Techniques for secondary metabolite production

Many drugs, beauty, and food products sold today consist of naturally produced compounds called secondary metabolites (SMs). They are mainly produced by plants and have a wide range of functions that makes them interesting for different industries.  Due to this, their commercial importance has increased, resulting in a growing demand for them.

Initially, chemical synthesis was assumed to be the primary method of producing them. However, SMs are complex in structure and therefore difficult and costly to synthesize chemically. If you are new to this topic, it might be interesting for you to learn more about secondary metabolites. You can read about it in our article "Secondary metabolites – An overview".

Now, how to produce SMs to meet industrial demand? Since 1960, plant cell, tissue, and organ cultures have provided an alternative source of SMs production. Naturally, plants synthesize SMs for free, but in little quantities. As a result, many approaches for enhancing and promoting SMs production via plant metabolism have been devised.

Let us now discuss the available techniques to produce plant secondary metabolites:

Conventional methods

Plant secondary metabolites production in natural systems depend on environmental conditions. This is because SMs are produced as a response to different types of stress that a plant experiences. Due to this, field cultivation of plants to produce SMs faces several difficulties associated with seasonal and environmental variations that result in unstable production.

With this in mind, the first attempts to promote SMs production under in vitro systems were directed to the optimization of:

• Media composition (carbon source, nutrients, and growth regulators); and
• Culture conditions (light, temperature, pH, agitation, and aeration).

Based on these optimizations, there are two tissue culture methods used for producing SMs. These are:

• Cell suspensions: This is a method of growing cell culture in a liquid medium. This method also serves as a tool for the selection of specific plant cell lines that can produce similar or even greater quantity of compounds than a field-grown plant.  More about cell suspensions here.
• Somatic embryogenesis: This tissue culture method has been used extensively for SM production as somatic embryos accumulate large amounts of secondary metabolites which leads to higher yield. More about this topic here.

Non-classical methods

Secondary metabolites in plants are structurally and functionally diverse. This diversity is due to the multiple mechanisms and pathways that each plant species possesses. As a result, having a fundamental understanding of the components involved in the development of SMs has proven critical. Why? Since the economic value of secondary metabolites has expanded, new ways to encourage the synthesis of specific metabolites are essential. Subsequently, using what we now know about plant secondary metabolism, novel and sophisticated technologies have been developed. These are:

Elicitation

It is the process of in vitro exposing plants to trace levels of elicitors in the growth media in order to promote metabolite production.

An elicitor is a substance that stimulates plant defense and consequently boosts secondary metabolite biosynthesis to protect the cell and the plant as a whole. These compounds can be abiotic (jasmonic acid, heavy metals, UV light, etc.) or biotic (chitosan, yeast extract, etc.).

Ultimately, we "trick" the plant with stress signals to activate its defensive system. In fact, elicitation is one of the most effective ways for altering the quantitative and qualitative production of SMs.

Precursor feeding

Plants employ initiator or intermediary chemicals, known as precursors, as the foundation for the majority of secondary metabolites. So, the underlying concept of this technique is that any of these molecules, when added at the start of biosynthesis, can boost the creation of the final product.

Precursor feeding is the most cost-efficient technique for stimulating SM production and is effective in a wide range of plant species. Shikimic acid, jasmonic acid, and amino acids like phenylalanine and tryptophan are all frequent precursors. Depending on the physicochemical property of the SM to be generated, they can be introduced during medium preparation or at intervals of growth periods.

Biotransformation

It is defined as the tissue culture's capacity to convert compounds provided in the medium into a distinct compound with new properties. The substrates are transformed to a different one in this process by the activity of plant enzymes, which may catalyze a range of reactions. Many plant species undergo biotransformation, which is a possible technique for producing new active components or modifying natural and manmade compounds. Scopolamine, for example, is produced in tobacco by the biotransformation of hyoscyamine (a substrate)!

Plant cell immobilization

This method involves immobilizing plant cells in a suitable medium, such as calcium alginate or agarose. Plant cell immobilization increases SMs production due to cellular cross-talk. This technique offers various advantages, including extended cell viability in the stationary stage, enhanced product accumulation, and the capacity to conveniently collect end products by absorption on a suitable resin.

Are plant tissue culture techniques enough to achieve market demand?

Currently, plant tissue culture techniques are not sufficient. Therefore, they are supplemented by genetic and metabolic engineering methods to increase SMs production output. As a result, we now have two crucial techniques: hairy root and shoot cultures. A biological vector is utilized in both cases to deliver a gene of interest into the plant genome. We may use it to not only stimulate the production of SMs that the plant does not naturally synthesize, but also to overexpress enzymes involved in biosynthesis and to block competing processes.

Why produce secondary metabolites using PTC techniques?

When creating secondary metabolites in the laboratory, you can use a variety of methodologies and procedures. Although each methodology has its own set of pros and cons, manufacturing SMs in vitro provides significant advantages over traditional methods. Some of them include:

• Production is unaffected by weather/seasons or geographical location.
• PTC provides for simpler access to the cellular machinery of plant cells and tissue, facilitating the production of recombinant compounds of commercial interest.
• The different techniques are sustainable, cost-effective, and encourage the production of SMs in very little time.
• It enhances the likelihood of producing unique synthetic substances that are not found in nature.
• Compounds extracted from tissue cultures are easily purified due to simple extraction techniques and the absence of significant quantities of pigments, which likely reduces production and processing costs.

Currently, all approaches are being scaled up utilizing bioreactors capable of producing large amounts of metabolites required by various sectors.

We hope this post has given you a better understanding of the many ways accessible to you to begin obtaining secondary metabolites!

For more interesting articles on different aspects of plant science, keep checking this space.

By Valeria Franco Franklin | 23/05/2022

References

• Bhatia, S., & Bera, T. (2015). Classical and Nonclassical Techniques for Secondary Metabolite Production in Plant Cell Culture. Modern Applications of Plant Biotechnology in Pharmaceutical Sciences, 231–291. doi:10.1016/b978-0-12-802221-4.00007-8
• Fazili, M.A., Bashir, I., Ahmad, M. et al. (2022). In vitro strategies for the enhancement of secondary metabolite production in plants: a review. Bull Natl Res Cent, 46: 35. https://doi.org/10.1186/s42269-022-00717-z
• Gonçalves, S., & Romano, A. (2018). Production of Plant Secondary Metabolites by Using Biotechnological Tools. In R. Vijayakumar, & S. S. Raja (Eds.), Secondary Metabolites - Sources and Applications. IntechOpen. https://doi.org/10.5772/intechopen.76414
• Hussain M, Fareed S, Ansari S, Rahman M, Ahmad IZ, Mohd. Saeed. (2012). Current approaches toward production of secondary plant metabolites. J Pharm Bioall Sci, 4:10-20. doi: 10.4103/0975-7406.92725
• Wawrosch, C., Zotchev, S.B. (2021). Production of bioactive plant secondary metabolites through in vitro technologies—status and outlook. Appl Microbiol Biotechnol, 105:6649–6668. https://doi.org/10.1007/s00253-021-11539-w