Biotechnological Advances in Artemisinin, Hyperforin, and Taxol Production

[Music] [Music] welcome to nptl online certification

course on pharmacognosy and metabolic engineering so this is lecture 47 so where I will briefly cover timinin hyper

forine and taxol the three promising candidates for biotechnological exploitation so Concepts covered will be

arimin hyperforin and taxol these are the molecules of pharmaceutical interest uh so rine is basically a TR

tarpine so we have studied the tarpo biosynthesis so there I have mentioned about about this

uh monot tarpine sarpin dipine tarpine so with timin what happens that two c typin unit joins together and

finally form this structure and this is found in the plant of asteres family which is called warud or the scientific

name is timia anua and artim is a very important compound because it is now used as an antimalarial drug so so

biosyn so the purpose of this whole class is just to give you an overview so here I'm not going to do a detailed

board work or explanation uh so that you can get an idea that's is the only thing so don't get afraid about the structures

and all these things so this is just uh to give you the concept so as you see that parile d phospate uh so the fpp and

which is the first enzyme is basically the the tarpine synthes so it is also called this

amorion synthes as well and that leads to the formation of this Amor 411 D and then by several cytochrome p450 enzymes

uh the structure underg goes different transformation and eventually double bond reductors and it forms the dihydro

arimic acid dihydro timic acid got converted into timinin this is basically a non- entic

conversion so uh it is you're showing here dihydro arenic acid then this is basically Auto

oxidation uh which makes toine so what we see here the ads ads is basically the uh what I have shown the tarpine synthes

so amorodian synthes so that is why it is called ads ads is an important enzyme so this enzyme has been characterized at

the molecular level and several attempts were made to ere Express this and as a result of this what happens to the final

product so amorpha 411 Dian by subsequently by cytochrome p450 and then reduct is it forms arenic alcohol that

will be converted into an alide by a dehydrogen and then in one way it goes to timic acid another de hydrogenous or

there will be another reduct which makes dihydro arimic alide and then another dehydrogen makes this dihydro arimic

acid and then finally it makes timinin so from the arimic acid it makes art artinun uh and but from this one dihydro

arimic acid it makes timinin so this is in general brief the biosynthetic pathway and uh this is

basically the same same thing only uh it's more clearly showing the product formation so I can skip this one and

other important Point as you know that csar pins are basically localized in the cytool as this is a tripin originated

from cpin scon so most of the pathway is basically operating in the cytool and one important Point here I have written

is is the triome so basically the leaf contains this fine ha like structure what I have mentioned in one of the

previous classes when I talked about the biosynthesis of Menthol and there I said that Menthol and other products

accumulate in the triome so here also this products finally it accumulated in the triome of this asy plant which is

Aria Ana so as you see the most of the steps are all basically occurring inside the

cytool so that is the con concept you should know and you should know what role Tricom plays okay now uh the

pathway and construction for engineering of artim in production in tobacco so tobacco was chosen as a model to

engineer the pathway and the pathway what is for the formation of arching is shown here on the top as you see here

this is the one written in red so these are the genes which were basically transferred into the tobacco and um that

finally leads to the formation of arine so the gene construct is shown here and uh the other one is the pathway

which is shown here so the work published in nature biotechnology in the year of

2011 that means the take-home message is that yes it is possible to produce timinin in tobacco provided you transfer

the whole cassets into it and the this is basically the experimental result that uh indeed

arimin is produced or not so they have checked the formation of arine by using this uhpc based methods so where you see

that where it is this ads trans gen here the detection of arimin is there so another trans gen detection of R whereas

in the control or or the or the transform plant lacking the gene only the empty Vector so their atan content

is very less and here is the same thing they have checked it with the standards so

okay uh next important point is that attempts were made to express this pathway in the East system so in the

East system this this gits that is the reductase and cytochrome p450 gen SE to

or three so these genes were transferred into East and as a result of that what

we see that the formation of timic acid so timic acid was detected so timinin was not but timic acid was detected so

that serve as the precursor and then subsequently using a semisynthetic approach timinin can be made so here is

basically the gcms chromatogram which is showing the product formation from the uh transic East as well as only the

control and as well as the uh artim found in the timia Ana plant so this is also published in nature so that means

take home message is this yes it is possible to produce the ultimate precursor for timin in

East so from here I mov to hyper forine so hyper forine again it is a part this is it is basically you can group it

under Tarpin because there are a lot of penile chains attached to it and it involves uh this DM p as well as gpp you

will see okay so what is basically hyperpin hyperpin is uh bicyclic poly

prated SI flool so this I can write it for you so it is

basically bicyclic poly prated asile

Flor pH p h l o p l o l o l

floro gluc null

derivative so this is what is called hyperin so hyper Forin is a very very important molecule because it is a

proven anti-depressant and and is safe to use therefore in the European market this is in the harbal shops it is sold

at as St John's oat so St John's oat is available it's available over the counter so anyone can buy it and use it

and this is the plant in the pistil uh the the hyper Forin accumulates but apart from that in the p

there are tiny dots which are Reddish in color so they are hyperin accumulates so I'll show you some of the nice pictures

now the pharmacological activity is basically one most important point is anti-depressant that is why the molecule

is so important but apart from that it has anti-inflammatory antibacterial neuroprotective anti-

tumoral antimalarial and so many so we are not going to discuss these things only to tell you that how important is

this molecule now because of its importance so scientists they wanted to produce

this product in in cell and tissue culture approach so there so that all around the year the plants can be grown

otherwise and moreover this is specific to tempered region only so in the tropical area these plants cannot grow

so if you have a culture system then it is possible uh to use biotechnological intervention to produce this now these

are the leaves with this dots and this is so and here a mature Leaf it's basically red in color and this red

color is because of the hyperin accumulation so and the translucent dots are also there where the hyper forine

accumulates so structurally these are different as you see there are lot of phenolic Rings joints and form hyp

but that also form by polyte synthes activity hyperin is also by polyte synthes activity so that way there is a

relationship between the between these things so dark nodules accumulate hyperin where

do translucent gland accumulate hyperin what I said so this is for the hyperin and this is for the

hyperin uh so root cultures of hypericum perforatum was established and these root cultures were

shown to accumulate hyperforin so this is the plant and this is the root

culture very interesting and then cell supension cultures of related species was also used to study the biosynthetic

pathway and their cell suspension cultures was treated with mythy jasmonate I have mentioned in the

elicitation class and then what happened that upon elicitation it accumulates hyper zenthon not specifically hyper

porin but that itself was appeared to be of pharmaceutical interest and um that is why I mentioned here that cell

cultures can also upon elicitation produce different interesting compounds now the proposed biosynthetic

pathway is basically like this so here what happens that three molecules M malony KO this is malony KO joins with

isoil KO so this is basically isoil qu b u t y r y l is qu this is Alon qu and it forms this

Flor Flor is a big name this one I write it here pH l o r i s Flor Flor

ISO buty Ro phenon

flu isoo phenon so which is the product and this is by the enzyme again it is a polyketide synthes so

PKS but subsequently this is PKS means polyketide synthes when we talk about phenolic

compounds we'll mention about this in little bit more details and here specific polyte synthes has been

identified and this was term as B so bu stands for isoy phenon

synthes so this was reported in the famous Journal phytochemistry by professor ludgar vus and his team and

the paper published probably in 2005 okay I have uh yeah okay sorry I have not put the reference here but reference

can be provided later on and next step is basically the the pration so this is you see the penile chain one

two then three so uh and here and you see here DM is required first one second one again a third one is again a gpp and

the fourth one again DM so basically these are all pren transfer ises so these tips are all catalyzed by pts or

PT you can say penile transfer so which transfer the penile side chain into it so uh and uh the first pration step is

catalyzed by a soluble dimethy alile transfer so here basically this appears to

be a soluble enzyme normally penal transfer is are membran BR enzyme but the first one what

they found to be a soluble enzyme okay and the major constituents accumulating in hyper parum which includes hyper

forine hyperin along with several janon which are all the polyte derivatives so this is in brief the biosynthetic

pathway which has been characterized only at the biochemical level okay not at the molecular level

yet it's very complex and then hypericum perum invitro root cultures were established this is invitro grown plant

and this is the root cultures and then root cultures are grown and and a fully grown root cultures here and these are

basically the freeze dried root cultures which are used for subsequent extraction and

Analysis okay so hyperin H parisin production was also checked uh from this uh root cultures established so this is

the normal plant and uh in vitro grown plant from their cising and then from root formation then Roots started

growing and this is a fully grown roots and then this is basically the dried Roots frz dried Roots like stack

coins okay and then what they have done they have compared the uh hyperm production in shot cultures and root

cultures so the shot cultures basically shown in the red red is from the shoot cultures and the blue is from the

uh root cultures what you see here is the SEO hyper forine which is number two I have marked

uh and then hyper Forin number six so here you see that the production of hypop porin is

higher in root okay

whereas uh say hyperin is more in the shoot okay and number eight is basically the add

hyperin which is uh almost undetectable in the root apart from that there are other compounds like number five which

is more in the root uh okay also number one which is also more in the root so this indicates

that root cultures are indeed very promising for the production of these compounds and for subsequent study

uh with this now I move into the taxol so taxel is a very important anti-cancer drug particularly for treatment of uh

ovarian and breast cancer so this is the figure of the bark of Texas brav Folia

the original uh U Pacific U tree so these barks are harvested and what is important that 5 to 600 year old trees

are required to treat one patient so the amount of this taxo is so low that you record so many trees subsequently they

found that actually Taxas brav f is restricted to certain areas so uh but another species which is more widely

available is txas Bata so and scientist also found txas Bata also accumulating taxol not only that much later they

found that the tax uh needles are also accumulating taxol although in low amount and of course much later they

found certain endide txom misis uh which are basically living in the soil or where tax are growing that species are

also uh capable of producing taxil although the content is less and then lot of efforts are being made to make uh

tax from the micrell sources now one important point is this the taxol as you see here the registered trademark is

there so the drug is called taxol but the original chemical compound is basically ply

taxel so the Bly taxel is the chemical compound taxol is the drug similarly the dosy taxel is the generic name and the

drug is called taxer so that I will come yeah it is written here you see the register trademark is

there so this is basically produced by ro poen so the the product is taxo but the original molecule is Dos taxel

similarly taxel is produced by Bristol May and the product is ply taxel so this you must know so when you say tax ta

taxel it is not correct either we should tell Backle taxel or tax or tax or dosy taxel yeah but we are more uh familiar

with taxel rather than back taxel that is our problem okay and so this is basically the structure

of taxol you see how complex the molecule is so it is basically again this tarpine derivatives and this is the

taxo okay and what is showing here in the left side is a three-dimensional

structure of taxol and this is basically the taxol

the the txin ring is here and the St side chain uh which are basically important

for imparting the cytotoxicity and unique effect of microt tubules okay this will

t t okay I and and and uh as uh as harvesting trees are not

very feasible so the scientist they look for alternative sources for uh tax all taxor production so one is the cell

cultures and cell cultures particularly using this coronatine as elicitor that showed promising effect in

producing uh back taxel so I'll will not go into details of it and little bit of

biosynthesis so why I because it is basically starting with the ggpp general General py hospit then

there is where taxin synthes and then it produces this taxa 411 D similarly what we found this

amorpha something D in case of timn acid then several intermediates and then it finally forms betin so betin is an

important precursor and from betin pexel is formed by the joining of this from philein the phyan coil be formed and

that it joins and ultimately it makes the pxel so and de atile betin from betin

deetle betin is formed and that is used as the source for semisynthetic different d derivatives of taxol

production so this is taxol this is taxo this is the atile betin so betin is important and uh and the biosynthesis is

very complex although biosynthesis has been or the has been resolved mostly uh chemical synthesis was also but uh

producing ply txell chemically is not visible okay and that is why scientists are looking for biotechnological

approach uh first with the empirical approach by sell and root cultures to produce that and later to uh still yet

years to come and when there will be a time when maybe the pathway can be expressed in East and then what we have

seen in case of other alcoh that maybe another 10 years or so that the no production of uh taxol in the East may

become a reality but still now it is not we have to wait and you see there are uh multiple so enzymes like synthes then

hydroxy then acle transfer then again hydroxy so many hydroxy lases there and these hydroxy lases are mostly membrane

bound okay then oxidase and then again atile transfer so this forms the betin then from okay and then uh it forms

other products and eventually the dexol so this indicates how complex is the

biosynthetic pathway but it is very interesting and lot of work has been going on so years to come we'll see some

more uh outstanding achievements so with this I end this class and with this I have

given you a brief overview of timinin which is very important antimaterial drug hyperin which is

basically basically a anti very strong anti-depressant and safe anti-depressant and third one is the taxol or or or or

pxel uh so which is basically a proven anti-cancer drug against ovarian and breast cancers so with this more or less

I finish the uh tpid group and the remaining classes I'll concentrate on the phenolic compounds

thank you very much