**5. Scale-up production**

Owing to the importance of some plant secondary metabolites, efforts have been made to study the feasibility of their production at the industrial scale. This is not always a simple process because plant cells have a relatively unstable productivity, a high shear sensitivity, a slow growth rate, and low oxygen requirements [14]. The scale-up involves the use of bioreactors of varying sizes and features, and cell suspension culture is the better culture system having several advantages in comparison with the other. The simplicity, predictability, and high efficiency at which the metabolites can be isolated from biomass or cultivation media are some of these advantages. Nevertheless, there are some examples of the use of differentiated tissues like shoots and somatic embryos [47].

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

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

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

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 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

Owing to the importance of some plant secondary metabolites, efforts have been made to study the feasibility of their production at the industrial scale. This is not always a simple process because plant cells have a relatively unstable productivity, a high shear sensitivity, a slow growth rate, and low oxygen requirements [14]. The scale-up involves the use of bioreactors of varying sizes and features, and cell suspension culture is the better culture system having several advantages in comparison with the other. The simplicity, predictability, and high efficiency at which the metabolites can be isolated from biomass or cultivation media are some of these advantages. Nevertheless, there are some examples of the use of differentiated

the economic viability [38].

86 Secondary Metabolites - Sources and Applications

**4.2. Metabolic engineering**

always improve production [16].

pathways.

**5. Scale-up production**

the most successful and recent application [43, 44].

tissues like shoots and somatic embryos [47].

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.

for hairy and adventitious root cultures [14]. The interested reader can find more important details about the scale-up process in the works by Murphy et al. [14], Yue et al. [13], and Isah et al. [3].
