Elementary plant Biochemistry and Biotechnology (2+1)

In vitro Secondary metabolites production

 
• Secondary metabolites are those compounds which are not directly involved in the primary metabolic processes such as photosynthesis, respiration, protein and lipid biosynthesis etc. Secondary metabolites include a wide variety of compounds such as alkaloids, terpenoids, phenyl propanoids etc. These substances do not participate in vital metabolic functions of the host plant tissues in the same manner as amino acids, nucleic acids or other primary metabolites but appear to serve as a chemical interface between the plant and its surrounding environment. These are used to protect against pests and to attract pollinators. They may also help in combating infectious diseases.


• Higher plants are valuable sources of industrially important natural products which include flavors, fragrances, essential oils, pigments, sweeteners, feed stocks, anti microbial and pharmaceuticals (about 25 % of the prescribed medicines are solely derived from plants) (Table 1). Many of the pharmaceutical compounds have complex structures which makes their chemical synthesis economically unattractive. Cultured cells of many plant species produce novel biochemicals which have so far not been detected in whole plants. For example, cell suspension cultures of Rauwolfa serpentine have been shown to produce highly polar alkaloids which are novel glucosides of ajmaline and its derivatives.

Extraction of certain secondary metabolites from cell lines has definite advantages.

• Plant cells are relatively easy to grow.
• The rate of cell growth and biosynthesis of secondary metabolites under in vitro cultures is quite high and the product may be produced in a short period of time.
• Plant cell cultures are maintained under controlled environmental and nutritional conditions which ensure continuous yields of metabolites.
• Suspension cultures offer a very effective way of incorporating precursors into cells
• New routes of synthesis can be recovered from deviant and mutant cell lines which can lead to production of novel compounds which are not previously found in whole plants.
• Some cell cultures have the capacity for bio transformation of specific substrates to more valuable products by means of single or multiple step enzyme activity.
• Culture of cells may be more economical for those plants which take long period to achieve maturity.

Disadvantages:

1. High production cost
2. Lack of basic knowledge on the biochemical pathways.
3. Often cultured plant cells do not produce compounds of high value

Improvement of secondary metabolite production through cultured plant cells

• Biochemical yields from cultured plant cells can be improved by increasing cell biomass yield per unit volume of culture and biochemical content of the cell biomass.

1.Enhancement of Biomass yields:

• Most of the biochemicals are produced in differentiated cells or organized tissues. Therefore, these biochemicals can not be produced in rapidly growing cell cultures and the culture conditions favoring growth suppress biochemical production and vice-versa. Hence, the production strategy consists of two phases: i. Growth phase- for cell biomass accumulation ii. Production phase - for biosynthesis and accumulation of the biochemicals. Biomass accumulation can be improved by using optimum culture conditions of which nutrient medium and inoculum size are particularly important. For example, Lithospermum erythrorhizon suspension culture yielded Shikonin of only 6.8 g cell dry weight/l on White’s medium, while the yield on LS (Linsmaier and Skoog) medium was 16.8 g/l. Biomass production can be markedly increased by the use of a larger inoculum size, to give higher initial cell density, in combination with proportionately enriched nutrient medium. For example, twice the concentration of normal medium for a two-fold increased inoculum size.

2.Improving Biochemical production:
Biochemical production by cultured cells can be increased by;

• Devising a suitable culture medium and conditions:
E.g. Auxins, especially, IAA increase the Shikonin production by Lithospermum cells.

• Development of high producing cultures:
All the clones of Lithospermum seedlings did not produce Shikonin. Hence, it is necessary to identify high producing clones among the several clones produced by the single genotype.


3.Use of elicitors
The molecule which stimulates the synthesis of secondary metabolites are called as elicitors (the phenomenon is called as elicitation). Eg. Biotic elicitors : 1. Endogenous elicitors- pectins, cellulose etc., 2. Exogenous elicitors – chitin, glucans etc. (microbial origin). Abiotic elicitors such as UV light, salts of heavy metals and chemicals that disturb membrane integrity can also be used. Cell wall pectins (100 mg/l of medium), increased anthraquinone production by Morinda citrifolia to 5.6 times that of control after 14 days incubation.

4.Use of organ cultures.

• For example, the monoterpenes in Mentha spp. are synthesized and stored in epidermal oil glands of leaves. Shoot cultures of Mentha grown on growth regulator free medium accumulate monoterpenes. In some cases, the secondary metabolites may be released in the medium, where they may be degraded or their synthesis may be regulated (suppressed) by feedback inhibition. In this case, the biochemicals are removed from the medium by adding certain compounds which absorbs (Charcoal), dissolves (Miglyol) or encapsulates (β-cyclodextrin). Such a culture system is called as two phase system. The culture medium constitutes one phase, while the adsorbant compound makes the second phase.