Unraveling the Missing Enzymes in Vindoline Biosynthesis Pathway

[Music] [Music] welcome to nptl inline certification

course uh on pharmacognosy and metabolic engineering uh this is lecture 28 where I will

discuss the missing enzymes of vindoline biosynthetic pathway this is based on one of the latest discoveries made uh

with this biosynthetic pathway so what I'm going to cover first is the revision of the established root to tarpo Indo

alkaloid biosynthesis so we'll recapitulate the pathway again then we'll talk about further clarification

of three T3 oxidase and T3 reductase in vindin formation and then we'll talk about the

biosynthetic steps to tabarin and catharanthine formation in catharanthus Rosas

so let's go to the slide so uh this is what I have covered in one of the previous classes where I have shown you

that uh tab personin converted to vindoline uh through multiple step

reactions uh sequentially formation of 16 hydroxy tonin then 16 methoxy tonin and

then uh it forms 16 methoxy 23 dihydro three hydroxy taronin and this St

requires two two enzyme activities one is tonin 3 oxidase and other in tonin 3 reductase and then subsequently other

steps like nmt d4h and d8 formed so uh what I would like to talk now is about this step that

means this issues related to T T3

o and t3r

activities that contributing

to vindoline formation

so 16 methoxy tonin okay so let me put it in this

way tabar sonin and uh six eoy

tonin and in between of course 16 hydroxy tonin is there but that I am not

showing if you say I can add it also no problem that is

16 hydroxy tonin so the enzymes

are t16 H

then second one is T

16 omt so this is also now called t16 H2

now what uh what we have discussed in the last class is this that

these 16 methoxy stonin converted into three

hydroxy 16 uh

aoxy 23 dihydro

T sonin and this was

subsequently converted into sorry

this acetoxy tonin uh uh what color I use I use this

color this acetoxy

taronin so the enzyme which is T T3

o or T3 r o here is oxidase or here

is reductase now uh T3 o and

t3r so they if they work separately then the step cannot be forwarded towards the formation of this

dis oxy dis oxy uh this acetoxy tonin okay suppose if it happens like that

that out of this oxidation and reductors one of them is not functional in that case what will

happen that this may leads to formation of three

hydroxy 2 three dihydro

tonin and this the next step it

forms this acetoxy window

voro C D and

this by the action of uh d4h

and d a t Okay the space is less I need to give l space

said this will be vindor scene actually there is a mistake

here deile vindorosine and finally it forms uh vindorosine v i n Vin d o r o s i n e

by the action of uh d a t

d4h so here what happens what I mean to say that only T30 is active so therefore in order to make

tonin sorry in order to make

vindoline so I put vindoline in this color these two enzymes will work have to work these two

enzymes have to work together okay so that what I have mentioned

that it should remain in the form of a metabolon so therefore one product one

it catalyzes one reaction immediately it catalyzes the other reaction so uh otherwise what will happen that

normally uh if this enzyme T3 oxidase uh is used separately and a substrate uh is going is it if if the

reaction is catalyzed by if a invitro reaction can be carried out sorry invitro reaction can be carried out with

t3o so it will never produce a proper substrate for t3r uh I think so if

t3o reaction can be cared out in vro then the product of t3o cannot be used as a

substrate for t3r so that is what the point is so therefore t3o and t3r are working as a

group uh and therefore ultimately it forms as a result it forms this product and what will happen if t3o is only

working t3r is not there then it leads to the formation of vindorosine not vindoline that is what uh I I'm telling

you uh okay that is for your information now uh for your information also that this t3o is basically a

cytochrome uh p450 enzyme

and the t3r is basically an alcohol

dehydrogen so the message again I write product of

t3o is an epoxide

and are not used

by d3r therefore coupled

action is required in order to have this activity so this was

discovered uh in the year 2015 and the paper published a famous Journal called proceedings of the

National Academy of Science of pns 2015 volume 112 page number 622 42

6229 and this is the first author is Michael Michael at all the group leader is Vincenzo

deuca from Canada so this is basically the uh question which I said at the beginning

that is clarification about the 32o and t3r in vindin formation now I go to the uh Missing step aspects that

is biosynthetic steps to taronin and cenin formation in catharanthus Rosas

so what we know from the biosynthetic

stps to to uh

to uh taronin and

centin formation in catanus

Rosas okay so if we write the step then the stto

sidin a glycon it forms

uh gasio cyrine is it forms this uh 421 dehydro

G and from here different pathway originat one root goes to

aisin uh and Serpentine and the other root which we

are now concerned with that this makes a product called stemar

denin s m m a d e n i n e

so this stadin so from stenin

to taronin or to cathan information it was not clear but the organic

chemists they have synthetically shown such reactions so but that actually uh helped now the

combinatorial uh chemist as well as the biologist or the synthetic biologist to work out the step so I will not narrate

the discovery of this rather I will just simply go through the steps so stemar denin

forms okay because of uh space shortage I will put the stemen in uh here in the

I put the stem here to start with s m m a d e n i n

e this stanine will be converted to steminine acetate

stenin acetate that subsequently converted

to pre condilo

carpin acetate this is an unstable

compound and this leads to the formation of

dihydro pre condilo

carpin acetate and

that subsequently leads to formation

of dehydro secin uh where I put it I will put it okay

here dehydr codin and from dehydr

acodin uh either catharin or

tonin is formed now if I put the arrow now the enzyme discovered I will show

here so steminine acetate to precond carpine acetate this is p s and this one is

dpas okay and and this

is uh CS

TS it [Music] releases

as and then so PA stands

for precon Dio

carpin acetate synthes and DPA stands

for same D Hydro

pre corn Dio Carin p

synthes so a scientist from uh led by Sara conod that time she was with she was

working at johnin Center UK so they collaborated with several institutions in Germany as well as in UK

other Institute like Oxford uh and they have elucidated this pathway so what

they basically identified they identified two mysic enzymes necessary for Vin blastin

biosynthesis so first one is basically uh an oxidase and a reduct is that isomerize tamarine

acetate into dihydro precond carpine

acetate which is then

de acetalated and cyclized to form

either catharanthine or taronin via to

hydrolyses so uh so that means

the uh so this is the basically these are the these two new enzy enzymes so these two new enzymes they

have characterized for the first time from catharanthus Rosas so in order to check the

activity uh so they have done uh virus induced gene silencing of tab t stands for taronin

synthes and CS stands for uh cathann synthes so when they did virus induced gen

silin this resulted in Mark reduction of tonin as well as

karantin level goes down that is one thing uh now another thing what they have done in order to confirm this

function of these enzymes so they uh reconstituted this tabarin and

catharanthine pathway in nikiana benina so I just go to the next slide because otherwise so

reconstitution of tonin

and cenin inana

menana so that means these jeans uh they have expressed so let me

try to show it in this way so so here the genes

for p plus

d plus CS so these three genes were

inserted and they

add steminine acetate here the fed and as a result of that

if it can produce the cathan

thing that will indicate that indeed these gen are functional similarly for the other one

what they have done they made the construct with

p plus DPS Plus Ts

they again do feeding of steam Den in

acetate and that led to the formation of

neveron so through this they have

confirm that indeed these are the missing enzymes of the pathway and this is the best way to

confirm the function of the newly discovered Gene in a heterologous system so the tobacco this pathway is not

operative so therefore they have tailored these three genes together stitch together and put it uh in the

nikiana benthamiana plant and then they have injected in the leaf steminine acetate and as I have shown in the

previous slide that steminine acetate will be taken up by the pass and it forms the precond carpin acetate if pass

is functional there then it makes precond carpine acetate then D pass will come and that makes dehydro pilocarpine

acetate and then similarly the catharanthine synthes or tonin

synthes through hydris activity it makes this uh either tonin or catharanthine so similar experiments They carried out

here and the results were confirmed so uh so the pathway showed how plants create chemical diversity and

this also enable the development of a heterologous platform for generation of stem deriv by active compounds so

stenin derived always problem with my

handwriting derived bioactive compounds can be generated so this paper

published in the

leading World leading Journal so one is nature another one is science I have mentioned this is published in science

in the year of 2018 volume 360 page number 1 to 3521 2

39 so the first author was capu at all c a p u t i caputi

at all and the group leader two group leader one of them is

Vincent Cod dult French from

uh TOS France and other one is basically Sara e conod uh from

this so this is indeed uh this appears to be very simple but this is indeed a very complex research and if you get a

chance to read the science paper with care and attention you will enjoy how they have carried out the

experiments and uh and uh through these experiments they have confirmed the pathway and with this the total pathway

of Vin blastin biosynthesis uh from uh tryptamine and cyanin is more or

less resolved so the interesting point is this that at some later stage the whole pathway may

be expressed in East system and denovo alkaloid production can be achieved what was possible in case of tropen alkaloids

or isoquinoline alkaloids which I told you in a few previous classes so with this I end this

class thank you