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In the pipeline script accessible from Github, see Code Availability below , the weights of the backbone versus tailoring enzymes can be changed. In addition, inconsistent production along with increased demand causes these drugs to often show up on the FDA drug shortage list. In Plant Secondary Metabolism Engineering: Methods and Applications , expert researchers provide detailed practical information on some of the most important methods employed in the engineering of plant secondary metabolism pathways and in the acquisition of essential knowledge in performing this activity, including the significant advances and emerging strategies. Hence, we can determine that the diversity of secondary metabolites inside section Nigri is similar to the diversity seen across the genus as a whole. This illustrates that we can detect homologous gene clusters over the genus. About this book Plants have evolved an amazing array of metabolic pathways leading to molecules capable of responding promptly and effectively to stress situations imposed by biotic and abiotic factors, some of which supply the ever-growing needs of humankind for natural chemicals, such as pharmaceuticals, nutraceuticals, agrochemicals, food and chemical additives, biofuels, and biomass. PLoS One.
In general, hairy roots are initiated from axenic plants by infection with A. Once initiated, they are maintained by transfer to selective media without the addition of plant growth regulators. The hairy root cultures are subcultured in the same medium approximately every 20 days. When the process is performed in liquid medium it is carried on under agitation in an orbital shaker rpm with 16 h- photoperiod using cool white fluorescent lamps. As we have said before, the synthesis of tropane alkaloids is produced in the pericycle of roots Hashimoto et al.
It is not surprising then that the establishment of hairy roots was considered as an alternative strategy for scopolamine production Oksman-Caldentey et al. On the other hand, undifferentiated cultures calli do not produce scopolamine proving the need of some degree of tissue specialization Moyano et al. Evidently, the tropane alkaloid synthesis requires root organization when scopolamine is the end product. Even though hairy roots are scopolamine producers the yields achieved are low, which has fostered the quest of an alternative production strategy.
The effect of the addition of GA 7 on kinetics of growth and alkaloid accumulation in two different Brugmansia candida hairy root clones have demonstrated that GA 7 10 -4 to 10 -1 mg. The term elicitor was first introduced to describe the action of biomolecules able to induce phytoalexin production. In general, elicitors induce defence systems and increase the resistance to pathogens in plants. Also, pathogens biomolecules derived from the pathogen cell wall exogenous elicitors , and compounds released from plants by the action of the pathogen, triggers that defence response Angelova et al.
Hence, it was evident that biotic elicitors produced by pathogenic microorganisms or released from their cell walls by plant enzymes were able to induce changes in secondary metabolites patterns.
Also, the release of certain compounds, such as cellulose or pectinase and molecules active in the signal transduction pathway salicylic acid, jasmonic acid , could also promote the plant defence response Benhamou, ; Guo et al. That response includes the production of phytoalexins pathogeneses related proteins , protease inhibitors and a variety of other defence compounds among them alkaloids. In vitro plant cell cultures have been used for studying the influence of elicitors on secondary metabolism Zabetakis et al. Non-conventional elicitors e.
Thus, elicitors represent a promising and inexpensive alternative to increase production of hyoscyamine and scopolamine by in vitro culture. Molecular biology, as a tool for DNA manipulation, could be used for engineering metabolic pathways. Figure 3 shows the usual strategies employed for overproducing a specific metabolite C. The strategies metabolic engineering uses to increase secondary metabolite production are a the overexpression of secondary metabolite precursors A ; b the overexpression of genes whose products are rate-limiting B , c the creation of new branches in the biosynthetic pathway E to C , d the blocking of reactions C to D redirecting them to the C pathway, among others.
Each of the strategies above mentioned has unpredictable consequences in the cell homeostasis. Overexpression of a key enzyme in the biosynthetic pathway is one of the strategies used to increase secondary metabolite production. In general, the overexpressed enzyme catalyzes a bottleneck step. In the tropane alkaloid pathway the key enzyme is H6H. As was mentioned above, H6H catalyzes two reactions, the hydroxylation at position 6, rendering an intermediate 6-hydroxyhyoscyamine; and the epoxidation that leads to the end product scopolamine.
Therefore, overexpression of H6H is an attractive strategy for poor scopolamine producing species with hyoscyamin-rich accumulation. In vitro shoot and callus cultures shown very low concentration of H6H and, hence, scopolamine production. On the contrary, hairy root cultures from scopolamine-producing species have high concentration of H6H. The difference in the expression of this enzyme could be attributed to the specialization of root pericycle for producing H6H. Several attempts were made in order to isolate and overexpress the H6H cDNA in hairy roots for scopolamine production.
The h6h cDNA was isolated from B. A strategy used is to introduce in low-producing scopolamine species numerous copies of their own H6H gene. All the protocols have several common steps. First, the h6h cDNA is cloned into the binary vector and amplified in Escherichia coli. Then, A. However, the results were not successful because the H6H overexpressing clones obtained did not have a significant increase on alkaloid production. Those results could be attributed to an upstream regulation of alkaloid biosynthesis, to a rate limiting speed of H6H or to a deficiency of precursors.
Another strategy focus on the lack of substrates in the tropane alkaloid pathway. Thus, the carbon flux is redirect from the primary to the secondary metabolism, being PMT the key and pivot enzyme between both metabolisms. There is a strong expression of the pmt gene in the pericycle of Atropa belladona roots that is suppressed by the addition of exogenous auxins Suzuki et al.
There are several protocols describing the establishment of PMT overexpressing hairy roots.
Nevertheless the results obtained are as disappointing as those obtained overexpressing H6H. Tracer-feeding studies with radioactive aminoacids demonstrated that putrescine is the precursor of tropane alkaloids. Sato et al.
Plant Secondary Metabolism Engineering. Methods and Applications. Editors: Fett-Neto, Arthur Germano (Ed.) Free Preview. -Provides an easily accessible. Plant Secondary Metabolism Engineering: Methods and Applications (Methods in Molecular Biology): Medicine & Health Science Books.
However, the alkaloid profile remained unchangeable. On the other hand, the overexpression of PMT from Nicotiana tabacum in Duboisia hybrid hairy roots yielding scopolamine produced an increase of pmt gene expression that is not reflected in the alkaloid production Moyano et al. Also, there are reports of heterologous tests with non-tropane alkaloid producing species such as Solanum tuberosum Stenzel et al.
The overexpression of PMT in N. However, when the pmt gene was overexpressed in the tropane alkaloid producer A. The overexpression of only one enzyme in a complex metabolic net could not be sufficient to increase some secondary metabolite expression. Particularly, there are several works about the overexpression of more than one of the enzymes involved in the alkaloid tropane pathway Zhang et al.
However, not significant scopolamine yields were attained. Then the topology properties of this network were computed by functions in igraph package in R, and network motif analysis was carried out by FANMOD. Frequent regulatory patterns were defined as the regulatory interaction pairs significantly simultaneously occurred.
The significance of co-expression relationship between signaling pathways and TFs were tested by fisher. Finally, the signaling pathways with adjusted P-value less than 0. Plant Sci. Hartmann T: From waste products to ecochemicals: fifty years research of plant secondary metabolism. Biotechnol Adv. Broun P: Transcriptional control of flavonoid biosynthesis: a complex network of conserved regulators involved in multiple aspects of differentiation in Arabidopsis.
Curr Opin Plant Biol. Plant Physiol. Trends Plant Sci.
Funct Plant Biol. Plant J. Plant Cell. Mol Plant Pathol. Vogt T: Phenylpropanoid biosynthesis. Mol Plant. Koes R, Verweij W, Quattrocchio F: Flavonoids: a colorful model for the regulation and evolution of biochemical pathways.