Decoding Strictosidine Biosynthesis: Enzymes, Pathways, and Biotechnological Insights

[Music] [Music] welcome to nptl certification course on

pharmacognosy and metabolic engineering this is lecture 33 so where I will narrate the recent

discovery of stricking biosynthetic pathway and this discovery was reported again in the famous journal Nature last

year that is 2022 so let us go so the concept covered simply I have written stck Nam

biosynthesis deed that means how stricking is biosynthesized in plants so let's go to

the slide number one so our topic of discussion is bio synthesis of

stricking now what is stricking stricking is basically a

poisonous alkaloid obtained from the plant called strios nox

bom I'm sure most of you or almost all of you are aware about a popular homeopathic drug nox

Boma so the KNX Boma the Homeopathy drug is basically obtained from a very very diluted extract either seed or root

extract of stnos noxb plant so because of this [Music]

uh poison poisonous nature of the alkaloid that actually fascinated the scientist or inspired the scientist for

the past 200 years to determine the complex skeleton of this molecule and they

reconstructed the molecule in the lab so that means the synthetic organic chemist

partly made the skeletons in separate event and then

they finally joined and made the synthetic stricking but that is not uh uh

commercially feasible if you want to make stricking because the the organic synthesis reaction are so complex and

expensive and at the same time it was not known that how in the nature plants synthesize stricking so that remained a

mystery until recent now so scientist have solved this using uh chemical logic by

using chemical logic and a

study uh of messenger RNA of

stnos Nas bom eventually uh L

to the uh or help to decifer the

whole pathway of

strict s sorry strict in

biosynthesis operating in plants so this discovery again published in the

famous jordal nature in the year of 2022 uh by Sara Conor's group so the volume

is 67 607 and page number 617 to

622 so the first author was Hong hongal and the group leader was again

Sarai konor from this time he moved from johnis Center to the max plank Institute for chemical ology in Germany so the

paper basically uh the affiliation given to this institute

now so we are going to see what is it okay so let's go to the okay

uh before that I will also tell few

things because stricking is a bioactive molecule it derivatives could have pH pharmacologically useful properties

without being poison so why this is important because once you know the biosynthesis of St

stricking once you know the enzymes and genes of the pathway and once you can synthesize the stricken in the

laboratory then it is possible to make deriv derivat or derivatives of stricking and it and these some of these

derivatives may not be poison but have more pharmaceutical

acceptance so knowing the biosynthetic roote could help the chemist generating the stricking scaffold

and possibly will be making in future such derivatives using the synthetic

biology and what is basically the ultimate rational for doing this project

okay now let me go to the pathway okay now another interesting point I tell before I go to the pathway that once

this paper was published so several news agencies they took interview and one of the interviews the Hong who

actually the senior post doctoral researcher and the first author of this paper what is said that they basically

failed to produce stricking because that was ending uh with the formation of uh

preening so I just write it here pre strict p r e s Str r y c h n i n e so spr pre- stricking is the

penultimate molecule of the pathway and from pre- stricking stricking will be synthesized and what I said that this is

a very toxic molecule okay so this

conversion that means the enzymes responsible for this conversion uh they fail to really

identify the enzymes or they fail to convert preing to stricking finally what they said that

when they left the pricking in the laboratory in one Friday and by mistake or whatever maybe and

when he came back in next Monday and started seeing that before throwing it out that time they found that this PR

stricking got converted into stricking that means the some amount of pre- stricking got converted into

stricking that means the solution containing the pre stricking when they analyze they found that some form uh

some portion of prein got converted into stricking that means from there it was clear to them that basically uh

spontaneous conversion is taking place which may not be

entic so may not be enzyme and eventually by doing several experiments they indeed confirm that this last step

is basically spontaneous in nature so with this now let us go to the next page so the

biosynthesis so how I will tell this first I will tell the basic biosynthesis uh what has been discovered

and then I will tell that how they have confirmed this biosynthesis using nikiana

species so again this is actually the part of this indol alkaloids because the the

intermediate we will start from gasio siine so let us start from there gasio sizin

gasio sizin will be converted to through a unstable intermediate which

is dehy dra okay so don't worry student you don't have to remember this difficult names this is just for your

information and that makes n

fluro curin are a

ren so the steps

so the enzymes are snv stands from strand for stck no

knb go gasio in oxidase so they have used another species so that they have mentioned in the paper that is stto in

species so they put it in this way SP and this they are also go uh they have also confirmed now the second species

they that is not producing stricking but producing another compound okay so this is number

one this this is number two and then finally uh from

this uh by uh two reactions this nor flurin is formed so therefore I put two arrows

here and the enzymes are snv

ns1 and snv

ns2 that means nor fluocin synthes one nor floc in synthes 2 and this in the publication they have marked as

four I'll simply follow their uh process so this in the next step we

will be converted into 18 hydroxy nor

neuro curin so this was marked as five and this in the next

step will be converted into to a compound called we

land gich alide

and the enzyme is is snv

Ws that is we Le gich synthes so this they have marked as number six this now this is one of the major

intermediate from there one pathway emerges leading to the formation of a compound called

diabol this they have given the name uh number

eight okay and the root which

moves towards formation of the formation of uh stricking that makes next product as

restrict pre stricking uh they have marked as number nine and pre stricking in The Next

Step makes stricking

this they have numbered as 10 and as I said this one is pre strien to

strien this is a spontaneous it releases a molecule of carbon

dioxide along with a molecule of water and

this produces stricking along with ISO

stricking so this number is 11

now the enzyme responsible for this reaction that means we and kich alide towards pre stricking that is

snv 0 atile transfer so next uh this trickin will be converted

into uh 10 hydroxy stricking and that in the next step will be

converted into beta

Colin beta Colin and the enzyme responsible for this reaction

is snv 10 H that means 10 hydroxy and this one is

snv omile transference and the next one

converts this to 11

dthy Bruin and that finally converted

into Bruin so Bruin will give the name

15 and Coline will give 13 okay diabol I'll use a different we can use a different

color sorry diabol in so when I put diabol in better I use don't use this

one okay so diabol accumulates in another species not in strict n buum but another

strict species where stricking is not produced instead it produce daolin that means there the pathway moves from whe

and gum aldhy One Step by a acetel transference it makes daolin whereas in case of strios no Muma the pathway moves

in this direction uh towards the formation of sprin and stricking and then subsequently it produces the other

alkaloids now the enzymes for this last steps I must put it that is the last one the

Bruin formation this is also snv othy transference and this one is basically

snv 11 hydroxy

and what is Bruin Bruin is basically the D meox related derivative of

stricken so Bruin is basically the D

myoxy derivative of

stricking and it accumulated in the roots of strios found

in stnos nox bom

Roots so this is in brief the biosynthetic pathway leading to the formation of stricken

Bruin and diabol in the other species so they have have used both the species for this study so basically they have done a

very detailed transcript analysis from both the species and then they uh

did uh indepth uh Gene annotation and

subsequent Kake pathway analysis and whatever analysis required that is beyond the scope of discussion in this

class and from there that then leads to the the discovery of these enzymes now what I am I am straight away writing

that SNB go SNB ns1 but what is important after this that this needs to be demonstrated in an another

heterologous system to confirm its function so and uh just for your information that

uh the gasio is basically it's coming from the indol alol pathway which started

from gpp and cpop hand if I say at that point so gpp is basically the tarpo

route which ultimately makes aanin and tryptophan is coming from the sikim route and that ultimately contributes

towards the formation of strictosidine and then gas ozine is formed and then the reaction proceeds

okay now there are some interesting uh observations which I need to tell you so first of all so let us go

to the next slide so now the validation of the function so what we write

that valid ation of

these genes or rather we should write validation of

the functions of this genes

so for that what they have done they have used as uh as I said the heterologous species that means they

have used uh nikiana nana n i c o t i a n a b e

n t h a m i a n a so that that means here they try

to reconstitute the

stricking bio synthetic

pathway so uh so they achieved what I I should write it they

achieve transient expression

of all enzymes that is starting with s NV

go comma s s NV ns1

snv n o snv

WS this is w please WS then

snv at then

snv 10 h and snv

omt okay that means they have transformed nikiana vamana plant with all the genes

that means the pathway will be operating but now you should ask yourself whether the nikiana benana will

produce the stricking or not what will be the answer yes or no no it will not produce because nikana

Bentham will not get gasos okay gisin is basically the product of the Indo pathway that is not

operating so then what needs to be done what needs to be done that you have to take a

transgenic Leaf expressing Nikos Bena and there you do infiltration

infiltration of gisin is s s o s c h i z i

n and and also you require Malon net source and D sorry simply iite and malonic

acid that was supplied in the form of disodium malonate so when you infiled in a live

plant simply inject it and allow the plants to take it up then after one week you

harvest and check for the check for the

alkaloid contents and what you will find or what they have

found is basically they found the presence of

stricking so I write it here presence of

stening ISO stricken

then Coline and

Bruin so they found this in the so when you find this this but you

see I have kept a I have kept a blank space here actually what happens that when

they transferred all the genes it did not produce the stricking colan bruing products so what happened

basically that the malonic acid what you are supplying so that malonic acid will

joins with a qu there may be and then it forms a malony Quay and melano Quay should be acceptable to the

sv80 that means the sv80 which is the

this step this step so is in order to have the sv8 function you need to have

the uh you need to have melony qu added in it so okay then only it make the pre-

stricking now what happened that snv at when you put the snv at then SNB 10 that means the all

the genes of the pathway uh it is not producing the stricking if stin is not produced it

cannot make beta Coline or rusin so then they found that basically the snv is not getting the accession of

the mile KO so in order to get that what they have done they have transferred here a gene from arabidopsis taliana so

which is a a AE that is added here so at AA is

basically asile activating cytosolic enzyme this is basically the asile activating

cytosolic enzyme that produces melony KO accessible to

cytosolic that uh that

produces melony qu accessible

to cytosolic snv 0

okay so that means uh a a a to be added here in the pathway and then the pathway will function and eventually it makes

the stricking okay so basically some alony transfer is is there and uh this finally

makes the thing so uh and therefore now what we can say the H ously reconstituted pathway I just write it

here one sentence the

[Music] ously re

constituted pathway matches

matches the physiological sorry matches the

physio logically relevant

Sol relevant pathway

in strios KNX

H plant okay so that means they have reconstituted

the pathway and it basically produces the

product now so uh to make this story uh short so what they have done that

initially they are not they have not transferred all the genes together they have done stepwise so they made it in

two combination in one combination they have INF filtered say uh spgo SP ns1 SPN and spws why that also I need to tell

because what I said that that is an the stri no species other species which is producing diabol but not stricking for

that what is required that if you go to the pathway if you go to the pathway suppose the pathway uh is meant for the

formation of uh diabol in the other species of strios then what we need to show here is this that

SPG then SP ns1 so there will be another SP ns1

then there may be SP n o which I have not shown okay and then there will be

SP WS right so if you

take these genes right and and of course this one then what we'll see that it produces the or if you

if you don't take up to this if you take only up to this much then what will happen this

basically leads to the formation of whe and gum alahh that

means expressing SPG spns1 SPN and spws leads to the formation of whe and gam alide so first they have confirmed so in

the another uh another combination what they have done they have taken SPG spns2 so that means uh what you can also

write here by putting a slash here that is sp ns2 so this

one for example I will use another color Okay color is not sufficient

this one so uh the second combination may be this this then

this and this and along with this that leads to

the formation of daolin okay so in order to produce the like

this way they have checked the uh formation of this short pathway which is operating in one stri no species and

also they have checked the formation of Will and galich aldhy which operates in both the species and next what they have

done uh The Next Step they have added all the genes what I have shown in the next slide that all this and eventually

they express all these genes in osiana Bena this leads to the reconstitution of the stricking pathway and still it did

not produce stricking unless you add a a AE asile activating cytosolic enzyme that actually allows the malano qu get

accessible to snv so only that is only one enzyme you have to add from another source uh it

because it is a Nana system it's not the strick noos so whatever you wanted to do plant will not

do okay so they are CL so you have to conquer so that is why they have conquered and eventually they made the

stricking and this stricking what they said this heterologously reconstituted pathway matches the physiologically

relevant pathway in strios KNX Boma so this is basically all about it but I thought that I will draw some structure

but I keep it in for the next class so next class I'll make a brief overview of whatever I have taught in this indor

alkaloids um in all this Starin noid indol Alid along with the uh stricking so this all I put a brief overview with

slides and showing the I'll show you the plants how they look like and and then I'll will show you the schematic diagram

with the enzymes and genes and also I'll show you some hemical localization some uh what people have done as well as the

uh immunochemical localization immunohistological localization of this enzymes all these things will come in

the next class with that I will end the uh Indo alol and once I finish that subsequent class I will talk about

another interesting alkaloid which was also discovered in 2022 the pathway which is called

cesin and once I finish the cesin all alcco will be covered so with this I end this

class thank you