Special Techniques used in the Production of Secondary Metabolites

This article throws light upon the three special techniques used in the production of secondary metabolites.

The three special techniques are: (1) Elicitors (2) Hairy Root Culture and (3) Bio-transformations. These special techniques not only provide a method to enhance the production of secondary metabolites but also clearly understand the mechanism under lying the production of secondary metabolites.

Technique # 1. Elicitors:

Strictly speaking elicitors are compounds of biological origin involved in plant microbe interaction. In the context of product accumulation by plant cell cultures, elicitors are mediator compounds of microbial stress (biotic elicitors) or are stress agents like UV light, alkalinity, osmotic pressure, or heavy metal ions (abiotic elicitors).

Molecules, which stimulate secondary metabolism leading to the induction of stress metabolites, are called elicitors and those derived from fungi may be referred to as fungal elicitors. The discovery of ‘elicitation response’ in 1972 by Keen and co-workers demonstrated that enhanced production of phytoalexins may also be achieved by treating plant cells with extracts prepared.

It has been demonstrated repeatedly that fungal-induced stress of normal, intact plant tissues leads to the induction and accumulation of phytoalexins. Although alkaloids have not generally fallen into the phytoalexin category, in the mid 1980s a number of investigators demonstrated that the production of certain alkaloids in plant cell cultures could be induced by treating cells with elicitors derived from a variety of pathogenic fungi.

The ability of cultures plant cells to produce certain metabolites in response to molecules isolated from fungi appears to be a general phenomenon, and must be correlated with physiological stress imposed on the cells.

Chemically defined elicitors (actinomycin-D, arachidonic acid sodium salt, chitosan, nigeran, poly-L-lysine and ribonuclease-A) or fungal preparations (whole extract or cell wall fraction) from common pathogenic fungi (Pythium, Alternaria, Helminthosporium, Fusarium, Colletotrichum, Sclerotinia etc) are used. Fungus is grown on fresh culture medium from stored stock cultures, homogenized and autoclaved at 121°C for 20 minutes and suitably diluted fungal preparations or chemicals are added in to medium to evaluate the elicitation effect. Certain examples of elicitation are given in the Table 29.5.

Technique # 2. Hairy Root Culture:

In the production of hairy root cultures, the explant material is inoculated with a suspension of Agrobacterium rhizogenes. This culture is generated by growing bacteria in yeast maltose broth (YMB) medium for two days at 25 °C with rotary shaking, pelleting by centrifugation (5 x 10³ rpm, 20 min.) and re-suspending the bacteria in YMB medium to form thick suspension (approximately 10¹⁰ viable bacteria per ml).

Transformation may be induced on aseptic plants grown from seed, or on detached leaves, leaf discs, petioles or stem segments from greenhouse plants followed by sterilization of the excised tissues. In some species a profusion of root may appear directly at the site of inoculation, but in others a callus will form initially and roots emerge subsequently from it.

In either case, hairy roots normally appear within one to four weeks. The susceptibility of species to infection is very variable. Addition of acetylsyrigone, the compound produced during the wounding response of plants, activates the Vir genes of Agrobacterium adding plasmid T-DNA transfer.

An alternative approach to transformation is to use cocultivation of plant protoplasts with A. rhizogenes. Cultures may be cleared of bacteria by several passages in media containing 200 mg/l cephalosporin and 500 mg/l ampicillin. The infection of plants with A. rhizogenes causes one or both of two pieces of T-DNA (Tt and Tg) to be inserted into the plant genome. Integration alters the auxin metabolism of transformed tissues in such a way that the hairy root phenotype is expressed and amino acid metabolism is modified in such a way that specific metabolites such as opines are produced.

Both TL-DNA and TR-DNA have rhizogenic functions but in most species the TL-DNA appears to be more important in determining the hairy root phenotype. Although synthesis in opines (manopine or agropine) is the fine indication that roots are transformed, the expression of the opine genes in hairy root tissue may be unstable over time. Thus, detection of the T-DNA by Southern hybridization is necessary to confirm that the cultured root tissue has transformed.

Hairy roots are characterized by a high degree of lateral branching, a profusion of root hairs and an absence of geotropism. Hairy root cultures do not require conditioning of the medium. A feature of hairy root systems of paramount importance for their commercial exploitation is their stable, high-level production of secondary metabolites. Although the productivity of untransformed root cultures may be similarly exploited, establishment and maintenance of such cultures is difficult and the auxin supplement needed for optimal growth can depress productivity.

The transformed hairy root cultures utilized in the production of secondary metabolites are listed in Table 29.6. It is important to note that several products are synthesized in roots (e.g., nicotine) and in such cases hairy root cultures offer a unique fast-growing organized culture system.

Technique # 3. Bio-transformations:

A bioconversion can be defined as the conversion of one chemical into another, i.e., of a precursor (or substrate) into a final product, using a cell suspension acting as biocatalyst (Fig. 29.6). The biocatalyst can be microorganisms, plant or animal cells, either growing or in a quiescent state, or an extract from such cells or a purified enzyme.

The biocatalyst may be free, in solution, immobilized or on solid support or entrapped in a matrix. In bioconversion by whole cells or extract a single enzyme or several enzymes may be involved. By means of recombinant DNA technology genes encoding relevant enzymes can introduced in the host cells.

Each individual cell contains many enzymes, which can display different catalytic properties depending on the conditions to which they are exposed. A plant cell contains 800-1000 different enzymes belonging to primary and secondary metabolism. An enzyme has two properties similar to catalysts; the ability to speed up a reaction and to remain unchanged at the end of the reaction, they ‘recycle’ in the reaction.

The principle for this process is that enzymes efficiently bind to the transition state of a precursor. Consequently, the free energy of activation for the reaction is strongly reduced and the bioconversion facilitated. The reaction is speeded up without changing the equilibrium. Once the product is formed it is released and the active site becomes available for further bio-catalysis.

Since each enzyme has its own specific site, enzymes are selective towards their precursors (substrate specificity). However, a number of enzymes with broad specificity are known, e.g., phenol oxidases, lipases, and esterase’s. Most biocatalyst is active at room temperature ensuring stability of the precursor and the product. Freely suspended cells from the simplest bio-catalytic system, as precursors can be supplied directly to the cultures. Often batch-grown cultures consisting of undifferentiated plant cells are used. The only barriers a precursor has to pass are the cell wall and cell membrane.

A wide range of potentially valuable compounds has been produced by adding precursors to various culture species. To produce one particular compound the best way is to use plant cells that can perform a one-step bioconversion. Unfortunately, often the precursor undergoes more than one bioconversion resulting in complex mixtures of (unknown) products. Nevertheless, a number of one-step bioconversions by freely suspended cells have been described. The bioconversion rates have been calculated in terms of mg product formed per liter culture per day (mg 1^_1 d^-1) this way of calculation enables comparison of bioconversion rates of freely suspended with those obtained by immobilized cells.

The bioconversion of papaverine (an opium alkaloid with spasmolytic properties) has been studied by Dorisse et. al. (1988) using cell suspensions of Glycyrrhiza glabra. After 21 days, 31.5% of this precursor was hydroxylated, mainly into papaverinol. The bioconversion rate of 3.7 mg 1^-1d ¹ was rather low.

Addition of gitoxigenin (belonging to the cardenolides with action on the insufficient heart) to a culture of Daucus carota yielded a single bioconversion product, 5(3-hydroxygitoxigenin (Table 29.7). The precursor was added at 0 h or at 72 h after inoculation of the cells. In the first case 50% of the precursor was completely hydroxylated into 5β-hydroxygitoxigenin after 51h, in the latter case this level was already reached after 24h, corresponding to a high bioconversion rate of 50 mg 1 ^-1 d ^-1.

Besides hydroxylation, glycosylation is one of the most interesting bioconversions. Glycosylation, particularly glucosylation (glucose coupling), occurs readily in plant cells, but only on a limited scale in micro-organisms and the organic chemist has problems with sugar coupling as well. Consequently many glucosyltransferases with different substrate (precursor) specificities for several hydroxyl bearing compounds have been investigated.

Examples are glucosylations by cells of Rata graveolens, Perilla frutescens, Catharanthus roseus, Lithospermum erythrorhizon, Mallotus japonicus and Eucalyptus perriniana. Glucosylations by cells of Glycyrrhiza echinata, Aconitum japonicum, Coffea arabica, Dioscoreophyllum cumminsii and Nicotiana tabacum have been performed as well.

Examples of other glycosylation reactions are the fructosylation of ergot alkaloid (ergotamine is used against migraine, ergometrine provokes uterus contraction) by cells of Claviceps purpurea (a fungus of class ascomycetes). Other examples are listed in Table 29.7.

Podophyllotoxin from Podophyllum resin (podophyllin is highly active against several cancer cell lines but its general toxicity is too high to allow clinical application. Chemical modification of the naturally occurring lignan has led to the clinically applied anticancer drugs teniposide and etoposide.

The organic synthesis of podophyllotoxin-like lignans is difficult, particularly with regard to their chiral centres and, therefore, alternative routes to lignans are needed. More than 30 lignans are known to exhibit powerful cytotoxic action.

Lignans are clearly candidates for plant cell biotechnological production. For the formation of podophyllotoxin and 5-methoxypodophyllotoxin by cultures of Podophyllum hexandrum and Linum flavum respectively, a series of bioconversions in the cells are required.