Elucidating the Complete Biosynthetic Pathway of the Alkaloid Cesin

[Music] [Music] welcome to nptn online certification

course on pharmacognosy and metabolic engineering so this is lecture 35 where I will discuss about the recent

discovery of cesin biosynthetic pathway so cesin cesin is basically an alkaloid

Amin alkaloid it is produced by the plant called gloriosa Superba s p e r b super bar

and another species called Chum autal I put species which

basically produces this alkaloid now why should we study cesin cesin has Cal applications including and including

pharmaceutical applications so the most important one it is used as a drug for treatment of goer that is a severe form

of arthritis and not only that cisin is basically used for cytological studies

in order to make uh polyploid uh in order to induce polyic

cesin is used so cesin plays an important role

uh for not only for the pharmaceutical application but but also to De for the fundamental plant

biology okay now cesin the structure of CES is pretty complex and uh Only The enhan shio

Selective synthesis so I put a color enhan

[Music] Co selective synthesis was only achieved in

that of in the year of 2017 but it was so complex that it cannot be applicable

at in industrial scale production it cannot be applicable to

produce at industrial scale therefore the demand for cultes in to be made

from natural resources available and that sometimes makes

the supply chain unreliable so

therefore there is a need for understanding the culture

biosynthesis uh so that uh later the molecular tools can be applied leading to more production of cin in

automl or in in gloriosa or in other or in some model plants also maybe in future it is possible to produce cusin

dinovo in another eukariotic system such as East

so uh now what is the uh what is the status of biosynthesis okay so in order to study the biosynthesis one needs to

know uh or should have the idea of a precursor molecule

fortunately for cesin such molecule was indicated by the organic chemist so scientists have

chosen the precursor molecule or the start molecule

as one f e

eile ISO quinoline one feny isoquinoline so this

was uh considered to be the uh important precursor for the formation of uh cesin now how this one Phile isoline to

be produced in Plants that's again another question so we will address that a bit later let's go to the next slide

so next slide is that the cesin biosynthesis

thesis so this has been done by uh so the by the use of uh by combination of

knowledges by combination of transcriptomic metabolic

logic and pathway

reconstitution so these are the important steps uh to be considered in order to

understand the cusin biosynthesis okay now now when we talk about the transcriptomic what they have

done that one thing was clear to them that cesin accumulates in the in the ryome that is the underground

part so r that is the underground part of gloriosa

Superba so what they have done they have done a comparative transcriptomic study d n c

i tox of leaf

steem Rome okay and then what they have done from their uh they try to study the

differential gene expression we try to understand the differential and this transcriptomic was

denovo that is no information available earlier about the transcription profile of this plant so

from there they could get an idea that what could be the candidate

genes for cisin biosynthesis so that means the

gene expression analysis will tell you particularly as I said that as the ryome is the part so definitely when you

analyze the uh differential gene expression in ryome you'll be able to find

several uh transcripts which are not present in the other part now these transcripts is properly analyzed from

there an idea can be generated that what will be the potential genes or enzymes responsible

for the pathway so actually that was the starting point and then uh once they come up with the idea that this could be

the enzymes and then they basically work out the chemistry putting the putative substrate and putting the enzyme and

there what could be the product formation so again for example I have discussed long

back so as I have said the substrate more or less is known from the organic chemist

from the study made by the organic chemist so and say you have discovered the

enzyme Z enzyme

y enzyme X that means the subsequent the genes are Gene X

Gene sorry so the X gen will in X Gene en code enzyme X maybe y Gene en code

enzyme Y and Z Gene and code enzyme Z so then once they understand this one that is

what are the putative candidate genes XY Z then then what enzyme they will encode that is XY Z enzyme and then if such

enzyme is there say methy transfer so you know a substrate then if a methy transported work then it will make uh

potential product so which could be B okay then there may be a hydroxy for example which may be involvement of Cyro

450 so the product will be a some sort of hydro hydroxate product so that will be C and similarly D like that way they

theoretically created the concept and once they created the concept then they uh characterize this genes

they uh clone it and they studied the functional expression of these genes individually in an nuarc system to

confirm that indeed Gene X converts A to B say Gene y converts B to C and then g z converts C to D So based on all this

knowledge uh what they have come up is basically the pathway which I am going to put it here so one one pheny

isoquinoline as I said that is the starting point so let us put that one that is the part of I should what

will be the heading The Heading will be part of part of

the biosynthetic pathway

of cusin so one

F eile ISO

quinoline Q UI okay the board is withit disturbing [Music]

q [Music] q

[Music] new q u i n o l i n e so this will be converted

into several uh step reactions and finally it will produce a compound which is called

s a terminal so s a u t

u m n a l i n e is automine so aamal is the species

from there perhaps the name given is aalin now this involves six

steps and including

four methy transfer ises and one p 450

enzyme and in The Next Step this by the action of one unusual enzyme or one unusual reaction that is 1

p450 that job was to do the phenol coupling p h e n o l phenol c o u p l i n g coupling it

makes the product which is called ISO Andros simin

ISO andro s

bin now ISO Andro s bin subsequently by a two-step reaction uh where uh it's basically two

steps so one is basically one methy transference another is one

p450 and that this is called basically the uh ring expansion and this leads to the

formation of in

horile d d

Callin All Greek names in formal dein

so it was uh subsequently predicted that in formal D Colin will be converted

into Colin through subsequent steps which may involve

uh uh demiles a deformes okay I think Def mileage we should write before which may

involve uh du for my

then dethy and in atile

transfer transfer is leading to the formation of our

Target molecule which is cesin

now this part is still not confirm at the molecular level yet to

be confirmed but up to

in formal deos cin this is confirmed so that means after to This Much from

here this is called the near

completion step of cesin

synthesis so this paper I will discuss again the steps a little bit more detail this paper published in the famous

journal Nature in the year of 2020 volume

500 84 page number 140 8 to

153 and the first author is netol n Ryan s

netol and this was published from Stanford University and this paper is so

impactful the editor asked another scientist with a similar expertise to write a News and Views article so

uh News and Views article also published in the same issue of nature

2020 volume 584 page number 49 to 50 so and they

have given a nice name for this News News and Views article that is the chemical Kno

how of the sorry

of the flame Lily so to sum up what I have

said that the starting point for net and his colleagues is basically based on a proposed biosynthetic pathway

uh given by organic chemists several years back so there what they considered that

one pheny isin is probably the starting point of cesin biosynthesis and this one pheny isoline

is probably derived from amino acid phy alanin and tyosin and this intermediate that is one fthy

isoquinoline this to be converted to n form demolin in N step

reaction the first intermediate that contains the molecular scaold of

cesin so this conversion basically involves two unusual bioch chemical reactions so number one is the rare

transformation which is known as phenol ring coupling so which

is this one and and on precedented reaction that leads to the expansion of the molecular

ring so that is somewhat unusual reaction which was not reported in the plant system before

so that means pheny isoquinoline is initially made from amino acids Phile alanin and

tyrosin it is then converted into aalin in six steps by four methy transfer and one cytochrome p450

enzyme uh then two other p450 catalyzes the unusual biochemical reaction f one is a phenol coupling and the second

one uh phenol coupling and as a result of this first one reaction it produces uh

ISO Andro simin and then a ring expansion which

produces in for mile demolin now a fif methy transference is also needed to convert

ISO andrin into the substrate for expansion so the enzymes for the final three

steps in which the Nile demin is thought to be converted into cesin is not yet confirmed at the molecu level but it was

predicted that deformes dyes and in atile transference will do the job so okay now this is in

brief what has been elucidated by net and colleagues and this paper published in nature what I said now let us go to

the uh some of the early steps of the pathway now uh if I put this in a sequence I

will not put I will not write the name of the each and every substances because it will be

rather pretty heavy so to start with say I what I said that the one

F eile ISO quinoline so one feny is Queen so this

is considered as a one that makes two two makes three three makes 4 four makes five five makes six then six

makes 7 7 makes 8 8 makes 9 9

Max 10 so the enzymes

are omt1 then nmt

then cyp 75 a 109 that is hyron p 450 then again

omt 2 then again a cyp 75 a 1 109

then omt then again Cy P 75 a 110

then omt 4 and then again cyp 71

FB 1 so these all constitutes the pathway so

the last one is the uh

in for mile D

Colin the penultimate step was O meile

and draw sing and that of previous one is number

eight was ISO Andro so

in and then the seven was autom nin and then subsequently previous steps so this is all the steps

so these are all characterized now now once they have characterized

this the next point was basically they want to check the biochemical function of all these candidate gen so they have

used the they confirm the biochemical function

in of the enzymes encoded a function of the enzymes

encoded by the nine candidate gen so there here they have used the

model plant which is a relative of the tobacco nikiana

B Manana so what they have done they have transferred

all nine genes

by Agro filtration along with that they have

done feeding of substrate which is the one which is uh

one fin ethy

ISO quinoline and

and then what they have done so then they and uh they check for the production of this uh product which is

the in form Mile demolin and all genes worked well and as a result of

that uh what they found that that there will be the uh they achieve total biosynthesis of so

what I put it in another color uh

upon transient expression

in nikiana B Manana Leaf they found

total biosynthesis of

in form mile D Colin

Callin call so that means

successful reconstitution of

cesin biosynthetic pathway in

osana Bena Miana now they are not happy with this

they wanted to also Express the early genes of the pathway that leading to the formation of the first product that is

one fenile isocon so what they have done in this this case they have used three different

modules so in the next one metabolic engineering

of ctis pathway

in osana v

m so what they have done they also inserted the primary uh me they they aim

to they aim to achieve that the primary metabolic products will be utilized directly by the plant so that it

produces the uh substrate uh which is the starting point

for the cesin pathway so they have used three module so in the module one where they have done so they have

taken several enzymes from different sources including gloriosa Superba which starting with DH P

synthes that is the starting point of this uh cumate pathway and eventually it makes phy

alanin and this subsequently by the action of multiple enzymes for

example GS spel uh GS 4

CL uh GS a e r

GS c4h G uh GS uh

CCR that leads to the formation of 4 h DCA so 4 H DCA and uh here one thing I would like

to tell that is this okay I'll tell later so this is

module one now they have also done the module

2 where El tyosin was taken and

GS d t r DC or D

DC they have taken another plant from another source BV CP

76 85 this is from Barbarian Volaris and that ultimately makes

dopamine 4 HDs 4 hdcs stands for four

hydroxy dihydro Camal deide that is why the CCR was

used okay and AER e stands for alkenal reduc

so now the module two completed so

this and this will join together by the action of

CJ NCS Nolin synthes and that leads to the formation of

one Fen eile isoquinoline

so this subsequently a module 3 which

involves so many enzymes which I have already listed before so I'm not putting all the

enzymes so n and eventually it makes

n form mile D

Callin and this subsequently by three successive

reaction eventually will produce cesin so what I said that these three

steps are not characterized yet but the penultimate product of cesin has been achieved so that

means the they characterize the nine genes from gloriosa suburba using transcriptomic metabolic logic

uh as well as the uh chemical as well as the PA pathway

reconstitution so pathway reconstitution they have done in uh nikiana bamana where starting from one Phile

isoquinoline until the formation of De form demolin and what I said file demin will be converted into cin by three

steps which have not yet characterized at the molecular level now uh uh but it is confirmed that this is the

near completion of the pathway so as I said the genes were characterized from gloriosa

Superba and functionally expressed individually uh in the another hus system then they put all the genes

together and they reconstituted the pathway in nikiana and then what they did they

also reconstitute the whole pathway starting from the primary metabolic products that is glycer phosphate and

arthros phosphate leading to the formation of uh dhp by putting dhp

synthes uh from gloriosa Superba and subsequently the genes for the simate pathway leading to formation of Phile

alanin and and then several larly gen of Phile propanoid including pal 4cl CCR c4h and AER that ultimately led to

the formation of 4 hdca that is four hydroxy dihydro Camal deide and this joins with dopamine now dopamine also

they produce through module 2 by putting the genes of dopamine formation hiding from gloriosa as well as from barbaris

that produces dopamine now this dopamine and hdca joins by Narco cloudin synthes and uh which they have hired from CJ

means coptis Japonica and it makes one FY isoquinoline and subsequently all all nine genes inserted and that makes the N

formal D Colin that means starting from the primary metabolism the end product

achieved so this is really an wonderful work and that is why this paper published in

nature so we have to wait for few more years when three uh uh penultimate and ultimate enzymes will be characterized

so that whole cine can be produced and that time maybe dinovo cine in East such interesting papers will come out in

years to come with this I end this class thank you