Introduction

Metabolism is the closely coordinated series of enzyme-mediated reactions in a living organism such as a plant (1). This complex biochemical network consists of metabolic pathways in which compounds that have direct importance for the vital functions of an organism are synthesized and utilized. This collection of processes is defined as primary metabolism and the compounds involved are primary metabolites, e.g., sugars, amino acids, fatty acids, nucleotides, and the polymers derived from them (polysaccharides, proteins, lipids, DNA, RNA, etc.). Furthermore, in all plants more specialized biochemical pathways exist, known as secondary metabolism, in which a wide range of so-called secondary metabolites are produced, e.g., alkaloids, anthocyanins, flavonoids, quinones, lignans, steroids, and terpenoids (2). Secondary metabolites play a role in the interaction of the plant with its environment, such as attraction of pollinating insects via color and scent and protection against prédation by herbivores and insects or against infection by microorganisms (2-4). These secondary metabolite pathways are often restricted to an individual species or genus and might be activated only during particular stages of growth and development or during periods of stress caused by, e.g., wounding, attack by microorganisms, or limitation of nutrients. Many intermediates are utilized in both primary and secondary metabolism. Therefore, the dividing line between primary and secondary metabolism is not always distinct. These overlapping roles of intermediates cause a close interaction between primary and secondary metabolism. Therefore, the regulatory mechanisms in plants that provide control at the cellular, genetic, protein, and molecular levels of secondary metabolism are the subject of considerable research.

The major interest in plant secondary metabolites is due to their functions for the plant, which are often connected with their impressive biological activities (5) and are the basis for commercial applications such as pharmaceuticals, insecticides, flavors, colorants, nutrition, spices, or fragrances (2,4,6). Because of various difficulties involved in growing plants in the field or collecting them in the wild (geographic location, seasonal variation, pests and diseases, risk of species extinction, political instability of the country where the plantations are, etc.), researchers are focusing on plant cell and tissue cultures as a production system as this system can be used under controlled conditions for the production of secondary metabolites of commercial interest. It is widely recognized that cultured plant cells represent a potential source of commercially valuable plant metabolites (6-15).

Some successes in production of secondary metabolites by plant cell cultures have been achieved, e.g., shikonin production by cultures of Lith-ospermum erythrorhizon Siebold & Zucc. (Boraginaceae) (16), berberine production by cultures of Coptis japonica (L.) Salisb. (Ranunculaceae) (17,18), and ginsenoside production by cultures of Panax ginseng C.A. Mey. (Araliaceae) (19). Taxol is the latest accomplishment with an increase of 10-to 20-fold in productivity compared with the average Taxus (Taxaceae) cultures (20). However, there are some major obstacles to the production of secondary metabolites by plant cell cultures: the spectrum of compounds produced is quite different from that in the plant, the yield of the product(s) for economically viable production is too low, and they suffer from heterogeneity and/or instability during long-term culture (21). As in many cases productivity is too low for commercialization, various strategies to improve productivity are being followed:

Cell line improvement: screening and selection for high-producing cell lines

Optimization of growth and production media (phytohormones, nutrients, precursors, antimetabolites)

Optimization of culture conditions (inoculum size, agitation, temperature, light, pH, shaker speed, immobilization, permeabilization, two-phase systems, two-stage systems, elicitation) Cultures of differentiated cells (in vitro shoot or root cultures, hairy roots) Metabolic engineering

Although the production of differentiated cells has resulted in improved productivity of the desired compounds (e.g., for tropane alkaloids), for large-scale industrial production the requirement for very special bio-reactors is a major economic constraint. Therefore, there is great interest in the last mentioned approach, metabolic engineering, the more so as it has the potential for application to plant cell cultures, microorganisms, and the plant. Also, it has the possibility of application to isolation of enzymes. Further applications of metabolic engineering of secondary metabolism are new compounds with biological activity, new flower or food colors, new taste or fragrance of food, improved nutritional effect of food, decreased levels of undesired compounds in food and fodder, and improved resistance against pests and diseases (6). For increasing the production of desired compounds, different strategies can be considered: increasing the carbon flux through a biosynthetic pathway by affecting, e.g., rate-limiting enzymes, feedback inhibition, and competitive pathways; increasing the number of producing cells; and decreasing catabolism. Besides the previous efforts using random mutation and selection, current genetic engineering offers the tool for a focused approach to improve secondary metabolite production along these lines (Fig. 1) (22).

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