are discussed.
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•For the first time a multi-class method for the analysis of over 20 antibiotics in faeces is presented.•The presented method is fully validated according to 2002/657/EC.•A comprehensive survey was carried out in pig and calve production.•A high number of antibiotics were detected in faeces.•In 34% of the samples more than one antibiotic was detected in animal faeces, up to a mixture of eight different compounds.
Biosynthesis of many classes of secondary metabolites in plants is induced by the stress hormone jasmonate. The gene for ORCA3, a jasmonate-responsive APETALA2 (AP2)-domain transcription factor from ...Catharanthus roseus, was isolated by transferred DNA activation tagging. Orca3 overexpression resulted in enhanced expression of several metabolite biosynthetic genes and, consequently, in increased accumulation of terpenoid indole alkaloids. Regulation of metabolite biosynthetic genes by jasmonate-responsive AP2-domain transcription factors may link plant stress responses to changes in metabolism.
Cross-talk between jasmonate (JA), ethylene (ET), and Salicylic acid (SA) signaling is thought to operate as a mechanism to fine-tune induced defenses that are activated in response to multiple ...attackers. Here, 43 Arabidopsis genotypes impaired in hormone signaling or defense-related processes were screened for their ability to express SA-mediated suppression of JA-responsive gene expression. Mutant cev1, which displays constitutive expression of JA and ET responses, appeared to be insensitive to SA-mediated suppression of the JA-responsive marker genes PDF1.2 and VSP2. Accordingly, strong activation of JA and ET responses by the necrotrophic pathogens Botrytis cinerea and Alternaria brassicicola prior to SA treatment counteracted the ability of SA to suppress the JA response. Pharmacological assays, mutant analysis, and studies with the ET-signaling inhibitor 1-methylcyclopropene revealed that ET signaling renders the JA response insensitive to subsequent suppression by SA. The APETALA2/ETHYLENE RESPONSE FACTOR transcription factor ORA59, which regulates JA/ET-responsive genes such as PDF1.2, emerged as a potential mediator in this process. Collectively, our results point to a model in which simultaneous induction of the JA and ET pathway renders the plant insensitive to future SA-mediated suppression of JA-dependent defenses, which may prioritize the JA/ET pathway over the SA pathway during multi-attacker interactions.
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