**4. Strategies to improve the production of secondary metabolites**

In the commercial exploitation of plant cell cultures for the production of high-value secondary metabolites, it is fundamental to achieve high yields and consistent productions. The production of secondary metabolites in plants is genotype-dependent; thus, the first step to initiate cell or organ cultures is the choice of the parent plant containing higher contents of the secondary product of interest for callus or organ induction, and the selection of high-producing cell/organ lines [14]. The selection is made by analyzing cell/organ growth and then by quantifying the desired product by chromatographic and spectroscopic techniques [14]. Nevertheless, even selecting a highly productive line, the production yields are not always adequate, and after long periods of cultivation they lose their production efficiency. Thus, many alternative strategies can be used to stimulate the production of secondary metabolites and obtain efficient yields including traditional and metabolic engineering strategies [3, 19].

#### **4.1. Traditional strategies**

There are several factors that can be optimized to improve the growth and metabolites productivity of the *in vitro* cultures. Among them, the following can be appointed: the culture medium composition, the medium pH, the inoculum density, the culture medium environment (e.g., temperature, light density and quality, etc.), the agitation and aeration, etc. [3, 14, 15]. The culture medium strongly affects the biomass and metabolites productivity, and thus the selection of the suitable culture medium formulation is an imperative step [3]. It must be selected according to the physiological requirements of the plant species, and there are several parameters that can be optimized, namely nutrients composition, salt strength, nitrate and phosphate levels, plant growth regulators type and concentration, carbon source, etc. For instance, carbon source plays significant roles in the signal transduction systems through regulating gene expression and developmental processes [3].

advantages of hairy roots [22]. Also, they can accumulate metabolites in the aerial parts of the plant. However, the difficulties in cultivating hairy roots in an industrial system limit

Although there are many studies reporting the production of secondary metabolites using callus cultures and differentiated tissues [3, 14, 23], in most cases, undifferentiated cells are the preferred culture system [13]. Cell suspension culture is a simple and cost-effective method that has been extensively used to overcome the problems of large-scale production. Plant cell is biosynthetically totipotent, which means that under suitable conditions, each cell has theoretically the capacity to produce compounds identical to those present in the parent plant [13]. Plant cell cultures have more immediate potential for commercial application than tissue or organ cultures [21, 24]. They are considered as a stable system for the continuous production of secondary metabolites of uniform quality and yield. Another great advantage of plant cell cultures is the possibility to synthesize novel products not usually produced by the native plant [25, 26]. This is the preferable biotechnological platform to produce highvalue secondary metabolites, as taxol [27, 28], resveratrol [29], artemisinin [30], ginsenosides

their commercial use to produce valuable plant secondary metabolites.

**4. Strategies to improve the production of secondary metabolites**

In the commercial exploitation of plant cell cultures for the production of high-value secondary metabolites, it is fundamental to achieve high yields and consistent productions. The production of secondary metabolites in plants is genotype-dependent; thus, the first step to initiate cell or organ cultures is the choice of the parent plant containing higher contents of the secondary product of interest for callus or organ induction, and the selection of high-producing cell/organ lines [14]. The selection is made by analyzing cell/organ growth and then by quantifying the desired product by chromatographic and spectroscopic techniques [14]. Nevertheless, even selecting a highly productive line, the production yields are not always adequate, and after long periods of cultivation they lose their production efficiency. Thus, many alternative strategies can be used to stimulate the production of secondary metabolites and obtain efficient yields including traditional and metabolic engineering

There are several factors that can be optimized to improve the growth and metabolites productivity of the *in vitro* cultures. Among them, the following can be appointed: the culture medium composition, the medium pH, the inoculum density, the culture medium environment (e.g., temperature, light density and quality, etc.), the agitation and aeration, etc. [3, 14, 15]. The culture medium strongly affects the biomass and metabolites productivity, and thus the selection of the suitable culture medium formulation is an imperative step [3]. It must be selected according to the physiological requirements of the plant species, and there are several parameters that can be optimized, namely nutrients composition, salt strength, nitrate

[31], and ajmalicine [32].

84 Secondary Metabolites - Sources and Applications

strategies [3, 19].

**4.1. Traditional strategies**

Secondary metabolites are produced by plant cells in response to environmental stimuli or as defensive mechanisms against invading pathogens. In this sense, the strategy available to improve the productivity of secondary metabolites, elicitation, aims to misguide the cells or tissues for a possible biotic/abiotic attack by using agents that trigger the defense response [33]. Elicitors have the ability to control an array of cellular activities at the biochemical and molecular level since they induce the upregulation of genes [33]. The elicitors can be biotic or abiotic and may comprise signaling molecules like methyl jasmonate, salicylic acid, microbial cell wall extracts (e.g., yeast extract, chitosan), inorganic salts, heavy metals, physical agents (e.g., UV radiation) among others [1, 34]. Methyl jasmonate and its related signal molecules, and salicylic acid are probably the most extensively used elicitors [5]. The combination of some elicitors with physical factors (e.g., UV light, temperature regime, and pulsed electric field) yielded good results for secondary metabolite production [35]. As reviewed by Giri and Zaheer [5], cell suspension culture is the most used culture system for elicitation treatment and secondary metabolites production. Due to its inherent characteristics of hormone autotrophy, uncontrolled growth, biosynthetic, and genetic stability distinctiveness, hairy root cultures have proved to be also a valuable culture system for elicitation experiments. In addition, there are some secondary metabolites that are synthesized only in the roots [14, 36, 37]. Multiple shoots culture is a less used culture system for elicitation treatments for the production of secondary metabolites which is particularly useful in the case of metabolites present in the leaves [5]. The elicitors can change the secondary metabolites production quantitatively and also qualitatively [5]. For extra information, consult the recent reviews on this subject [1, 5].

Nutrient and precursor feeding are also used to improve the yields of secondary metabolites production. Nutrient feeding involves the replenishment of nutrient medium, and in precursor feeding, plant cell cultures are used to convert precursors into products by utilizing preexisting enzyme systems [14]. Immobilization of plant cells is another strategy used to overcome problems of low shear resistance and cell aggregation. This procedure can be done by several methods, and the most widely used are surface immobilization or gel entrapment. In this technique, the cells are entrapped in a specific gel or a combination of gels. Examples of matrices used are calcium alginate (the most used), agarose, gelatin, carrageenan, or polyacrylamide [14]. This strategy has several advantages, such as the extension of cells' viability in the stationary stage, the simplification of downstream processing, the high-cell density within small bioreactors reducing the costs and risk of contamination, an increased product accumulation, the minimization of fluid viscosity, among others [38].

The permeabilization of plant cell membranes with chemicals, the use of electric field stress, and ultrasound techniques are strategies used to facilitate the removal of secondary metabolites from vacuoles and membrane systems of the plant cell, facilitating the secretion of products into the culture medium and thus simplifying the purification process [14, 17].

The cultivated cells have the capacity for biotransformation of supplied compounds, which are not necessarily natural intermediaries of plant metabolism, into high-value compounds. This can occur through different reactions as hydroxylation, oxidation of hydroxyl group, reduction of carbonyl group, hydrogenation of carbon-carbon double bond, glycosyl conjugation, and hydrolysis, catalyzed by plant enzymes [14]. This is probably one of the most commercially realistic approaches; however, in some cases, the costly precursors may limit the economic viability [38].

Some important milestones in the production of secondary metabolites by plant cell cultures are the production of shikonin [48] and ginseng [49], and the most successful example of the scale-up process is probably the production of taxol by Phyton Biotech Company (Germany) to supply part of the demands of Bristol-Meyers Squibb Company during the year 2002 [50]. Phyton Biotech operates the largest cGMP plant cell culture facility in the world designed for large-scale production of Taxanes in 75,000 L-size bioreactors that run up to 880,000 L per year [51]. Berberine, ginsenosides, shikonin, scopolamine, and rosmarinic acid are also examples of plant secondary metabolites presently produced at the commercial

Production of Plant Secondary Metabolites by Using Biotechnological Tools

http://dx.doi.org/10.5772/intechopen.76414

87

Several factors should be considered in scaling up the production of secondary metabolites using bioreactors, namely the optimization of culture conditions, biomass production measurement (especially with tissue and organ cultures), and so on [52, 53]. Several bioreactor designs have been tested and used for plant cell cultures. Some of them as is the case of stirred tank reactors, bubble column reactors, airlift reactors, and ebb and flood reactors are merely extension of microbial culture. For plant cells with a high shear sensitivity, Wang and Zhong [54] develop the centrifugal impeller bioreactors that are based on the principles of a centrifugal pump. Mechanically driven "wave reactors," "slug bubble reactor," and "undertow reactor" are also adequate for high shear-stress-sensitive cells [14]. On the other hand, airlift bioreactors are suitable for not highly shear-sensitive cells and

**Figure 1.** Structures of some relevant plant secondary metabolites produced on a commercial scale.

scale (**Figure 1**) [3, 17].

### **4.2. Metabolic engineering**

Metabolic engineering offers a new perspective to understand the expression of genes involved in the biosynthesis of secondary metabolites through overexpression studies allowing the alteration of biosynthetic pathways [39, 40]. This involves the study of enzymatic reactions and biosynthetic processes at gene, transcriptomic, and proteomic levels, and the manipulation of the genes encoding the critical and rate-limiting enzymes in the biosynthetic pathways [41, 42]. Theoretically, the secondary metabolites productivity of plant cell cultures can be improved through the overexpression of genes encoding regulatory enzymes involved in their biosynthetic pathways [16]. However, the overexpression of certain genes may not always improve production [16].

Metabolic engineering approach also uses the inhibition of competitive pathways to increase metabolic flux of targeted biosynthetic pathway intermediates for a higher production through a variety of approaches. Certain steps in the biosynthetic pathway could be inhibited to induce the accumulation of preceding intermediates. The understanding of phenylpropanoyl biosynthetic pathway that is involved in the biosynthesis of several plant secondary metabolites is the most successful and recent application [43, 44].

The in-depth understanding of the biosynthetic pathways is still a barrier to the practical use of this strategy to enhance production [45, 46]. For the large-scale production of important secondary metabolites to meet industry demand, more studies are needed to identify ratelimiting steps and regulation along with bottlenecks on the lack of clarity of their biosynthetic pathways.
