Environmental Regulation of Indole Alkaloid Biosynthesis in Catharanthus roseus

[Music] [Music] hello welcome to pharmacognosy and

metabolic engineering this is nptl online certification course so now come to lecture 23 where I will talk about

indal alkaloids the environmental factors that regulate indol alkaloid biosynthesis and under this what I'm

going to cover basically the role of light and elicitor in upliftment of indol alkaloids in

culture and Sh root culture system for indol Alco production and to study the

metabolic pathway and finally we'll also see the elicit induced expression of early pathway gen in indol aloid

biosynthesis that means how elicitors can upregulate the IND alcol production so I would like to tell that that so far

our discussion uh we are uh mostly uh dealing with some of the only early steps of Indo alcol biosynthesis but the

pathway is very complex and in the subsequent classes we'll look into it but it is interesting also so don't get

daunted so now go to the let's go to the board so board is this that the uh

plant cell and tissue culture

system for indle alkaloid

production I have already mentioned that cell cultures of Cenas Rosas was

exploded but uh it failed to produce the late enzymes of the pathway so let briefly remind you the final stages of

the biosynthesis that means what I said that ultimate Target is basically to find to

find in blastin or vin Christine from catharanthine and vindoline

and that eventually coming from strict toid

in and which is basically coming from SEO lanin and

crypin and then in between also there are products

like as micine and other product camine Al so on so

uh so cell culture failed to produce the alkaloids like vindoline and catharanthine which are monomers

monomeric indol alkaloid and I have made it clear in the earlier

class then Vin blastin and Vin Christin is a

diic so uh then so sale culture

failed to produce windin or even karantin so from there scientist thought that

maybe differentiated cultures plays important role so they go for differentiated

culture one good example of differentiated culture is basically Sho culture so

and another one will be the here root

culture with here root culture there are

reports which are not always consistent uh with the findings from different groups for example one group

in United Kingdom they have developed hary root culture of catharanthus Rosas and using imuno based detection they

could detect that VIN blastin in the herud culture detection

of in blastin UK group whereas the group from from

Netherlands or in USA they did not find any pin blastin

okay but then what they have found in hary root cultures hary root cultures are capable of producing some of the

early Indo alkaloids and also her cultures are also uh capable of

producing a few l IND alcol of the pathway which is for which is called

taronin so where tonin will come tonin will come here so I'll write tab sun in

now with the pen so let indol colors for

example tab sonin detected

but this is detected in herot cultures when they are grown

under dark condition okay but no vindoline was

found okay and some cathan thing are also detected

now let's see what is happening with the multiple shot cultures so the multiple sh cultures appears to be a very

promising system and here scientists they have detected

vindoline accumulation in substantial amount and not only

that one of the multiple truit cultures also reported

of Vin blasting accumulation in this multipot culture so now where lies the difference

so multip shot cultures they are basically uh when you when we are talking about shots shots cannot grow uh

in under dark condition shots cannot grow under dark condition so in order to grow shoots you have to put light that

mean the question comes here the light light plays important role in this vindoline accumulation or

not okay so when that is the case one interesting experiments were conducted by the group from Rice University us

so what they have done they they already developed very stable herot culture these herot cultures they made them

photoheterotrophic that means they slowly reduce the uh percentage of

sugar and instead they are giving them sufficient light illumination and under that condition

what will happen these her root cultures will turn green so their idea is

basically the whether green hay root culture are capable of producing vindoline or not

if they are producing vindoline then that is good so that may be a serve of serve as a good source for vindoline and

then catharin can already be can already produced in herot Culture by elicitation so if the green herot cultures produced

vindoline then then vindoline can be harvested but to their surprise GRE

herot culture uh are

not are not producing any vindoline instead they are producing the other

alkaloids so what happens that the green hay root when the

normal hay root cultures are grown in dark what I said they produces taronin and then they checked what

happens to the tabarin condition in the green hairy root culture what they have also found

that no taronin that means in green hary root culture taronin

formation is not there if tonin formation is not there vindoline cannot be formed so from there what is clear to

us that tabin requires a dark condition tonin synthes cannot synthesize so under light tonin may not

synthesize so that is why when hay root cultures are grown under dark it accumulates

Ronin but green hary root cultures cannot produce vindoline the reason is that it is not producing tonin so how

can pendolin will come okay so this is an important

point so uh next what we should see that what happens when plants are getting

light but that that basically uh that we will cover but uh I am not not covering that

aspect now because when I bring these issues here then that leads to the dissection of the late steps of

vindoline formation from tabarin to vindoline and Beyond so that I will look in one of the subsequent classes so what

I'm going to see now that let us go to the concept of cell culture so the cell culture failed to produce vindoline

catharanthine but they are producing aisin ageline serpentin all these things so these alkalides are also

important catharanthus Rosas producing

so on so next what they did the scientist they try to produce more of this in cell culture so how they can do

that so in one of the earlier classes I mentioned about elicitation so that means we will bring

that issue again here in this class and is elicitation

of sell and organ culture so this organ culture also includes herot

culture for enhancement

of alkaloids so here I will uh before I go to the

elicitation mechanism I will give you some good example of the Practical work so that means this uh one US group so

what they have done so as I said that they have a very stable here root line so

here root culture of catharanthus Rosas was was

made uh and was treated with different elicitors what are these elicitors These elicitors are are they have chosen one

is pectines another one is

kaitin and third one is jasmonic acid

so I will write here elic treatment now here something I have to

tell that this elic treatment how it to be done so once you have the cell culture or organ cultures growing and it

h it reach the lit this is l

list exponential phase so at that stage

uh uh elicitors elic elicitors can be applied and the duration may be for 48 hours and

then cultures to be harvested and look for the uh our detection

of oh my hand is sleeping mostly the hlc based methods are using

so they use TLC hlc dad and sometime they use Mass spectrometry to confirm the products so this is how it has to be

done so pectines is basically an endo galao unes so upon application of ptinus what happened that it increased the

level of taronin so

tabarin level increased

by 2.5 * than control culture okay upon application of kiteen

kiteen is basically a component of fungal cell wall so when you apply kiteen with the the system uh will think

that they are under fungal attack so they will induce the specialized metabolism and as a result you will see

the enhancement of alkaloids so here basically this lead to increase of aalin

level up to 50% when you compare with the control

when jasmonic acid was used so jasmonic acid is basically a signal uh transducer in in defense

response so it basically increase specific yield of

aumen by 80% and

Serpentine by 60% not only

that they have also found two alkaloids which are normally uh the late step alkaloids

which is called Lo nine l o c h i c i

n and or so I just check the lock net

Larin and hor hammerin so these are basically the late St of Indo alids which are produced only in Roots so they

also found that there is an enhancement of these alids as well when treated with jasmonic acid so jasmonic

acid was found to be the most suitable elicer for upliftment of alkaloids and this is interesting once you treat with

the El it the pathway uplifted so the enzyme activity can be studied and subsequently genes can be isolated so in

the next board I will show you what has what happens upon this elicitation so apart from uh this jasmonic acid uh even

the an fungal extract even East elicitors can also East exts can also used as

elicitors so we we also study the biosynthetic pathway and we have

also used the East elicit for our own experimental work particularly for upliftment of phenolic compounds so uh

so that means that East extracts filter sterilized East extracts uh or autoclip distex can be used as elicitor so what

happen so in this elicitation process so on that I would like to tell but before I will tell that what happens upon this

uh uh in the in that in the signal transduction process upon jasmon net treatment so there are two things so let

me draw this uh model for at least sitter

leading to s g

expression okay this is the plas my

membrane here lies the receptor so

where IIT molecu it come and sit over here

and that leads to wining of ion

channels and influx of calcium this is

cytool this is inside the nucleus so

here this is j e

r just more T response element then there's a tataa and

then the Gen for S Str is here so if it works that

means or from

lias through this octad deid pathway just monitor

synthesized so here lies

the Orca so

and and so what happens that

uh when any me apart from jmet any other elor is used okay say it could be fungal extract extract so that binds and that

opens the calcium channel and that calcium channels directly influence the ba area that is where the elicited

response elements where this one transcription Factor binds which is called CR BPF 1 so the CR BPF

1 CR stands for Cenas rosers BPF 1 this is catharanthus Rosas P box

binding Factor one homologue catharanthus Rosas P box binding Factor one homologue so this is one and the war

ca3 Orca this is octoid responsive catharanthus ap2 domain protein so this

again I will mention in the next class as well so so what happens there are two ways

one is via calcium channel it activates one transcription factors which bind in the elicited response element and other

one by the activation of the jasmonate pathway which activates the jaspon receptor and then protein kyes plays

role and then what happens that activates a specific transcription factor which is called Orca octoid

responsive catharanthus ap2 protein I will mention that later and that binds uh to the particular jasmon elicited

response element here and then as a result of that it initiates the transcription and we see that Str Str

genes are get transcribed so uh normally what happens sometimes the

specialized metaboli levels are very low in cell and organ culture and that level can be enhanced by adding the

elicitation so elicitation is basically activating the pathway so this is the early step uh and as a result of that

there will be more transcription leading to more enzyme protein and depends on the substrate availability It ultimately

leads to more product formation product

formation in higher

amount so I will end this class here and the next class we look into more details uh at the molecular level how

this uh this um specialized transcrip factors bind and modulate the process and once we

finish that then we'll move into the late steps of Indo alcco biosynthesis that will be the

subsequent classes so with this I stop here thank you very much