Eric Gontier

Production of plant secondary metabolites: a historical perspective

Abstract Studies on plant secondary metabolites have been increasing over the last 50 years. These molecules are known to play a major role in the adaptation of plants to their environment, but also represent an important source of active pharmaceuticals. Plant cell culture technologies were introduced at the end of the 1960s as a possible tool for both studying and producing plant secondary metabolites. Different strategies, using in vitro systems, have been extensively studied with the objective of improving the production of secondary plant compounds. Undifferentiated cell cultures have been mainly studied, but a large interest has also been shown in hairy roots and other organ cultures. Specific processes have been designed to meet the requirements of plant cell and organ cultures in bioreactors. Despite all of these efforts of the last 30 years, plant biotechnologies have led to very few commercial successes for the production of valuable secondary compounds. Compared to other biotechnological fields such as microorganisms or mammalian cell cultures, this can be explained by a lack of basic knowledge about biosynthetic pathways, or insufficiently adapted reactor facilities. More recently, the emergence of recombinant DNA technology has opened a new field with the possibility of directly modifying the expression of genes related to biosyntheses. It is now possible to manipulate the pathways that lead to secondary plant compounds. Many research projects are now currently being carried out and should give a promising future for plant metabolic engineering.

Biosynthesis of coumarins in plants: a major pathway still to be unravelled for cytochrome P450 enzymes

Coumarins (1,2-benzopyrones) are ubiquitously found in higher plants where they originate from the phenylpropanoid pathway. They contribute essentially to the persistence of plants being involved in processes such as defense against phytopathogens, response to abiotic stresses, regulation of oxidative stress, and probably hormonal regulation. Despite their importance, major details of their biosynthesis are still largely unknown and many P450-dependent enzymatic steps have remained unresolved. Ortho-hydroxylation of hydroxycinnamic acids is a pivotal step that has received insufficient attention in the literature. This hypothetical P450 reaction is critical for the course for the biosynthesis of simple coumarin, umbelliferone and other hydroxylated coumarins in plants. Multiple P450 enzymes are also involved in furanocoumarin synthesis, a major class of phytoalexins derived from umbelliferone. Several of them have been characterized at the biochemical level but no monooxygenase gene of the furanocoumarin pathway has been identified yet. This review highlights the major steps of the coumarin pathway with emphasis on the cytochrome P450 enzymes involved. Recent progress and the outcomes of novel strategies developed to uncover coumarin-committed CYPs are discussed.

Molecular Cloning and Functional Characterization of Psoralen Synthase, the First Committed Monooxygenase of Furanocoumarin Biosynthesis

Ammi majus L. accumulates linear furanocoumarins by cytochrome P450 (CYP)-dependent conversion of 6-prenylumbelliferone via (+)-marmesin to psoralen. Relevant activities, i.e. psoralen synthase, are induced rapidly from negligible background levels upon elicitation of A. majus cultures with transient maxima at 9-10 h and were recovered in labile microsomes. Expressed sequence tags were cloned from elicited Ammi cells by a nested DD-RT-PCR strategy with CYP-specific primers, and full-size cDNAs were generated from those fragments correlated in abundance with the induction profile of furanocoumarin-specific activities. One of these cDNAs representing a transcript of maximal abundance at 4 h of elicitation was assigned CYP71AJ1. Functional expression in Escherichia coli or yeast cells initially failed but was accomplished eventually in yeast cells after swapping the N-terminal membrane anchor domain with that of CYP73A1. The recombinant enzyme was identified as psoralen synthase with narrow substrate specificity for (+)-marmesin. Psoralen synthase catalyzes a unique carbon-chain cleavage reaction concomitantly releasing acetone by syn-elimination. Related plants, i.e. Heracleum mantegazzianum, are known to produce both linear and angular furanocoumarins by analogous conversion of 8-prenylumbelliferone via (+)-columbianetin to angelicin, and it was suggested that angelicin synthase has evolved from psoralen synthase. However, (+)-columbianetin failed as substrate but competitively inhibited psoralen synthase activity. Analogy modeling and docked solutions defined the conditions for high affinity substrate binding and predicted the minimal requirements to accommodate (+)-columbianetin in the active site cavity. The studies suggested that several point mutations are necessary to pave the road toward angelicin synthase evolution.

Ruta graveolens L.: a promising species for the production of furanocoumarins

Psoralen and its derivatives (linear furanocoumarins) are used in dermatology, and are also very promising in the treatment of other diseases. These secondary metabolites occur naturally in plants. Ruta chalepensis, R. angustifolia, R. graveolens and R. montana (Rutaceae) have been studied to evaluate their potential for the production of furanocoumarins (psoralen, xanthotoxin, bergapten, isopimpinellin). The Ruta species contained from 4 to 17 mg g−1 dry weight (DW) of furanocoumarins (FCs). These concentrations were higher than those found in other families known to produce the same compounds (Moraceae, Apiacae and Fabaceae). Among the four species tested, R. graveolens exhibited high concentrations of FCs and the best biomass production. Sixteen different R. graveolens origins were cultivated in order to find the best candidate for FC production. Plants contained an average concentration of 10 mg g−1 DW of FCs, ranging from 2.8 to 17.5 mg g−1 DW, but there was no statistical difference due to origins. FCs allocation was studied within R. graveolens in different parts of the plant. Fruits had a greater FCs concentration, five–tenfold higher than roots, stems or leaves. Isopimpinellin, a minor FC in other organs, was highly concentrated in roots, where it reached 25% of the total FCs. Potential yield of FCs is the result of both biomass and secondary metabolite production. These parameters were affected by the phenological stage of plants, with greatest production from fruits, which had the highest concentrations, biomass and therefore yield of FCs. R. graveolens plants cultivated in field could possibly produce 12 kg of FCs per hectare, mainly in fruits and leaves.

Optimized culture conditions for the production of furanocoumarins by micropropagated shoots of Ruta graveolens.

Ruta graveolens in vitro cultures are a potential source of secondary metabolites (furanocoumarins) of significant medical interest. Experiments led in our laboratory showed that micropropagated shoots were richer in furanocoumarins than any other plant material. In order to optimize the molecule production by such cultivation systems, several factors related to the culture medium were studied. Effects of medium composition on biomass growth and furanocoumarin content were analysed and optimal conditions were determined for phosphate (300 mg l−1 of NaH2PO4), nitrate (2527 mg l−1 of KNO3), carbon source (10 g l−1 of sucrose) and phytohormones (2,4-dichlorophenoxyacetic acid (2,4-D) 50 μM and benzylaminopurine (BAP) 50 μM). Ruta shoot growth and furanocoumarin production were compared for optimized and standard culture conditions. Specific medium gave better results in terms of growth (tD equal to 6.9 days against 8.6 for standard conditions) but no significant differences appeared concerning metabolite concentrations. However, the present study opens the way to scale-up studies with bioreactor cultivation systems.

Hairy root and tissue cultures of Leucojum aestivum L.—relationships to galanthamine content

Galanthamine, an isoquinoline alkaloid acetylcholinesterase inhibitor, is an important agent used all around the world for the symptomatic treatment of senile dementia of the Alzheimer’s type. The production of this metabolite and the availability of the plant are limited and prompted the search for an alternative way to obtain this valuable metabolite using in vitro cultures of Leucojum aestivum L. It is known that cell differentiation level shows a major influence upon the accumulation of alkaloids. For this reason, tissue cultures of L. aestivum showing different stages of morphogenesis controlled by exogenous growth regulators were established. Agrobacterium rhizogenes strain LBA 9402 has been tested for its capacity to induce hairy roots of this monocotyledonae plant.

Development and validation of an efficient low cost bioreactor for furanocoumarin production with Ruta graveolens shoot cultures

Despite efforts made to produce plant secondary metabolites from cell suspensions, only a few industrial applications have been successful. Generally, higher yields are obtained when cultivating organs (roots or leafy stems) instead of undifferentiated cells. In this case, new problems arise because of the structure of the plant material, and special bioreactors have to be built for such applications. Furthermore, the high cost of commercial bioreactors may limit the number available for the researcher to carry out many experiments in parallel. Because of this, we developed a very low cost system (i.e; bioreactors) that allows good growth of Ruta graveolens L. shoots and production of secondary metabolites (i.e. furanocoumarins). The development of a very simple auto-priming siphon allows the use of common jars ranging from 3 to 20 litres for temporary immersion cultures. The very low cost of such a home-made bioreactor allows scientists to run many different experiments at the same time. It thus saves time in optimising the culture medium parameters and in replicating trials before reaching the step of final culture system development with highly equipped (costly) bioreactors.

Tropane alkaloid profiling of hydroponic Datura innoxia Mill. Plants inoculated with Agrobacterium rhizogenes.

INTRODUCTIONnHydroponics has been shown as a possible way to produce high quality plant biomass with improved phytochemical levels. Nevertheless, effects of plant biotic and abiotic environment can lead to drastic changes and plant growth conditions must be optimised.nnnOBJECTIVEnTo evaluate how much microbes and Agrobacterium rhizogenes TR7 wild strain may affect the tropane alkaloid profile in Datura innoxia Mill. plants cultivated in hydroponic conditions.nnnMETHODOLOGYnDatura innoxia Mill. plants were cultivated in hydroponic with sterile or non-sterile conditions. For half of the non-sterile plants, Agrobacterium rhizogenes TR7 strain was added to the nutrient solution for hydroponics. The tropane alkaloid content of leaves and roots was analysed by UFLC/ESI-HRMS and MS/MS. The metabolite profiles were compared using partial least square-discriminant analysis.nnnRESULTSnIn sterile conditions, aerial parts contained more scopolamine than the roots. However, the diversity of tropane alkaloids was greater in roots. Furthermore, 21 known compounds and four non-elucidated tropane alkaloids were found. The tropane alkaloid profile was shown to be statistically different between sterile and non-sterile hydroponic conditions. The levels of 3-acetoxy-6-hydroxytropane and 3-hydroxylittorine were higher in plants inoculated with A. rhizogenes. Five other tropane compounds were found in higher amounts in non-axenic control plants. Hyoscyamine and scopolamine total contents were much higher in the whole plant co-cultivated with A. rhizogenes TR7 than in controls. Furthermore, the leaves and roots of axenic plants contained more alkaloids than non-sterile ones.nnnCONCLUSIONnIn hydroponic conditions, microbes induced variations of the phytochemical levels. Addition of A. rhizogenes TR7 into the nutrient solutions improved the total hyoscyamine and scopolamine production.

Agrobacterium-mediated transformation of Ruta graveolens L.

Agrobacterium tumefaciens is used to develop a genetic transformation method for a medicinal plant Ruta graveolens. The direct plant regeneration strategy is preferred to callus line establishment. In vitro seedlings, 2- -to 3-wk-old, are used to excise hypocotyls and co-cultivated for 3 d with A. tumefaciens strain C58C1Rif containing plasmid pTDE4 harbouring neomycin phosphotransferase (npt II, kanamycin resistance) and beta-glucuronidase encoding genes. The Southern blot analysis has shown that 78% kanamycin resistant plants contain gene encoding beta-glucuronidase. The GUS histochemical assay shows that 67% transgenic plants exhibit the corresponding enzymatic activity. Routine transformation efficiency of R. graveolens L. is 11% and could reach up to 22%. Transgenic plants are grown in the greenhouse within 4 months after the initial seedlings.

Datura innoxia plants hydroponically-inoculated with Agrobacterium rhizogenes display an enhanced growth and alkaloid metabolism

BACKGROUNDnThe production of secondary metabolites through the culture of entire plants is of great interest. Soilless culture, such as hydroponics, enables the control of plant growth and metabolism. Specific environmental conditions must be developed to maximize the productivity of medicinal plants used as efficient natural bioreactors.nnnMETHODSnThe nutrient solution of newly established hydroponic cultures ofDatura innoxia Mill. were inoculated with Agrobacterium rhizogenes (A.r.) wild strains (TR7, TR107, 11325 or 15834). Growth and the alkaloid contents of roots and aerial parts were analyzed. Axenic cultures were also performed with modified TR7 strains containing the egfp or gus reporter gene. In vitro isolated root cultures enabled the phenological and molecular demonstration of gene transfer.nnnRESULTSnA.r.TR 7 led to a greater improvement in plant secondary metabolism and growth. Positive expression of the reporter genes occurred. Isolation and subculture of some of the roots of these plants showed a hairy root phenotype; molecular tests proved the transfer of bacterial genes into the roots isolated from the plants.nnnCONCLUSIONSnHyoscyamine and scopolamine productivity is enhanced after A.r. inoculation in the nutrient solution of hydroponic plants. Transformation events occur in the original roots of the plants. This leads to chimeric plants with a part of their roots harboring a hairy root phenotype. Such semi-composite plants could be used for successful specialized metabolite bioproduction in greenhouses.