Plant cell cultures: Chemical factories of secondary metabolites

The production of plant secondary metabolites by means of large-scale culture of plant cells in bioreactors is technically feasible. The economy of such a production is the major bottleneck. For some costly products it is feasible, but unfortunately some of the most interesting products are only in very small amounts or not all produced in plant cell cultures. Screening, selection and medium optimization may lead to 20- to 30-fold increase in case one has producing cultures. In case of phytoalexins, elicitation will lead to high production. But for many of the compounds of interest the production is not inducible by elicitors. The culture of differentiated cells, such as (hairy) root or shoot cultures, is an alternative, but is hampered by problems in scaling up of such cultures. Metabolic engineering offers new perspectives for improving the production of compounds of interest. This approach can be used to improve production in the cell culture, in the plant itself or even production in other plant species or organisms. Studies on the production of terpenoid indole alkaloids have shown that the overexpression of single genes of the pathway may lead for some enzymes to an increased production of the direct product, but not necessarily to an increased alkaloid production. On the other hand feeding of such transgenic cultures with early precursors showed an enormous capacity for producing alkaloids, which is not utilized without feeding precursors. Overexpression of regulatory genes results in the upregulation of a series of enzymes in the alkaloid pathway, but not to an improved flux through the pathway, but feeding loganin does result in increased alkaloid production if compared with wild-type cells. Indole alkaloids could be produced in hairy root cultures of Weigelia by overexpression of tryptophan decarboxylase and strictosidine synthase. Alkaloids could be produced in transgenic yeast overexpressing strictosidine synthase and strictosidine glucosidase growing on medium made out the juice of Symphoricarpus albus berries to which tryptamine is added. Metabolic engineering thus seems a promising approach to improve the production of a cell factory.

Biotechnology for the production of plant secondary metabolites

ion and allylic carbon radical formation with subsequent triplet ground-state oxygen capture result ultimately in the formation of lipid hydroperoxides. The explicit mechanism depicted in Scheme 1 is supported by the work of Berman and Adams and others, and it was proposed that the damage caused to the parasite’s FV membrane leads to vacuolar rupture and parasite autodigestion.41 This mechanism is consistent with the observed oxygen dependence of antimalarial action of artemisinin and the morphological changes seen following artemisinin administration to parasites in vitro.44 The biological significance of hydroperoxides in relation to biological hydroxylation and autoxidation of, for example, lipids and membrane bilayers is well established. The generation of unsaturated lipid hydroperoxides provides a means of initiation of such processes. In contrast to these proposals, other workers in the field have suggested that membrane-bound heme may have a role to play in reducing the effectiveness of endoperoxides such as dihydroartemisinin. Further work is required to clarify the role of vacuolar membrane bound heme in the mechanism of action of endoperoxide antimalarials.45 An alternative nonlipid “artemisinin derived source of hydroperoxide” is discussed below. Although Scheme 1 would appear chemically plausible, several workers have proposed that the parasite death in the presence of artemisinin is probably not due to nonspecific or random cell damage caused by freely diffusing oxygen radical species but might involve specific radicals and targets, some of which are described later in this review. The following section details the specific “transitory” species that may be responsible for the antimalarial mechanism of action of artemisinin. Targets of these species are discussed and will conclude with the most recent studies by Eckstein-Ludwig who suggest that artemisinin derivatives target the PfATP6, the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) of the parasite.46 Transitory Species Mediating the Antimalarial Activity of Artemisinin: Carbon Radicals, Carbocations, Hydroperoxides, and High-Valent IronOxo Species. On the basis of the seminal work of Posner and co-workers in the early 1990s, the free radical chemistry of artemisinin is now very welldefined and has been shown to involve an initial chemical decomposition induced by heme Fe(II) (reduced hemin) or other sources of ferrous iron within the malaria parasite to produce initially an oxy radical that subsequently rearranges into one or both of two distinctive carbon-centered radical species.47 Scheme 2a summarizes the main radical pathways available for artemisinin following endoperoxide-mediated bioactivation. Since artemisinin is an unsymmetrical endoperoxide, the oxygen atoms of the peroxide linkage can associate with reducing ferrous ions in two ways. Association of Fe(II) with oxygen 1 provides an oxy radical that goes on to produce a primary carbon-centered radical (5a). A surrogate marker for the intermediacy of this radical species is the ring-contracted tetrahydrofuran (RCT) product 5b. Alternatively, association with oxygen 2 provides an oxy radical species that, via a 1,5-H shift, can produce a secondary carbon-centered radical (5c). Again, like the previous route, a stable end-product, hydroxydeoxoartemisinin (HDA) (5d), functions as a surrogate marker for this secondary carbon-centered radical species. It has been proposed that final alkylation by these reactive intermediates of biomacromolecules such as Scheme 1. Proposed Chemical Mechanism for the Observed Artemisinin Mediated Lipid Peroxidation of Cell Membranesa a Carbon radicals, generated from artemisinin, abstract allylic hydrogen atoms from unsaturated lipid bilayers to set in motion the downstream generation of a variety of reactive oxygen species by a classic lipid peroxidation mechanism. Heme or hematin/thiols associated with the lipid bilayer may be the catalysts responsible for initiation of these processes. Perspective Journal of Medicinal Chemistry, 2004, Vol. 47, No. 12 2947

A medicinal chemistry perspective on artemisinin and related endoperoxides.

Plant in vitro cultures represent an attractive and cost-effective alternative to classical approaches to plant secondary metabolite (PSM) production (the "Plant Cell Factory" concept). Among other advantages, they constitute the only sustainable and eco-friendly system to obtain complex chemical structures biosynthesized by rare or endangered plant species that resist domestication. For successful results, the biotechnological production of PSM requires an optimized system, for which elicitation has proved one of the most effective strategies. In plant cell cultures, an elicitor can be defined as a compound introduced in small concentrations to a living system to promote the biosynthesis of the target metabolite. Traditionally, elicitors have been classified in two types, abiotic or biotic, according to their chemical nature and exogenous or endogenous origin, and notably include yeast extract, methyl jasmonate, salicylic acid, vanadyl sulphate and chitosan. In this review, we summarize the enhancing effects of elicitors on the production of high-added value plant compounds such as taxanes, ginsenosides, aryltetralin lignans and other types of polyphenols, focusing particularly on the use of a new generation of elicitors such as coronatine and cyclodextrins.

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Elicitation, an Effective Strategy for the Biotechnological Production of Bioactive High-Added Value Compounds in Plant Cell Factories.

Manipulation of plant natural product biosynthesis through genetic engineering is an attractive but technically challenging goal. Here, we demonstrate that different secondary metabolites can be produced in cultured maize cells by ectopic expression of the appropriate regulatory genes. Cell lines engineered to express the maize transcriptional activators C1 and R accumulate two cyanidin derivatives, which are similar to the predominant anthocyanin found in differentiated plant tissues. In contrast, cell lines that express P accumulate various 3-deoxy flavonoids. Unexpectedly, P-expressing cells in culture also accumulate phenylpropanoids and green fluorescent compounds that are targeted to different subcellular compartments. Two endogenous biosynthetic genes (c2 and a1, encoding chalcone synthase and flavanone/dihydroflavonol reductase, respectively) are independently activated by ectopic expression of either P or C1/R, and there is a dose-response relationship between the transcript level of P and the degree to which c2 or a1 is expressed. Our results support a simple model showing how the gene encoding P may act as a quantitative trait locus controlling insecticidal C-glycosyl flavone level in maize silks, and they suggest how p1 might confer a selective advantage against insect predation in maize.

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Engineering Secondary Metabolism in Maize Cells by Ectopic Expression of Transcription Factors

From aseptically grown Artemisia annua plantlets, shoot cultures were initiated. Using different concentrations of auxine, cytokinine and sucrose, a suitable culture medium was developed, with respect to the growth of the shoots and their artemisinin accumulation. Nitrate concentration and conductivity appeared to be suitable growth parameters. The artemisinin content was measured gas chromatographically. The shoot cultures were maintained in the developed standard medium, consisting of a half concentration of MS-salts with vitamins, 0.2 mg l-1 BAP, 0.05 mg l-1 NAA and 1% sucrose. The growth of the shoots and the artemisinin content remained stable for a longer period. They showed considerable photosynthetic activity and generally contained ca. 0.08% artemisinin on a dry weight basis. The highest artemisinin content found was 0.16% in the above mentioned standard medium, but also on the same medium with 0.5% sucrose. Attempts were made to further improve the artemisinin production by varying the medium composition through addition of gibberellic acid or casein hydroly-state; by omitting plant growth regulators; by precursor feeding, i.e. mevalonic acid; by influencing the biosynthesis routing through inhibition of the sterol synthesis by miconazole, naftifine or terbinafine; by changing gene expression with 5-azacytidine or colchicine; and by elicitation, using cellulase, chitosan, glutathione or nigeran. Enhanced artemisinin production was found with 10 mg l-1 gibberellic acid, 0.5 g l-1 casein hydrolysate, 10 mg l-1 or 20 mg l-1 naftifine. Relative increases of 154%, 169%, 140% and 120% were found, respectively. Other additions caused the growth to cease and the artemisinin contents to drop.

Production of the new antimalarial drug artemisinin in shoot cultures of Artemisia annua L.

This short review tries to compile some results, which show the importance and the potential of plant cell and tissue cultures for a biotechnological production of natural products. On the other hand, it can not be denied, that a real breakthrough of this technique has not yet been achieved. The problems involved and possible ways to overcome these will be discussed.

Natural product formation by plant cell biotechnology

Rosmarinic acid biosynthesis can be stimulated in cell cultures of Coleus blumei by culturing the cells in medium with 4% sucrose. In enzyme extracts of these cells two new enzymes of rosmarinic acid biosynthesis were discovered. Hydroxyphenylpyruvate reductase reduces 4-hydroxyphenylpyruvate and 3,4-dihydroxyphenylpyruvate to 4-hydroxyphenyllactate and 3,4-dihydroxyphenyllactate, respectively, using NADH. Rosmarinic acid synthase transfers the caffeoyl moiety of caffeoyl-CoA to the non-phenolic OH-group of 3,4-dihydroxyphenyllactic acid, in course of which rosmarinic acid is formed.

Two new enzymes of rosmarinic acid biosynthesis from cell cultures of Coleus blumei: Hydroxyphenylpyruvate reductase and rosmarinic acid synthase.

Cell suspension cultures of Coleus blumei producing high amounts of rosmarinic acid were used to study the biosynthetic pathway of this caffeic acid ester. One of the involved enzymes, the hydroxyphenylpyruvate reductase is characterized in this paper

Hydroxyphenylpyruvate reductase from cell suspension cultures of coleus blumei benth

The paper discusses glycosylation of trans-cinnamyl alcohol to obtain the biologically active compound rosavin and possibly other cinnamoylglycosides. Cell suspension cultures of Rhodiola rosea were established from callus of leaf origin cultured under light in a modified Murashige and Skoog medium. Under these conditions, no rosavin was formed. However, when trans-cinnamyl alcohol (2.5 mM; in MeOH) was added to the medium, after 72 h incubation cells transformed over 90% of the cinnamyl alcohol into a number of unidentified products. The structure of potential rosavin accumulated in intracellular spaces was elucidated as [3-phenyl-2-propenyl-O-(6'-O-α-L-arabinopyranosyl) -β-D-glucopyranoside] by means of chemical and spectral analysis using TLC, HPLC, UV, LSIMS and NMR methods. Rosavin yields of 0.03–1.01% dry weight were obtained. The actual amount depended on the cell strain cultured and the biotransformation period.

A. W. Alfermann

Papers

Production of the new antimalarial drug artemisinin in shoot cultures of Artemisia annua L.

Natural product formation by plant cell biotechnology

Two new enzymes of rosmarinic acid biosynthesis from cell cultures of Coleus blumei: Hydroxyphenylpyruvate reductase and rosmarinic acid synthase.

Hydroxyphenylpyruvate reductase from cell suspension cultures of coleus blumei benth

Rosavin as a product of glycosylation by Rhodiola rosea (roseroot) cell cultures