Engineered Yeast for Sustainable Production of Anti-Cancer Drug Vinblastine Precursors

[Music] [Music] hello welcome to nptl online

certification course on pharmacognosy and metabolic engineering now we will go to lecture number 32 where I will talk

about engineered East BRS precursors of anti-cancer drug Vin blasting if you remember in the in a few previous

classes when we talk about tropen alkaloid as well as isoquinoline alkaloids they are also I have uh shown

you examples rather very uh positive achievements using e system to synthesize the either benzil

isoquinoline or tropen alkaloids dinovo so the similar attempt was also made with this benzenoid IND monotoring

indol alkaloids leading to formation of the precursors of Vin blastin so we will briefly look into this aspect today that

is denovo synthesis of dimeric monotoring indol alkaloid precursors in East cells let us go to the board

now well so the topic what I am going to write a microbal supply chain

for the production of

of the anti-cancer drug in BL so this work what I am going

to discuss now this work was published in the famous journal

Nature just last year and references volume number 609 page number 341 to

347 and the authors are Zang and

Kling so they are mostly based uh at Danish Technical University of Denmark uh okay

so what basically the uh Genesis of this work uh so that we will discuss first of all that why there is a need for

microwell supply chain for the production of anti-cancer alkaloids that is important to know so uh that means

that we will now outline the problem problem is this that the uh precursors of Vin blasting that

is catharanthine and vindoline as well as the final alkaloid V or vin blastin they are always harness

from the plant tissue therefore in order to extract this you need a huge supply of this plant material now because

of different reasons including natural calamities political changes in the political scenario sometime this

pandemic okay forest fire all this leads to the disruption of this this supply chain and if there is a disruption of

the supply chain there will be indeed shortage of this life saving drugs like shortage of drug means the shortage of

the raw material for the production of this drug uh therefore it is important to

ensure the stable supply chain for

this uh value added products now what is will be the solution

then solution will be using synthetic biology

approaches where if possible the whole pathway can be uh

transferred into a eukariotic system preferably East and so that uh the East will be able to produce the desired

products and also it is important that ideally one should be able to make this recombinant

East synthesized the desert alkaloids

denovo that means uh you should aim not to supply the uh expensive precursors rather you should only grow

the yeast with with basic sugars medium and amino acids and East will utilize that it will uh run its primary

Metabolism from there it will start the specialized metabolism and ultimately the pathway for this monotoring indol

alol leading to formation of the products so that is what it is so in order to make that so scientist they uh

they made uh their task uh jotted down and uh in three modules that is they should try to

complete each modules so the module one is basically uh the

dinovo synthesis of strictosidine then module two will

be denovo synthesis of cenin and module 3 will be denovo synthesis

of vindoline so each of these modules each of these modules must be

expressed in East and

tested individually to confirm

functional expression of

that portion of the pathway portion of the

pathway so and and from here

the final stren should

contain all the three modules leading

to the formation of of

catharanthine and GRE blastin

using glucose and amino

acid Crypt ofen well so this should be so let us

briefly uh draw the modules what needs to be operating in the East so

the module one say the strioed module if you say so I just first write

the sorry so this makes loganic

acid loganic acid will make

loganin loganin will make seanin and this subsequently will

make stripto Sid in and then it will

make stto in aglycon this all you know because I have said this repeated times but

uh uh this is required uh to tell you once again okay so this is

the first module that means from the janal to the formation

of to side like so this is the stricto in

module sorry s r i well so the next step it

makes G so Siz in and

then it eventually makes stemar dening this

Tarin will be converted into stemar denin

acetate and this will be converted into pre C pin

acetate now this is important because the scientist really they were waiting

until the two missing enzymes are discovered so these are P precond carpine acetate synthes and

dpas D Hydro precond

carpin acetate and this will leads

to the formation of taronin

so and steminine acetate also

contributes towards the formation of cenin

so the module 2 is basically from here that is stto side in aglycon

onwards until the formation of cathan and

okay now this is the module two so what you should write here that is tabarin karantin

module now the module three which is basically the vindoline

module so this will start from toning this all you know but still I would like to put it here

that is tonin will make 16

hydroxy tonin and then

16 myoxy tonin and then subsequently it

makes three hydroxy 16

methoxy 2 three dihydro

tasin these steps all we have studied and this is by the action of these two enzymes and this lead leading

to the formation of D acetoxy violin and

then de atile vindoline and finally it makes

vindoline okay so let us Circle it or let let us make

the border for this that is uh from anyway I'm not telling anything new so already I have discussed this but

just to uh recapitulate your concept I I have grown this once again

okay now three modules were individually expressed in the East and finally as I

said that uh finally the spinal strin they have constructed which contained all three modules and

sequentially and that if function that leads to the formation of catharanthine and

vindoline okay so the the question you should ask yourself why they basically stop there why not to add a par

oxides uh in the East system so so that both the catharanthine and pendolin produced in the East system can be

utilized for the production of V Christine that means if we put

uh line here that means cenin and vindoline they should

join and then they make in blasting and this is par oxid sometimes

people mention like prx1 but uh in spite of several attempts they failed to express

peroxidase functionally in the final East stren that means the final East

strengths were incapable of producing pin blastin now that is basically the

bottleneck that is why at this stage using this recombinant strain where all this uh where all the all together they

made 56 genetic edits so let me go to the next slide so that means in the

final East stren they

made 56 genetic edits

including 34 Gen

from plants and

and deletion of or over expression

of tangents of e

origin so this part we did not I I did not uh show uh show you in the previous

board because to make the things simplified and why all they did they did all this basically

to improve the generation

of key precursors in

the biosynthetic pathway okay so the what will be the so okay if

we put it in the form of a diagram say the while typ is this is while

type and from this while type is what was achieved at the first stage is basically

the D noo stto in this is

okay from there theid this

is denovo okay I think I should give the space

here from there the

next do know vindoline from thereo

karantin and Boling these are all easts so this is successful but

why the why they are unable they fail to

express par oxides in East

Str so uh so what they thought perhaps it requires more

compartmentation and what scientists did for isoquinoline alcal so basically they uh they utilize the paroxysm and they

put some enzymes like Narco cloudine synthes inside the paroxysm so now what they thought that perhaps they will put

the peroxidase enzyme inside the peroxisome and drive the pathway there so that that may be a

safer subcellular compartment which will allow the par oxidase to function and that eventually may lead to the

formation of Vin blasting but for in order to achieve that we have to

wait okay so that is basically the future Direction so the future Direction I will end this with writing future

direction to have the peroxides

expression inside sub cellular

organically and that probably help to couple the catharanthine and vindoline together and makes the VIN blastin so

whatever achieved until this that is to make stable production of vindoline and catharanthine so that they they have

achieved that itself is a very great achievement that is why the paper published in nature last year so with

this I am basically uh ending this class so the denovo formation of Vin blasting precursor in East

cell is now achieved with this I end the class thank you