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Pathway engineering in medicinal plants
Recent advances in metabolic engineering of both native and heterologous secondary metabolite producing organisms have allowed higher levels of production, direct synthesis of desired products, and the biosynthesis of novel products (Mijts and Schmidt-Dannert, 2003). Increasing the production of active phytochemical compounds is targeted through genetic manipulation of the metabolic biosynthetic pathways of the active compounds. Metabolic engineering utilizes knowledge of cellular metabolism to alter biosynthetic pathways and has many advantages over traditional methods of strain improvement through extensive screening. Although the metabolic pathways of active compounds are mostly poorly understood, and relatively few key genes have been isolated, there are some successful reports with regards to the engineering of biosynthetic pathways leading to improved breeding of medicinal plants (Ferreira and Duke, 1997; Charlwood and Pletsch, 2002). An example is the nine-fold enhancement in the production of the sedative compound scopolamine in hairy root cultures of Hyoscyamus niger (black henbane). This has been
performed by the simultaneous overexpression of two genes encoding the rate-limiting upstream and downstream biosynthetic enzymes (Zhang et al., 2004). A threefold enhancement has also been reported in the production of artemisinin, with anti-malarial and anti-cancer activity, in transgenic Artemisia annua plants through the overexpression of farnesyl diphosphate synthase, the enzyme involved in the process in the first biosynthetic step (Chen et al., 1999; Chen et al., 2000). In another example a 78-fold increase in flavonoid levels in the tomato peel was achieved by the overexpression of the Petonia chalcone isomerase (CHI) gene, an early enzyme of the flavonoid pathway, which was found to be a key enzyme in the increase of flavonol production (Muir et al., 2001). This technique can also be used for the induction of new compounds in plants. Isoflavones in legumes, for instance act as phytoalexins, that is, the biosynthesis of these antimicrobially active compounds are induced by microbial infection. By overexpression of isoflavone synthase, a cytochrome P450 enzyme, these compounds could be produced in Arabidopsis, tobacco plants and maize, which normally lack the ability to synthesize these compounds (Jung et al., 2000; Yu et al., 2000).
Antituberculosis activity
The radiometric respiratory technique using the BACTEC system (Becton Dickinson Diagnostic Instrument, Spars, md) was used for testing susceptibility of Mycobacterium tuberculosis H37Rv (ATCC 27264) using the method described by Heifets and Good (1994). Solutions of the cell suspension culture and compound 1 were prepared by maceration of a requisite amount of the sample in a known volume of dimethyl sulphoxide (DMSO) to obtain a concentration of 5.0 mg/ml for both the crude extract and compound 1. The solutions were stored at 4 ˚C until used. Subsequent dilutions were done in DMSO and added to 4.0 ml of BACTEC 12B (7H12 medium) broth to achieve the desired final concentrations of 2.0, 1.0, and 0.5 mg/ml for the crude extract and 200, 100, 50 µg/ml for compound 1, with PANTA (Becton Diskinson & Company), an antimicrobial supplement. Control experiments showed that the final amount of DMSO (1 %) in the media had no effect on the growth of M. tuberculosis. The radiometric respiratory techniques using the BACTEC 460 system (Becton Dickinson Diagnostic Instrument, Sparks, md) was used for testing susceptibility against M. tuberculosis as previously described (Mativandlela et al., 2006). Isoniazid (INH) (Sigma-Aldrich, South Africa) at a final concentration of 0.2 µg/ml served as the drug-control. The MIC was defined as the lowest concentration of the compound that inhibited more than 99 % of the bacterial population. The bactericidal effect (minimum bactericidal concentration, MBC) of the extract was assessed by plating the bacterial suspensions from the BACTEC vials, which exhibited a MIC effect, on 7H11 agar medium at the end of the experiment. The MBC was defined as the minimal bactericidal concentration, which effectively reduced by at least 99 % the viable counts in the extract or compound containing samples as compared with those in the control vials (extract and compound free vials). The experiment was repeated three times.
Chapter 1: Introduction
1.1 Medicinal plants past, present
1.2 Medicinal plants in South Africa
1.3 The genus Helichrysum
1.4 Aim of the study
1.5 Objectives of this study
Chapter 2: Production of galangin in the cell suspension cultures of Helichrysum aureonitens
2.1 Abstract
2.2 Introduction
2.3 Material and methods..
2.4 Results
2.5 Discussion
2.6 Conclusions
Chapter 3: Isolation, identification and bioactivity of a novel chlorophenol derivative from Helichrysum aureonitens cell suspension cultures
3.1 Abstract
3.2 Introduction
3.3 Materials and methods
3.4 Results
3.5 Discussion
3.6 Conclusions
Chapter 4: Biosynthetic pathway for flavonoids in cell suspension cultures of Helichrysum aureonitens
4.1 Abstract
4.2 Introduction
4.3 Materials and methods
4.4 Results
4.5 Discussion
4.6 Conclusions
Chapter 5: Isolation and characterization of cinnamate-4-hydroxylase (C4H) in Helichrysum aureonitens
5.1 Abstract
5.2 Introduction
5.3 Materials and methods
5.4 Results
5.5 Discussion
5.6 Conclusions
Chapter 6: C4H expression in Pichia pastoris
6.1 Abstract
6.2 Introduction
6.3 Materials and methods
6.4 Results
6.5 Discussion
6.6 Conclusions
Chapter 7: General discussion and perspectives
7.1 Introduction
7.2 Cell suspension cultures of Helichrysum aureonitens
7.3 Characterization of flavonoid biosynthesis in Helichrysum aureonitens
7.4 Characterization of cinnamate 4-hydroxylase in Helichrysum aureonitens
7.5 Further work
