Metabolic Reprogramming in Catharanthus Roseus for Non-Natural Indole Alkaloids

[Music] [Music] hello welcome to inline certific online

uh nptl certification course on pharmacognosy and metabolic engineering so we'll now go to lecture number

31 where I will discuss the concept of metabolic reprogramming uh particularly with the

monotaro Indo Alid biosynthesis so pernal is basically the English name of catharanthus Rosas let us go to the

concept which I'm going to cover first of all I'll talk about the silencing of tryptamine biosynthesis for production

of non-natural alcohols next I'll will talk about the con concept of metabolic reprogramming and finally uh we will see

that how metabolic reprogramming can be applied to catanas Rosia culture system so as to produce non-natural indol

alkaloids so let us go to the board so the question is nonnatural

indul alkaloids let me go to the previous one okay I'll put the indol term here

because we will talk only with the indol okay non-natural alkaloids means that the the alkaloids which are not produced

by the plants okay uh so for example uh triamine tryptamine is produced by the

plant but what about flot tryptamine or chorot triamine so this is not produced by the plant but

flot triamine may be useful but because that subsequently converted into fluro strictosidine and further Downstream

products and eventually a late indol alkaloid having a fluro substitution uh could be more

pharmacologically acceptable than the concept than the conventional one therefore there is a need uh for

exploring non-natural indol Alid production in the plant system so this is all about our topic of discussion so

under this context I will talk now the silencing of tryptamine

biosynthesis for production

of nonnatural alkaloids

in plant culture in fact this is the title of the publication

where this work appeared so this was published in This Journal called pns I have mentioned it several times

proceedings of the National Academy of Science USA this published in the year of

[Music] 2009 and volume number 106 page number

136 7327 8 and the group leader was again Dr

Saray Conor which I mentioned her name several times so what they have

done they what they have done they have silenced the tryptamine biosynthesis so how we all know that

tryptophan makes tryptamine right by the action of the enzyme which is called

tryptophan decarbox okay so and then subsequently tryptamine joins

with uh seanin and makes the alkaloid which is

strictosidine and then I have mentioned several times I'm not going to uh talk again about it

and about it so this is a CO name and then eventually it makes all the downstream

products so this one that means [Music] the

TDC so to silence tryptamine biosynthesis what needs to be done is basic Bally to block the TDC activity

and this group they have used RNA interference approach to block the tryptophan

decarbox and as a result what is going to happen so again they use the system as a plant culture so they have used a

hiot system this is because that is a organized culture stable system so they have used again the co

transformed they they raise the co transformed here root culture expressing and

RNA having the uh blocked TDC that is the having the construct uh that means with the TDC with the rni

construct or rather called hairpin construct that is the best word to use and that basically leads to formation of

this uh short interfering RNA and eventually it blocks the DDC

so trans genic har Roots will not produce

tryptamine okay but this will not hamper its normal growth so here root grown very well even with the blocking of

TDC okay now if the TDC is blocked what happens to the downstream enzymes whether Downstream enzymes will be

disrupted or not so as this pathway has already been characterized what scientist they did actually they uh

check the expression level of the downstream enzymes and what they found that Downstream enzymes are perfectly

active that means the MRNA for Str Str was produced mRNA for SGD was produced and

subsequent but this pathway is not operating or rather operating in a slow pace because the tryptamine supply is

interrupted so then how to use this culture so two ways this culture can be used one is that you can

do a feding so feeding of normal

tryptamine when you do the feding of normal stpine then normal Indo pathway will be

restored that is fine but this is not really very challenging so what happen Okay block a pathway at the uh add the

substrate and the subsequent enzymes will work so that is fine but it is nothing that much great

to to discuss or to uh make more of sensation on it so what they did basically they now did an uh outstanding

experiment so they did feeding both feing of nonnatural substrate

non-natural tryptamine what are this non-natural tryptamine for example they have they

have done feding of fluto tramin so if you use uh this fluto tryptamine uh then what is going to

happen that we are going to see now yeah fluro means Florin so that means feeding of non-natural

tryptamine that means you feed uh so let me go to the okay this slide I will use so this

time no pin uh

root with HP so these hadot cultures you have done

feeding with fluro tryptamine so when fluto tryptamine you

fed or what the when what they have done that means that if we if I put it here

what is going to happen that all

this inal colid pathway will remain will be active again sorry

uh then so so this leads to the formation of

fluro strictosidine so I will erase this F strict toid in and then

eventually it will make all the products for example you'll be able to

get fluro uh AG

Malin fluro serpentin

fluro catha ranin

even luro taronin so all these nonnatural Indo

alals can be produced so two things number one when you feed normal tryptamine normal pathway uh

normal pathway restored and when you feed the fluto

triamine that leads to Restoration of the uh that leads to

the formation of the new pathway leading to the formation of

of non-natural Indo alkaloids so as I said that these

non-natural indor alkaloids are sometimes better than their natural counterart for pharmaceutical

application so that is why this work has very significant impact uh for future uh

industrial application so this is basically in an Essence

the work described in this pnas paper from this concept we'll move into the metabolic reprogramming so what what is

metabolic reprogramming and this they have done in catharanthus

Rosas culture so the system was head root culture but this is what they put it in the title

so here let me explain uh this metabolic reprogramming concept for

example uh there is a pathway where a makes b b makes C and C makes D and the enzyme responsible for this

are enzyme X enzyme y enzyme Zed okay so the green denotes

enzymes and the blue denotes the so a is the substrate earlier substrate and D is the final product

now it has been found by the uh scientist that a d is important but a substituted D may be

more important that means If instead of D if one can produce a substituted D so this

is called substituted D so I have denoted with d

Prime so that makes a better drug than

the D so how to make this so D prime it is possible to

make uh one is that that uh then all the enzyme has to be changed or one way to think about it

that if we modify the pathway if a makes B if somehow if you can modify the pathway so instead of B if it start

making B Prime that means there must be an enzyme which

makes B2 B Prime and then you know it makes a to B Prime and

then B Prime will subsequently okay I'll use a different color to indicate that V Prime and V

Prime will be subsequently converted into C Prime and

then in principle to D Prime so that means there should

be enzyme y or Zed or or some

modified y or Zed if it can function now what happened the scientist did one

thing they did the feeding feeding the plant culture with B

Prime and then grow the culture and then check whether C prime or D Prime is produced or

not and when the culture uh when the cultures were fed with B Prime surprisingly what has been

found that these cultures were able to produce uh both C Prime and D

Prime but B Prime is very

expensive limitation to this procedure is this B Prime is indeed very expensive therefore uh this may not be

feasible so then how to do that so this so that means feeding B Prime into

the culture will fulfill the purpose because the enzyme y May accept the B Prime and

makes C Prime and the Z accept the C Prime and makes D Prime and D Prime is our product of Interest so only the hard

lies with the B Prime because B Prime feeding B Prime is not feasible therefore scientist thought of

making B Prime through the enzymatic reaction now how it is possible uh so the point here what I have discussed

that e makes

B by the enzyme X now if this enzyme X can be

mutated and the mutant enzyme if this mutant enzyme say the

mutant enzyme what will say the XS X Prime so this this mutant enzyme can

accept the substrate a prime which is eventually not that

expensive so that means the substrate e Prime substrate

is cheap so if the X enzyme is mutated and the mutant enzyme X Prime can accept a

prime substrate and which can easily be uh supplied through feeding so that it can makes the product B Prime if that

happens then the job is done then what is going to happen these subsequently

convert B Prime to C Prime and C Prime to D Prime that means what I mean to say that the

plant culture has the natural ability for this conversion

to to transform

B Prime to D Prime

through C Prime so that means

the enzyme Y and Z will do its function so the hardle what was making B Prime is

now sorted out by making a mutated enzyme of X which is X Prime and when you insert the X Prime enzyme into your

target plant and Supply a prime as the substrate then a prime will be taken up by the transformed or transgenic plant

expressing X Prime and subsequently it makes B Prime and B Prime makes C Prime so this is basically the concept of

metabolic reprogramming now keeping this concept in mind now I will tell the the metabolic reprogramming of

cus cultures so what is here again the normal

pathway a makes B makes C makes D and what I have shown the enzymes

are x y z and this is what is the normal pathway and

the reprogrammed pathway what will will what we are going to see is this

that if Prime will be converted into B Prime this will converted into C Prime this

will be converted into D Prime and if you put the arrow

so this is the X Prime and these two are the normal Y and Z I think with this it is clear and a

prime needs to be fed

feeding required I'm not writing more now now come to the real example the X enzyme

which is denoted here with blue X is basically the stripto in synthes which I have mentioned several

time St Str and uh X is s so we all know the S function

what it does it conver or it it it joins two thing one is

tryptamine and another is is cyanin and forms strictosidine and then from

strictosidine the pathway moves and formed different

[Music] indle alkaloids now s Gene has been mutated

and they made the mutated S as X Prime so what is X Prime X Prime is nothing but

the mutated s so what is this

mutation this mutation they have done by

uh changing the valin into methionin so the mutated ex uh X Prime is called

uh s Str

V 214 M that means that the 214 position of

the amino acid of the St Str protein valine was replaced with the methionine that is a single amino acid change which

makes a recombinant s year so they have done a series of experiments for 7 8 years and finally they are able

to uh find the most suitable one is basically this one so this St

Str uh that is this particular Gene what they have done they have transferred

or into cus culture and the approach was Co

transformed y root y

root culture so the cor transform herot culture expressing v214 M St

now question is that what happens to the normaler question is what happen to the normaler you are not disrupting the

pathway you are not disrupting the pathway so the normaler is there oh sorry I'll use a

different color to make it okay so the normal is tier is there X is there what you are doing you

are putting an additional copy of mutated Ester so this is the normal Ester this is the mutated Ester both you

both you are putting in the kathas Ros root cultures now you imagine the situation what is going to happen when

normal s is there the plant naturally producing tryptophan seanin it joins and it is

making strictosidine that stto subsequently converted into strictosidine

aglycone then through stanine it produces ultimately cenin and some of the

maybe some of the other alkal maybe vindoline or maybe the hor hammerin or loin all which are root specific

metabolites okay now you are putting additional copy as the mutated one so the mutated one normally the gene will

be expressed press but it will not function because it is not getting the substrate so what you need what needs to

be done that this Co transform transgenic hary root culture needs to grow in the medium normal medium but you

have to add the a prime what is a prime a prime is basically what are the what are the

substrates for S Str one is seanin one is tryptamine so what happened that here you add the

uh so they have also found that cyanin it did not have to do any changes so the tryptamine accepted site got mutated so

here you have to feed either floro so oh sorry feeding

of fluro chloro and uh and uh and what is the

methy methylated and another one is

uh bromo that means

that means flot triamine bromo triamine chorot triamine or methylated tryptamine if

these synthetic substrates were fed in separate cultures then what will happen

these will be accepted by V2 214 M leading to the formation of see uh

chloro strictosidine bromo strictosidine methylated strictosidine or floro

strictosidine and then that subsequently will be converted by the normal enzyme of the

pathway so what I mean to say that normal strictosidine as well as chorost strictosidine

both we will both will work side by side so that mean normal strictosidine is produced as a result of normal EST

chloro strictosidine is produced because you fed chloroamine and you have v214 M so it

produces chloro chorost strictosidine and say it could be floro also and what I what I am saying that both the pathway

moves side by side without interfering each other So eventually you are going to get the normal indol alkaloids what

you are supposed to get from the root cultures along with that you will get substituted Indo alkaloids which may be

more effective for pharmaceutical applications than the normal one uh this is the beauty of metabolic reprogramming

here both the normal pathway as well as the substituted pathway both are operative

only the changes what you have done at the very early level where the S Str was mutated with by v214 m and the mutation

happened in the site where it accepts the tryptamine so it no more accepts the tryptamine but it can accept the

chloramine so in this what uh what is that that it the pathway have uh the pathway has relaxed the substrate

specificity so that means what you write here one point let me write it for you all that

this V 214 m s Str that

has relax next substrate

specificity that means if you do if you take a normal root culture having strictosidine and there if you feed

fluro fluro bromo it may not accept okay or is the acceptance that will be very less but with this this

recombinant enzyme it basically relax the substrate specificity so it is ready to accept all sort of changes and makes

their subsequent derivatives so this is basically the beauty of metabolic reprogramming and this work demonstrat

the basically the capacity to make new molecules through a precursor directed approach so this also another important

uh phrase c r s o r right PR sorry

precursor directed approach so this work published again in

this famous Journal which is uh nature chemical biology so

uh Net G bile again led by it's a bithor

paper uh by Ruan and SAR konor so this paper also published in 2009 March issue of natur chemical

biology Volume 5 page number 151 to

53 so the authors are r u n g u Ruan uh Ruan maybe from Thailand and

okono so is basically idish

so they are the authors they published this paper and this paper was so important that the editor of nature

chemical biology asked uh two scientists of similar background to write an article for the non-specialist

readers and uh the article published in the same issue as alkaloid biosynthesis takes root and uh there beautiful uh

diagrams are given so that means so I will end up with uh that uh schematic diagram uh no I'll will not take much

time so the uh V 214 m s Str plant is expressing this and when

you are doing feeding of

chloro Camin romot

triamine and methy tryptamine of course fluto is there and that leads to the formation of

different products for for example cloro strictosidine

bromo strictosidine as well ased

to side in and this pathway moves further and produce the desired product along with the normal

pathway which operate side b side which is St which accepts nor Mal tryptamine along with

Cogan I'm not showing so it produces strictosidine and this pathway moves

together so uh this is what is this that the titoy in analogs can accept all the

downin enzymes through feeding experiment and that resulted in the formation of modified natural products

so and that that includes asalin tabersonine serpentin catharanthine all so if I write that

is aalin serpentin

tonin catharanthine all normal as well as substituted can be produced depending of

the type of uh uh substituted substrate you do the feeding so this is said that this is having a history it is said that

this is having a historical importance of generating modified natural products from altered precursors particularly

with the bacterial system but with the plan system this is for the first time and this is basically the precursor

directed approach and using this approach what has been found that the biosynthetic pathways have relaxed their

substrate specificities the pathway as a result of that it can produce all natural

non-natural uh indol alcol what you desire to get it for future pharmaceutical applications so with this

I end this class thank you