Production of Secondary Metabolite

Secondary Metabolite Production

Content

• General Introduction

• Factors affecting Secondary Metabolite Production

• Strategies to Enhance the Production

Objective

At the end of the lecture the student will able to:

• Discuss the advantages and production of secondary metabolite production

• Explain the strategies to enhance the production of secondary metabolites

• Identify the suitable media for the production of shikonin

Production of secondary metabolites

• Secondary metabolites derived from primary metabolites, do not have any role in the growth of the tissues/cells/plant

• Responsible for therapeutic activity

Advantages of Secondary Metabolite:

• Independent of environmental factors such as climate, geographical and seasonal variations, diseases, microbial contamination etc

• Ensures quality and yield of the product

• Well defined controlled system – facilitate improved product formation

• Possible to produce novel compounds of commercial value

• Cultured cells can be used to study the biosynthetic pathways

• Plant cultures are particularly useful in case of plants which are difficult or expensive to be grown in the fields.

The production time is less and labour costs are minimal

Factors affecting production of Secondary Metabolite:

Tissue origin genetic character

Culture conditions

Physiological factors

Selection and screening of high yielding strain

i. Tissue origin genetic character:

• Plant cells are genetically totipotent

• Under proper environmental conditions, any cell may be induced to produce any substance

• Sec metabolites are present only in specific part of the plant

• Eg: Tobacco alkaloids present in roots

• Such plant part should be chosen where there is highest concentration of desired product

ii. Culture Conditions

• Chemical composition of nutrient media influences the production of biomass and sec metabolite production

• Balance should be maintained b/n these two

• Excess increase in biomass reduces the yield of desired products

• Major chemicals that affect biomass are carbohydrates, nitrogen, potassium, phosphorous, trace elements, vitamins etc

• Plant growth regulators exert a remarkable influence on production Eg: Gibberellins increase berberine production, but decreases shikonin production

iii. Physiological factors

Temperature:Plays vital role in yield optimization

Eg: At 5 0C alkaloid production increases but reduced at 35 0C

Light: Secondary metabolite production in cell culture is influenced by photoperiodicity as well as quality and intensity

Eg: Cardenolide production in D. lanata is greatly influenced by light as well as intensity Blue light increased diosgenin content in D Deltoida and rede light decreased

iv. Selection and screening of high yield strains

• Different species of plant may vary widely in their biosynthetic abilities

• Selection of high yielding strain helps in optimization of yield

• Cell clones from better strain should be selected, it can be achieved by

- Mutagenic techniques

- Radio immune assay (RIA)

- ELISA, etc

Strategies to enhance the production of Secondary Metabolite:

Addition of precursor

Light intensity

Selection of high yielding strains

Isolation of product

Use of elicitors

i. Addition of precursors

• Improved metabolite production sometimes may be achieved by the addition of precursor to the culture media

Eg: (i). Addition of coniferin to Podophyllum hexandrum improved production of podophyllotoxin by 12.8 fold

(ii). Addition of L-tryptophan to cinchona ledgeriana cultures enhance the production of quinoline alkaloids

ii. Light intensity:

Light intensity and selective wave length have stimulating effect

Eg: Blue light increased diosgenin content in D Deltoida and red light decreased

iii. Selection of high yield strains

• Major factor in increasing the production of sec metabolites

Eg: High yielding strain of Catharanthus roseus on Zenk’s medium results in higher production of vincristine and vinblastine

iv. Isolation of product:

• For continuous production the metabolites should be released rather than retained with in the cells

Eg: Addition of silicone products extract the metabolites without disturbing the culture

v. Use of elicitors

• Elicitors in plant biology are extrinsic, or foreign, molecules often associated with plant pests, diseases or synergistic organisms

• Elicitor molecules can attach to special receptor proteins located on plant cell membranes

• These receptors are able to recognise the molecular pattern of elicitors and trigger intracellular defence signalling via the octadeconoid pathway

• This response results in the enhanced synthesis of metabolites and increase resistance to pest, disease or environmental stress

• Elicitors are of 2 types, namely biotic and abiotic

Elicitor	Type of elicitor	Culture	Sec metabolite
CuSO4	Abiotic	Lithospermum	Shikonin erythrorhizon
Colchicine	Abiotic	Valarian	Valepotecrates (60 fold)
Fungal extract	Biotic	Catharanthus	Indole alkaloids roseus
Yeast/yeast extract	Abiotic	Orthosiphon aristatus	Rosmarinic acid

       

Production of Shikonin

• Shikonin is napthaquinone red pigment obtained from the roots of Lithospermum erythrorhizon

• Shikonin is used as antimicrobial, anti-inflammatory, to treat burns, wounds and also as a dye in cosmetic preparations

• Chemically it is a napthaquinone derivative

• First process for the commercial production of natural plant product by the cell suspension cultures was developed in Japan

• Two-stage culture process was developed

• Cells are first grown in a growth promoting medium (MG-5) for 9 days, filtered and pumped to a second vessel in which the M-9 production medium is added

• Cells are cultured for 14 additional days, after which they are recovered by filtration, and the shikonin is extracted using n-hexane followed by hydrolysis with 2% KOH

• Purified by re - crystallization

Summary

• Protoplast –Cell without a cell wall

• Isolation of protoplast – Mechanical method and enzymatic method

• Enzymatic method –Direct method and sequential method

• Cell wall degrading enzymes – Cellulase, pectinase, macerozyme etc

• Purification by washing and sedimentation method and flotation method

• Protoplast culture - regeneration of cell wall on a suitable media

• Protoplast fusion -Spontaneous and induced fusion

• Secondary metabolite – Do not have any role in growth

• Independent of environmental factors, well defined system, ensures quality and yield

• Factors – Plant tissue genetic character, culture conditions, physiological factors and selection of high yield strain

• Strategies –Addition of precursor, light intensity, high yield strain, isolation of product and elicitors

• Shikonin – Red dye, Lithospermum erythrorhizon, anti-inflammatory, in cosmetics

• Production –Two stages, growth medium and production medium

• Process of initiation and development of an organ is called organogenesis

• Employed in micro propagation from bud and shoot material and in organ production from callus and suspension cultures

• Roots, shoots and flowers are the organs that may be initiated from tissue cultures