Datura innoxia Mill., une plante de la famille des Solanacées synthétisant des métabolites secondaires, dont les alcaloïdes tropaniques, a été utilisée comme modèle pour cette étude portant sur les cultures en hydroponie. Nous avons déterminé et hiérarchisé les paramètres environnementaux (biotiques et abiotiques) permettant d’améliorer croissance et production de métabolites. La réponse de la plante dépend de l’approvisionnement en oxygène de la solution nutritive, de la température perçue tant niveau des parties aériennes qu’au niveau racinaire. Une augmentation de l’intensité d’éclairage n’améliore pas la teneur en alcaloïdes de la plante mais l’utilisation de lumière orange engendre un changement des paramètres de croissance et des teneurs en molécules recherchées. Nous avons également montré que la présence de microflore dans le milieu de culture provoque une dégradation des molécules d’intérêt et altère leur accumulation dans la plante. Par contre, A. rhizogenes a un effet favorable sur la croissance de la biomasse et la biosynthèse d’alcaloïdes tropaniques dans la plante. L’état transgénique des racines, issues de co-culture hydroponique de Datura innoxia avec A. rhizogenes TR7, a été estimé par une vérification précoce et confirmé par des analyses moléculaires. Nous avons pu mettre en évidence les modifications du phénotype racinaire et les mécanismes d'interaction plante-microorganisme. L’inoculation des agrobactéries peut donner lieu à des transferts naturels de gènes induisant ainsi l’obtention d’un pool racinaire chimérique regroupant des racines normales et des racines transformées. Ces travaux ont ouvert de nouvelles perspectives en vue de déterminer la capacité de production de métabolites secondaires par des plantes en culture hors sol, de manière à obtenir une biomasse riche en molécules recherchées / Datura innoxia Mill. a plant species belonging to Solanaceous family, produces hyoscyamine and scopolamine as two main tropane alkaloids. It has been studied in the present work for the development of hydroponic based secondary metabolites bioproduction. More specifically, biotic and abiotic environmental factors have been analysed in order to improve growth and alkaloid accumulation in plant tissues. Oxygen concentration in the nutrient solution is proved to be important for both biomass and compound production in temporary immersion culture conditions occur as one optimum. Temperature has also a major effect, directly and most probably through oxygen solubility and root needs. We showed that artificial light may positively complete natural one but the kind of spectrum must be chosen carefully. From this point of view, orange light from High Pressure Natrium Vapour light showed better results than classical white light. Nitrogen (NO3-15mM) and pH (5 to 6) management may lead to culture optimization. We also show that well chosen wild Agrobacterium rhizogenes strains may be added to the hydroponic nutrient solution in order to improve growth and alkaloid bioproduction. This result is due to the multi occurrence of genetic transformation events. Our results lead to a first classification for impacts of biotic and abiotic factors. It opens new window for plant milking technology industrial development

Th.--Pharm.--Paris, 1870?

Th.--Pharm.--Paris, 1870?

No description available.

Datura metel is a herbaceous plant found in almost all tropical parts of the world. It belongs to the family Solanaceae whose members, viz. Duboisia, Atropa, Hyoscyamus and Datura plants are known to produce tropane alkaloids- hyoscyamine and scopolamine which are most noted for their therapeutic use as anti-cholinergic agents. Since these alkaloids are produced in very low amounts in plants, alternative sources and methods of production for these alkaloids have been crucial in meeting the demands for these drugs. Endophytic fungi inhabiting a plant may have the potential to produce the same compounds as the host plants. The aim of the present study was to search for tropane alkaloid producing endophytic fungal isolates from Datura metel. Eighteen endophytic fungi were isolated from various tissues of Datura metel and screened for the presence of three tropane alkaloid biosynthetic genes- putrescine N-methyltransferase (PMT), tropinone reductase I (TRI) and hyoscyamine 6β-hydroxylase (H6H) using PCR-based screening approach. Six endophytic fungal isolates were found to possess the PMT, TR1 and H6H genes. The fungi were identified using molecular taxonomy as Col letotrichum boninense, Phomopsis sp., Fusarium solani, Col letotrichum incarnatum, Col letotrichum siamense and Col letotrichum gloeosporioides and the identity was confirmed using colony and spore morphology. The production of tropane alkaloids hyoscyamine and scopolamine by the fungi has been ascertained using various techniques like TLC, HPLC and ESI-MS/MS by comparison with the authentic reference standards. The amount of tropane alkaloids produced by all six fungi in liquid cultures was quantified using HPLC analysis. Among the six tropane alkaloid-producing fungi Col letotrichum incarnatum gave the highest yields of hyoscyamine and scopolamine which were 3.906 mg/L and 4.13 mg/L, respectively. With an aim to characterize the tropane alkaloid biosynthetic genes in these fungi, the PMT gene was isolated from five of the endophytic fungi- Col letotrichum boni-nense, Fusarium solani, Col letotrichum incarnatum, Col letotrichum siamense and Col-letotrichum gloeosporioides for the first time and the sequence analysis showed high ho-mology (98%) to the Datura metel PMT cDNA. The gene was found to be devoid of introns in the fungi. Further phylogenetic analysis of the full length PMT sequence from the fungi strongly supports the hypothesis of horizontal gene transfer between the host plant and endophytic fungi. For further in detail characterization of fungal PMT, the Col letotrichum boninense PMT gene was taken as a representative. CbPMT gene was cloned in pRSET A expres-sion vector and heterologously expressed in E. coli and biochemically characterized. For optimal yield of soluble protein upon heterologous expression different conditions such as IPTG concentration, temperature and time post induction were optimized. Optimal yield was obtained by inducing the culture by 0.25 mM IPTG once it had reached and O.D. of 0.6 and incubating at 37◦ C for 3 h. The recombinant CbPMT enzyme expressed as histidine tagged fusion protein was purified using Ni-NTA affinity chromatography. Gel elution studies were carried out to determine molecular weight of the protein and it was found that the protein exists as a homodimer in solution with some amount also present as a monomer. Catalytic activity of the purified recombinant enzyme was studied for its dependence on both substrates putrescine as well as S-adenosylmethionine (SAM). The Km and Vmax values for putrescine were found to be 464 µM and 18.55 nkat/mg, respectively, while those for S-adenosylmethionine were found to be 628 µM and 18.63 nkat/mg, respectively. Optimum temperature for activity was found to be 37◦ C and optimum pH range was found to be 8-9. Fluorescence spectroscopy was used to study the binding affinity of both the sub-strates to the enzyme. Fluorescence quenching data for each substrate was analysed by using a nonlinear regression curve fit and Kd values were found to be 0.309 mM for pu-trescine and 0.118 mM for SAM, respectively. Circular dichroism spectrum of the enzyme indicated a pattern typical for alpha helix in the secondary structure. Binding of either substrate led to increase in ellipticity of the protein. Fluorescence quenching studies with collisional quenchers- acrylamide, potassium iodide, and cesium chloride indicated that the native protein is folded in a conformation that allows tryptophan residues to be acces-sible for quenching. The fraction of tryptophan residues (fa ) accessible for quenching by acrylamide (1.06) was found to be higher than that for potassium iodide (0.54) while that cesium ions was the least (0.38). The neutral quencher acrylamide could access all the tryptophans meaning that none of tryptophans are completely buried inside hydrophobic cores. the differential accessibility to the charged quenchers, however, indicates that more of the tryptophans are surrounded by positively charged amino acids. The unfolding of the protein was studied with the aid of chaotropic agents guanidine-HCl and urea and thermodynamic parameters were determined. The denaturant m-values were found to be 2.313 kcal/mol/M for Gdn-HCl and 2.345 kcal/mol/M for urea respectively. The free energy of unfolding was estimated to be 2.635 kcal/mol for Gdn-HCl and 4.630 kcal/mol for urea. Since no reports are available about the thermodynamics of folding and unfolding of PMT from any plant source, this study contributes towards the understanding of protein stability. Although a lot of reports are available on the biochemical characterization of PMT from different plant sources, the crystal structure of PMT is not yet available. In the current work, homology based modelling studies on CbPMT were carried out to get some idea about the protein tertiary structure. Homology based modelling studies showed that a significant amount of protein is present as α-helices which are present on the surface while the β-sheets are present in the interior of the protein. Each monomer of the protein is capable of binding both the substrates and hence the dimerization property of the enzyme could be a purely structural one leading to more stability and solubility of the protein. In conclusion, this study has shown for the first time that endophytic fungi have significant potential to be used for tropane alkaloid production and six such fungal strains have been identified. Although the production of tropane alkaloids by endophytic fungi is not very high, it can be scaled up by over-expressing the biosynthetic gene putrescine N-methyltransferase in the highest producer- Col letotrichum incarnatum to further increase the yield. These endophytic fungi have significant potential to be applied in fermentation technology to meet the demands for these drugs economically.