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.
Metabolic engineering of plant secondary metabolism
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. 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. The unsuccessful results could be attributed to the transgenic transformation processes itself. The mechanism of integration of transgenes into plant DNA is poorly understood, the integration of many genes at one or a few loci could not happen by chance.
Evidently, h6h played a more important role in stimulating scopolamine accumulation than pmt. Nevertheless, when pmt redirects the carbon flux to the tropane pathway and h6h is overexpressed there is an accumulation of scopolamine Zhang et al. The genetic transformation with numerous in-tandem genes could be troublesome. For hairy root induction and pmt and h6h cDNA insertion, at least three tandem transformations.
The second round of PCR PCR 2nd Reaction is carried out with the M13 forward and reverse primers in order to amplify a cDNA fragment encoding the sequence of interest flanked by the att left and right recombination sequences. New and simple DNA transfer methods simplify the process as, for example, the Gateway system, based on bacteriophague Lambda site-specific recombination system Karimi et al. Figure 4 shows a protocol designed in our lab for the production of a PCR fragment for h6h cDNA ready to recombine into the destination vector independently of the antibiotic gene resistance.
In this chapter, we have reviewed some of the most relevant strategies for improving tropane alkaloid biosynthesis such as the establishment of scopolamine overproducing organ cultures, the elicitation and the genetic transformation with homologous genes. Nevertheless, the knowledge generated and the strategies in use have demonstrated that the tropane alkaloid metabolism is immersed into a complex net of metabolic pathways with a delicate equilibrium quite difficult to be manipulated.
Modern system biology tools, like elicitation and overexpression, allow the carbon flux redirection with some limitations. These margins cannot be overcome and decelerate the development of a competitive and sustainable production platform. Those troubles and limitations have fostered new strategies based on functional genomics Goossens et al. Biotransformation is one of those new strategies. The production of scopolamine and other alkaloids was studied in engineered N.
Moreover, it was also evident an enhanced production of various nicotine alkaloids suggesting that the regulation of the alkaloid production is probably more complex than presently known. Another approach was the bioconversion of hyoscyamine to scopolamine using recombinant Saccharomyces cerevisiae that expresses the h6h cDNA isolated from B. Transformed S.
However, the results have shown a low ability of hyoscyamine conversion to scopolamine Cardillo et al. In the last two decades plant biotechnology has made considerable advances in the quest of a scopolamine and other tropane alkaloids productive process. Several groups have explored a wide spectrum of strategies that have led to the exhaustive knowledge of the tropane alkaloid pathway, its limiting steps and some of the regulation pathways. It is evident that genetic transformation is a promissory tool for engineering tropane alkaloid biosynthetic metabolism in order to produce high amounts of scopolamine.
Combining genetic transformation and metabolic engineering would be a powerful strategy to re-direct the metabolic flux towards that biosynthetic pathway. However, the yields obtained up to now did not reach those of the current scopolamine productive process. Future research could be done considering the higher structural diversity of tropane alkaloids that could be functional to create new metabolic pathways and biological active products.
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Our readership spans scientists, professors, researchers, librarians, and students, as well as business professionals. NL Received 6 May ; Accepted 10 May Key words: genetic modification, metabolic engineering, phytochemicals, plant cell biotechnology, plant cell culture, plant secondary metabolism Abstract The technology of large-scale plant cell culture is feasible for the industrial production of plant-derived fine chemi- cals.
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.
Introduction — New tastes or fragrances of food. First of all, — Lowered levels of unwanted e. These compounds Barberan , but also for the production of fine are not involved in the basic metabolic processes chemicals such as drugs, antioxidants, flavors, fra- of the living cells, but are involved in the interac- grances, dyes, insecticides and pheromones.
In the tion of the producing organism with its environment. Particularly, the possi- resistance against insects e. Each plant species has perspectives for the exploitation of the biosynthetic its own specific set of secondary metabolites. About capacity of plants and plant cells. Possible goals for , compounds are now known from plants, with genetic engineering could be: about new ones being discovered every year — Increased levels of fine chemicals.
Verpoorte The largest group consists of the ter- — New compounds for screening for biological activ- penoids, comprising more than one third of all known ity. The second largest group is formed by — New flower colors or food colors. Isolated enzymes can only be used for biocon- versions, i. This approach can be useful as part of a synthetic produc- tion, in which difficult steps e. Thus for the biotechnological production of com- plex natural products plant cell cultures seems the Fig.
Ajmalicine, with the present state of the art 0. Each cell in such a culture has the full set of genes necessary for all the functions of a plant, including secondary Mankind has been quite efficient to find uses and metabolism totipotency. For the application of such abuses for such compounds. They range from poi- in-vitro cultured cells for an industrial production sons for hunting or fishing, to medicinal uses. But there are two major questions to be answered.
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But many secondary metabolites also give — Is the economy of the process competitive with color to our daily life and taste to our food. Bitter existing production methods.
Metabolic Engineering of Plant Secondary Metabolism
Shear forces in stirred bioreactors are determining the quality of these products. Some often cited studies in the maceutical application is one of the most important s claimed that plant cells grow better in low-shear uses. Compounds amongst others, Meijer et al. The steroids are probably the best example of in stirred bioreactors. This is confirmed by several the latter.
In volume the production of such plant com- reports on large-scale culture of plant cells in biore- pounds for the pharmaceutical industry is small. Some actors, e. The technology is thus feasible. Some compounds are produced cell biotechnology based process have been made in amounts of several hundreds of tonnes a year e. But in terms of acreage, the production of Stepan-Sarkissian , Goldstein et al. Gulik et al. We have This makes such products rather vulnerable for fluc- calculated that at a production of 0.
This has raised the Gulik et al. The most interest in developing biotechnological processes for important factor is the high investment cost, whereas the industrial production of such fine chemicals. Depreciation costs possibilities for a biotechnological production: of the large bioreactor facilities are the major costs. The fold higher yield has been pathway of the products involved and have the genes reported for berberine Figure 2 in Coptis japonica available. There is no theoretical reason why plant cells should produce less.
Shikonin, the only commercially produced secondary plant in secondary metabolites. Examples are the production metabolite, using plant cell cultures. This shows that also plant cells are capa- ble of diverting a large part of the metabolic flux into secondary metabolism. In conclusion, production in plant cell cultures is economically feasible for certain compounds, pro- vided that cell cultures do produce them. In fact this turns out to be the major bottleneck.
Most econom- ically important natural products, are produced only at very low levels or not at all e. As a result, so far only a few products of plant cell biotechnological processes have found their way to the market. Taxol, the production of this compound in cell cultures has been optimized considerable, bringing it close to an industrially , the only pure chemical so far produced on feasible process. Other products are gin- seng roots and certain polysaccharide mixtures from cell cultures.
Biosynthesis, function and metabolic engineering of plant volatile organic compounds
Products that came close were rosmarinic Whether this costly procedure will be done is not clear acid and sanguinarine, but as these eventually did not yet. Taxol Figure 4 is the role during the development of new plant derived latest success story. One has been able to increase compounds as a drug. Plant cells may provide the productivity 10—fold if compared with the average necessary amounts during drug development, when Taxus cultures Jaziri et al.
However, for the a agri horti cultural production is not yet available.
Secondary metabolism and metabolic engineering (/)mycamesejy.gahnology-University of Verona
Here we will discuss some of the strategies used for improving productivity. Particularly metabolic en- gineering will be discussed in more detail. Strategies to improve productivity Screening and selection, medium optimization In the past years a number of different strategies have been followed in order to improve productivity of plant cell cultures. The first efforts were clas- sical approaches known from microbiology for the optimization of for example antibiotics.
These com- prise screening and selection of high-producing cell lines and the optimization of growth and production media. The problem with the first approach is that Fig. Hairy roots. Never- theless, it was shown for certain plant species that Differentiated cells extensive screening programmes can lead to stable high-producing cell lines, such as in case of berberine Because of the lack of success in producing some Figure 2 production in Coptis japonica cell cultures of the major compounds of interest by using the ap- Sato et al , Verpoorte et al.
The first and logical approach effective means to improve productivity. Particularly was the culture of differentiated cells. By definition, developing production media is a useful approach, as secondary metabolism is a form of differentiation.
In elaborating a medium that is both suited for growth plants there is a clear correlation with cellular differ- and production is much more difficult. In case of a pro- entiation and secondary metabolism. Glandular hairs, duction medium, the effect is directly observed after and flowers are the most obvious and visible examples. Indeed, such cultures often produce the ber of subcultures.
This was shown for example for same secondary metabolites as the intact plant, and Tabernaemontana divaricata cell cultures. The effect by medium optimization even higher levels have be of replacing the auxin 2,4-D with NAA in the medium obtained in certain cases. This is followed by a decrease, and interest is the transformation of plants with Agrobac- eventually stabilisation after about 10 subcultures at terium rhizogenes Doran These soil bacteria a level that was not much higher than in the original are capable of infecting plant cells and cause the pro- medium Sierra et al.
An advantage if compared with normal by combining these approaches. However, an impor- root cultures is that hairy roots can grow without tant conclusion one might draw from all the work done growth hormones in the medium. Hairy roots have in these classical approaches is that it only works if a similar production profiles as normal roots. A clear basal level of the desired compound s is present in the example of success in obtaining high productivity is cell culture. If no product is formed, e. Twenty times zero remains high levels in root and hairy root cultures Oksman- zero.
This approach was among other applied in At- ropa belladonna hairy roots in which the enzyme that converts hyoscyamine into scopolamine was overex- pressed Yun et al. Sanguinarine, the production of this compound can be in- A major disadvantage of differentiated organ cul- duced in cell cultures of among others poppy, by adding fungal tures is that they are difficult to grow on a large elictors. For example, hairy roots require specialized bioreactors, such as rolling drum fermenters or mist Elicitation fermenters for a review see Su In these biore- actors the working volume is quite large if compared Elicitors are compounds that are able to induce the de- to the active biomass.
Consequently production costs fence response against infections, and in particular the are very high. The importance of such cultures is prob- production of phytoalexins Smith We make use of the latest methods in molecular biology, metabolic engineering e. Go directly to: Content Search box Breadcrumb. Scope of the project: The goal of this project is to engineer Saccharomyces cerevisiae for the sustainable production of high value and industrially relevant plant secondary metabolites, such as pigments natural colours , terpenes and plant hormones.
Twitter Whatsapp Linkedin Email. The role of secondary metabolites in the interaction of plants and insects Metabolic engineering of pharmaceutical compounds in plants Source-sink relation in plants The mechanism and control of desiccation tolerance in seeds The molecular dissection of seed quality The mechanism and regulation of seed dormancy and germination Genetical genomics of plant stresses Subcellular transport of metabolites Investigating the role of stromules in scent monoterpene emission and hormone secretion by plants Subcellular transport of proteins The biophysical basis of seed longevity Molecular analysis of Castor bean germination and seedling establishment Functional characterization of ribosomal protein Metabolic engineering of yeast for the production of plant-derived secondary metabolites.