Chemical Production through Plant Cell Culture
Chemical Weekly – 14th Dec. 1999
Over four fifth of about 30,000 known natural products are of plant origin. Some of these can be synthesized in quantities sufficient to be economically useful as chemical feed-stocks or as raw materials for various scientific, technological and commercial applications. The area, which has received maximum attention from scientific community, is the potential use of plant-derived compounds as medicine or source of medicines, aromas and fragrances. The recent developments in plant tissue culture techniques coupled with genetic engineering, has shown the promising results. Apart from the economic production, it is the saving of the environment by avoiding sacrifice of the plants and trees, which is great advantage of the new approach.
The Need and The Potential –
In the past century pharmaceutical industry has used ‘reductionist’ approach – ‘isolating single active molecule’. Western medicine uses a rationalistic, mechanistic approach for treatment. But, today there is growing interest in herbal remedies – use of plant extracts and plant derived products. The scientific study of traditional medicinal plants – ‘ethnopharmacology’ is becoming increasingly popular in search of new drug sources. The pharmaceutical companies of UK, USA and Europe are interested to go in a scientific way for such sources. This develops the need of easy and continuous production, extraction and recovery of chemicals from plants and therefore the scope for plant tissue culture.
The demand for plant-derived chemicals is largely from developed countries while they are available in Third World Countries. Political instability, plant diseases may interrupt the supply of these chemicals suddenly. Also consistency in quality and quantity of useful chemicals produced is important. These were some of the causes for efforts for development of plant tissue culture for chemical production. Over 1500 compounds are identified in plants every year and some are suggested to be produced by cell culture.
Plant cell cultures may be used for synthesis of complex substances like alkaloids, flavonoids, terpenes, steroids, glycosides, which are otherwise difficult for chemical synthesis. Plant cell cultures can be thought of for production of novel metabolites. Plant cell cultures may also be used for bio-transformations. Thus, hydrogenation, dehydrogenation, isomerization, glycosylation, hydroxylation, opening of ring and addition of carbon atoms are some of the reactions which can be performed with plant cell cultures.
Economic Aspects of Plant Tissue Culture for Secondary Metabolite Production
Production by cell culture is justified for products whose world annual potential market would be US $20-50 million with a minimum selling price of about US $400-500 per kg. Thus Ajmalicine and jasmine oil in-spite of their high selling price – US $1500 and $5000 per kg are still not suitable because of their small market of US $8 million and $500,000 respectively. In another estimate it is thought that US $500 per kg for the production of drug and yields of 1 gram per litre will be economical. Products costing more than US $1000 per kg are suitable for production by plant cell culture as per another estimate. According to the study carried out in Japan, any substance of plant origin with a value exceeding or equal to US $80 per gram could be profitably produced by cell or tissue culture.
The estimated annual market value of pharmaceutical products of plant origin in industrialised countries was over US $20 billion in mid 1980s. The annual market for codeine and anti-tumor alkaloids vinblastine and vincristine was about US $ 100 million per product. The world market for aromas and fragrances has been estimated to be US $6 billion in 1990.
Pharmaceuticals are not attractive area for use of plant cell culture as production method, due to competition from synthetic routes. Still commercial production was envisaged for vincristine and vinblastine from Catharanthus roseus cell cultures (by Eli Lilly & Co.),, ubiquinone from Nicotiana, L-dopa fromMucunna puriens and digoxin from Digitalis lanata.
Prospects are more promising for the production of aromatic substances, flavouring compounds and food additives, and basic materials for fragrances by plant cell or tissue cultures. These products though less valuable than pharmaceuticals, have larger market.
Chemical Production by Cell Culture –
The understanding that every cell of every organism has complete genetic information to make the entire organism (plant or animal) has led to the development of tissue culture technique for producing a variety of products by growing the cells in correct medium. For the propagation of plant cells medium used is relatively simple. Small segments of any organ (leaf, root, stem or other) are stimulated to differentiate and divide, generating a disorganised mass of cells or callus. Callus can be placed in liquid shake culture to disperse and suspend the cell aggregates and grow them as fine suspension of cells. Such cultures could be used to produce secondary metabolites the same way as what bacterial and fungal cultures are used to give antibiotics and vitamins.
Higher plants are indispensable source of some drugs and raw materials. Several commercial products including morphinane, alkaloids, cardiac glycosides, essential oil, rubber are obtained from agricultural plants. With the application of cell culture method, perhaps, continuous production of chemicals may be possible and growth of whole plant (which takes number of years) may not be required for getting the products.
Stages of plant cell culture for chemical production –
- ·Selection from wild plants a high producing one
- ·In-vitro culture or Callogenesis (selection and stabilisation of producing calli with a view to identify high producing cell line.
- ·Maximising callus or cell suspension
- ·Culture conditions study and isolation of best-producing line.
- ·Industrial scaling-up
- ·Mass cultivation in bioreactors
- ·Downstream processing i.e. extraction and purification of compound sought.
Status of Achievements
In the course of 1980s, substantial advances were made in plant cell culture technology, notably in two areas; in large-scale “fermentation” technology, and in the understanding of some of the factors influencing levels of known secondary metabolites in plant culture systems. Plant cell culture was attempted on 90 species producing over 125 phytochemicals and the target drug was detected in 90% of the cases. Although yields were lower than those typical of plant in most of the cases, there were some examples like rosmarinic acid, sanguinarine and various acridones from Coleus sp., Papaver smoniferum and Ruta sp . accumulating more secondary metabolites in cell cultures than in the whole plant.
With the onset of the 1990s, Japan and western part of Germany were engaged in industrial production of secondary metabolites by plant cell cultures. In Japan, seven private corporations have created a common subsidiary in research and development on plant cell cultures. The Plant Cell Culture Technology (PCC Technology), has been set up with support of the Japan Key Technology Centre by Kyowa Hakko Kogyo Co., Mitsui Petrochemical Industries Ltd., Mitsui Toatsu Chemical Inc., Hitachi Ltd., Suntory Ltd., Toa Nenryo Kogyo Co. and Kirin Breweries Co. Ltd. Japanese scientists and companies are optimistic about future of plant cell culture.
In Europe, Canada and the USA, a number of companies do carry out research in the field of plant cell culture for production of chemicals. In France, Sanofi-Elf-Bio-industries has supported research work on the production of bioconversion of ellipticine – antitumor alkaloid by cell culture of Ochrosia elliptica. Mero company is involved in production of fragrance compounds. Nestle’s subsidiary, Francereco, carries on its research programme on the production of secondary metabolites by plant cells. The pharmaceutical company, Roussel-Uclaf is interested in the production of diosgenin. Rhone-Poulenc is also interested in plant cell culture work.
Achievements have been reported from Japan, Germany and US. Success has been achieved in manufacturing many plant derived chemicals, oral contraceptives and antiseptics such as diosgenin, digoxin and berberine respectively, anthocyanines, artificial sweetners, pyrethrins (insecticide) and flavours such as vanilla, capsicum and Shikonin – (to treat burns, red pigment to dye silk, in the manufacture of bio-lipsticks); ubiquinone 10 – (for treatment of heart disease); Ajmalicine – (antihypertensive agent) etc.
The synthesis of shikonins by cells of Lithospermum erthrorhizon is the only commercial process developed so far. Japan imported Shikonins from China and Korea. It costs $4500 per kg for pure natural product. In 1983, the first commercial process of plant cell culture was reported in Japan. The Shikonins (anthroquinones) are used in Japan for their antibacterial and anti-inflamatory properties and also as dye. Roots make up 2%dry weight of Shikonins but the plant takes several years to be of commercial use. Therefore, in 23 days fermentation process, plant cells are grown in 750 litres fermentor and 23%of dry weight Shikonins accumulates. The productivity of L.erythrorhizon cell cultures was 60 mg per gram of cells per week that is 1000 times higher than that of the plant roots, which required a longer time period of 5-7 years. The success of shikonin was due to the selection of cell line, which accumulated a ten-fold higher level of shikonin than that found in roots of mature plant. Cell cultures have now become a major way of commercial production of shikonin. Air-lift fermentor is suited for Shikonin production by plant cell culture.
Higher quantities of berberine are obtained from cell cultures of Coptis berberica. This plant species accumulates significant amounts of berberine in its roots in 4-6 years. Similar concentrations could be obtained in 4 weeks using tissue culture. Hara et al of Mitsui Petrochemical Industries Ltd. have isolated a cell line of Coptis japonicathat contains 10% of berberine (dry weight) and which produce about 1500 mg of this antibacterial and antipyretic alkaloid per litre in 14 days.
Sanguinarine is a benzophenantridin alkaloid extracted from the roots of Papaver somniferum. 3-4 years are required for plant maturation before the substance can be extracted. Cell cultures have been used to produce large quantities of this alkaloid. This alkaloid is used in toothpastes and mouth lotions to combat dental plaque and tooth decay. Commercial production is the joint venture of Plant Biotechnology Institute of the National Research Council of Canada, Saskatoon, and Vipont Research Laboratories Inc., Fort Collins, Colorado, USA. The manufacturing process uses eliciting power of extracts of a fungus Botrytisthat induces synthesis of sanguinarine and dihydrosanguinarine by plant cells. Production rate of alkaloid in semi-continuous cultures reached 3% of dry biomass in pilot plant experiments.
In West Germany, Alfermann et al of Boehringer Mannheim AG were able to grow cells of Digitalis lanatain 200 litre bioreactors and obtain 500 g of b-methyl-digoxin in 3 months. Bioconversion rate was 93.5%, if unused substrate is recycled.
Ulbrich, Wiesner & Arens cultured Coleus blumei cells in 42 litre bioreactor fitted with the module spiral stirrer. Using this system along with aeration, they reported high yields of rosmarinic acid (5.5 g per litre), representing 21% dry weight of cells.
Heble and Chadha reported successful cultivation of Catharanthus roseus cells in 7-20 litre capacity bioreactors, modified to provide air lift and agitation, in single and multiple stages. The cells produced high levels of total alkaloids comprising ajmalicine and serpentine as the major components.
For Ubiquinone 10, which is used in Japan for congestive cardiac disease, tobacco cells are grown in 20000 litres capacity fermentor. Catharanthus roseus plant produces a range of indole alkaloids such as Ajmalicine, Vinblastine, Vincristine. The last two in the list are useful in cancer therapy. Also the last two cannot be commercialised by tissue culture as the quantities produced are low and extraction is difficult.
There is demand for vinca alkaloids, Vinblastin and Vincristine. Vinblastine is marketed as Velban by Eli Lilly. Vinblastine is mainly useful for treating Hodgkin’s disease, lymphocytic lymphoma, histiocytic lymphoma, advanced testicular cancer, advanced breast cancer, Kaposi’s sarcoma, and Letterer-Siwe disease. Vincristine, is marketed as Oncovin by Eli Lilly. It is used mainly to treat neuroblastoma, rhabdomyosarcoma, Hodgkin’s disease, acute leukemia and other lymphomas.
Vindesine and Vinorelbine, are semi-synthetic derivatives of Vinblastine. Vindesine, which is marketed under the names Eldisine and Fildesin, is used mainly to treat melanoma and lung cancers (carcinomas) and, with other drugs, to treat uterine cancers. Vinorelbine seems to have a wider range of antitumor activity than the other vinca alkaloids.
Plant tissue culture can be used for biotransformation. 12 b-hydroxylation of digitoxin to produce digoxin (heart stimulant) can be carried out using the suspended culture or immobilised system of Digitalis lanata(foxglove).
The National Cancer Institute of the USA has awarded ESC Agencies Corp. of San Carlos, CA, a research grant of $800,000 to investigate the feasibility of scale up and economic production of anti-ovarian cancer drug Taxol by plant cell culture. Traditional production requires the bark of 3-5 mature trees (Taxus brevifolia) to supply enough of taxol for one person per year. The ESC agency is already having proprietary cell culture technology to grow cells from roots, leaves or stem in culture using fermentation that stimulated cells to produce taxol.
Taxol is an anti-cancer drug obtained from Yew trees. Currently it is made semi-synthetically by the acetylation of precursor extracted from yew tree needles. But there is a shortage of yew trees for regular bulk production. A process that could boost production without destroying trees has been developed by plant physiologists at the US Agricultural Research service (ARS) in Ithaca, N.Y. Cells extracted from yew seeds are cultivated in an aqueous solution of nutrients. Once the population has reached desired size, an elicitor compound – methyl jasmonate – is added to induce the cells to make taxol. The yield is 120 mg/l – well above the economic threshold of 100 mg/l and it is readily scalable. While direct comparison cannot be made with semi-synthetic route, the yield from cell cultivation is many times higher. ARS is seeking industrial partners to scale up and commercialise the process.
BARC researchers have long been experimenting with Vinca rosea, whose leaves contain the material for making anti-cancer drugs like Vincristine and Vinblastine. The researchers have isolated the active cell line from the leaves of the plant and extracted anti-cancer drugs. BARC researchers have pioneered methods to produce anti-cancer drugs. BARC has transferred this technology to Kabra drugs – a Indore based pharmaceutical company to set up large commercial bioreactors that can produce herbal compounds (such as ajmalicine, vindoline, catharathine etc.) and other drugs in bulk quantities. This is the first time BARC has transferred its technology to private company for commercialization in the area of medicinal biotechnology.
Scientists from Central Institute of Medicinal and Aromatic Plants (CIMAP) in Lucknow have devised tissue culture method for rapid multiplication of medicinal herb Centella asiatica that is used in several ayurvedic preparations. It is reported to be useful against leprosy, filaria, viruses, bacteria and insects. Natural populations of the herb are gradually getting depleted due to use of the extracts. Tissue culture offers scope for continuous supply of the same. Thus conservation and propagation of medicinal plants will be achieved.
Immobilized Plant Cells for getting Biochemicals
The use of immobilised plant cells for getting chemicals is under study. In this bio-transformation of cardiac glycosides is one. Capsacin, Buinine, Digoxin, Ajmalicine, Serpentine, Vinblastine also are being studied for immobilised tissue culture method for production. De novo synthesis of chemically complex molecule from simple carbon and nitrogen source may be possible. Energy cofactors are available in cell but permeability to substrate and release of product are to be achieved in multi-step reactions.
The researchers team in Tokyo University have developed a bioreactor for the production of codeine. Poppy cells have been immobilised in calcium alginate beads and they catalyse conversion of codeinone to codeine. This enabled them to overcome the drawbacks of plant-cell bioreactors, due to the instability of cells and the low yields of desired compounds. The size of cell cluster was decreased to 2.5 mm in diameter; thereby increasing lifespan and obtaining yields of codeine that were equal to that in non-immobilised cells.
The use of immobilised cells bypasses the direct extraction of the compounds from the biomass, as the products now appear in the medium itself. Examples of this approach are the production of caffeine, capsaicin and berberine. However, for the metabolites, which appear in cell vacuoles it is difficult to extract them in medium even in immobilised cells suspension.
Products being studied in plant cell culture –
- Alkaloids Ajmalicine, Vinblastine, Serpentine, Codeine, Quinine, Nicotine, Vincristine
- Quinones Shikonin, Ubiquinone, Anthroquinone
- Cardiac glycosides Digoxin, Digitoxin
- Saponins Ginseng
|Difficulties faced in actual commercialisation –Industrial production of useful substances by cell culture should take into account the following properties, some of which could be major constraints. These are : slow growth of cells with doubling time of 24-48 hours requiring usually 2-3 weeks to provide sufficient biomass; susceptibility to microbial contamination; use of axenic cultures; oxygen needs; and susceptibility to shearing stresses due to large cell size which on average is 200,000 times larger than that of bacteria. In general cell multiplication and metabolite synthesis are uncoupled, the latter occurring at the end of growth phase.
(i) Several, metabolites are released in cell vacuoles instead of in medium and therefore trapped.
(ii) Cells must remain viable for long periods to release the product continuously.
The knowledge concerning biosynthetic pathways of secondary metabolites is lacking. Therefore high-yielding cell lines cannot be obtained.