In this article we will discuss about the metabolic engineering of alkaloid biosynthesis and phenylpropanoids.
Alkaloid Biosynthesis:
Metabolic engineering of Alkaloid, belongs to the broad category of secondary metabolism. These nitrogenous compounds are used as active principle to combat various diseases. More than 10,000 alkaloids have been isolated and their structures elucidated.
They have very important functions in plants and also in animals. Historically, the use of alkaloids containing plants extract used as medicinal and poisonous. Several new drug discoveries have been made based on alkaloids.
With the introduction of biotechnology into the plant alkaloid field, alkaloid biosynthesis can be manipulated and also potential to alter the pattern of alkaloid accumulation in plants (Fig. 17.11).
Thus, alkaloid field is now an exciting area for metabolic engineering of tailor- made plants that accumulate increased quantities of desirable pharmaceutical or to produce food stuff plants with lower alkaloids content as in case of coffee without caffeine. Following are some of the examples of transgenic work were carried out in alkaloid biosynthesis based on their classification.
The most common indole alkaloids are antimalarial quinine from cinchona officinalis, strychnine, and anticancerous vincristine, vinblastin from catharanthus roseus. In the initial experiment, the cDNA for enzymes that catalyse biosynthesis have been isolated and heterologously expressed in bacteria but expression of long pathway is not feasable in bacteria.
Therefore, alkaloid biosynthetic pathway was too long to be engineered in microorganism could be modified in the parent plant using antisense or co-suppression technology. Implication of their novel technology provides accumulation of desired alkaloids by blocking side pathways or catabolic steps (Fig. 17.12).
One of the most drawbacks with these approaches is the requirement of complete knowledge of the pathway and involvement of enzymes. Thus, progress towards identifying enzymes of indole alkaloid biosynthesis has been characterized. The first successful cDNA cloning experiment into alkaloid was achieved with two cDNA encoding enzymes: tryptophan decarboxylase and strictoside synthase.
Tryptophan decarboxylase catalyses the decarboxylation of L-tryptophan to protoalkaloid tryptomine. Tryptomine can then serve as substrate for another enzyme strictosidine synthase, which catalyses the stereospecific condensation of the tryptamine and aldehyde moiety, secaloganin to form the first monoterpenoid indole alkaloid.
The tryptophan decarboxylase cDNA from Catharanthus roses has been heterologously expressed in tobacco plants. Introduction of transgene increases levels of tryptomine and tyrasimine. A fine example of metabolic engineering could be seen by the transformation of Brassica napus with the C. rosens tryptophan decarboxylase cDNA. Brassica seed has limited use as animal feed due to the presence of indole glucosinolates.
Expression of transgene for tryptophan decarboxylase redirects tryptophan pools away from indole glucosinolate productions and accumulates more of tryptomine. As a result the mature seeds of the transgenic B napus plants contain reduced level of glucosinolates and achieve a potentially and economically useful product.
In another transgenic approach, strictoside synthase from Rawalfia serpentina has been functionally expressed in microorganisms like E. coli and yeast. The same enzyme from C. rosens has been expressed in tobacco.
Tropane alkaloids are derived via arginine metabolism. In an attempt to produce tropane alkaloid 6β-4 hydroxylase and tropinove reductase, both of which are enzymes of scopalamine biosynthesis in Hyocyamus niger, have been cloned. One of the most medically important alkaloid is the scopalamine.
Currently, Duboisia is the commercial source of scopalmine. Certain tropane alkaloid producing plants such as Atropa accumulates hyocyamine instead of scopalamine as major alkaloid. Expression of transgene in medicinal plant could alter the alkaloid pattern such that pharmaceutically useful alkaloids, scopalamine, could be produced.
In order to achieve this, the cDNA encoding hyocyamin 6β-hydroxylase from H. niger was introduced into Atropa belladona as a consequence, resulted transgenic plant and hairy roots accumulated enhanced level of scopalamine. These successful transformation experiments clearly show that it has a distinct implication for the future of metabolic engineering of medicinal plants.
Later efforts to increase the tropane alkaloid content of deadly night shade by 35S driven over-expression of putrescine N-methyl transferase was found to be unsatisfactory. In the extended study, transformed Nicotiana sylvestris showed increased levels of nicotine, arising from increased supply of the tropane moiety.
Phenylpropanoids:
Metabolic engineering of phenylpropanoid path way received considerable attention. Phenyl propropanoids are generally a aromatic metabolities containing one or more phenolic hydroxyl functions. Many of these phenolics produced by plant can easily undergoes oxidation.
Many other phenylpropanoid metabolities acts as defensive role, lignin formation in woods. The flavanols found in grape and so in wine, tomatoes comprises of important antioxidant component. It also acts as anticancerous agent.
In the core pathway of phenylpropanoid, a key enzyme phenyl alanine ammonia lyase (PAL), play a central role, which catalyses the initial conversion of phenyl alanine to cinnamate. Further reaction was depicted in the (Fig. 17.13).
Secondary metabolities such as stibenes. Coumarins and flavanoids are resultant product of this pathway.
One of the earliest attempts made in this pathway is directed towards the anthocyanins involved in flower and coloration. The enzyme CHS was targeted in this concern by over expression under Cam35 promoter. However, flavonoids level was found to be decreased probably due to gene silencing.
In the later studies, over expression of chalcone isomerase (CHI), driven by Cam35 in tomato enhanced significant level of antioxidant flavanols in the fruit peel.
In another study, expression of cytocrome P450 isoflavone synthase (IFS) in Arabidopsis, resulting in the production of low levels of the isoflavone genistein. Manipulatism of enzymes for lignin production in this pathway have been successfully attempted.