Comprehensive Overview of Indole Alkaloid Biosynthesis and Metabolic Engineering

[Music] [Music] welcome to nptl online certification

course on pharmacognosy and metabolic engineering this is is lecture number 34 where I will make a final overview of

indol Alco biosynthesis along with metabolic engineering applications

so uh in previous classes four or five classes I have discussed detail about the tarpo Indo alkaloid biosynthesis and

metabolic engineering of the pathways leading to enhanced production of the alkal words

and also I have discussed about the role of transcription factors role of light in regulating the pathway and

finally I ended up with the concept of non-natural indol alkaloid production by manipulating the pathway and feeding of

non-natural substrate and also I talked about metabolic reprogramming concept where

the normal alkaloid biosynthetic pathway along with the modified pathway both can go side by side leading to the formation

of both natural as well as non-natural indol alkaloids in catharanthus Rosia hay Roots so in this class basically I

will uh show you the structures because if you remember the previous classes I did not uh show any structures in order

to make those Concepts simple because structures are pretty complex so so this is an overview class so I will here show

you the structures let's go to the next slide so basically regulation of indol alcohol biosynthesis so what we have

seen that uh the tryptophan synthes uh sorry uh tryptophan decarbox which converts tryptophan to

tryptamine so this is TDC and uh this is important en time for making tryptamine which is the substrate

which is one of the substrates for uh strictosidine synthes which is called St by joining

tryptamine and seanin so the tryptamine is coming from the simate pathway uh whereas the cyanin is coming

from the uh tarino pathway originating from pyro and gly alide 3 phosphate and geranial T Hydro hydroxy plays an

important role because it makes 10 hydroxygen anal and finally it makes lanin and loganin converted into seanin

by seanin synthes and once strictosidine is formed then stoin glucoside will work and it will make stoy in aglycones and

that leads to formation of different uh Pathways so one is camine

which uh leads to the formation of aisin and then of course Serpentine uh whereas the other roote

goes towards the formation of catharin and the another route goes towards the formation of

taronin and then taronin finally forms vindoline and cenin vindoline joins and make this structure which is a diic

indol alol VIN blastin so these things we'll see in more detail in the next slides so

uh what I have also mentioned that the late step of vindoline biosynthesis that is starting

from taronin till the formation of vindoline so this is the late step

now in the aial part that is where the leaf are situated so there taronin is converted into vindoline by the and this

requires the several enzymes seven so t3o there will be t3r so on so all together seven enzymes

are involved towards the formation of vindoline and then this vindoline and catharanthine joined by the action of

peroxides and makes an hydrin blastin and so on however in The Roots taronin is converted into Lo

nin as well as hor hammerin and then 199 no atile horam medicin so these and the subsequently

the enzymes responsible for this pathways are also characterized like t9h uh like

uh sorry like t9h like Matt these are well characterized uh another important point

to tell here that the stto in aglycon makes the dehydro gasio sizin so that is an important compound from there the

Starin is formed and then from St to tabin and what I have also shown in on the one of the previous classes that the

missing step of uh Missing step between St steminine and taronin that I have also

mentioned okay uh now so this is again an overview so the as you see here that

uh uh the two uh enzymes which basically complete the BIOS sythetic steps one is this Pas and another is the DP so that

was characterized recently and the paper published in the famous Journal science in the year of

2018 and uh this is one and this actually outlines the late steps and where you see that this t3o

t3r that those are also much later discovered enzymes these are also there and this is the first steps of iridoid

pathway leading to the formation of loganin and the remaining things are also present here one important point is

that from the camine uh this Tetra hydro alstonine and finally alstonine can also

synthesized so this is a hypothetical view of indol biosynthesis in catharanthus so here

basically the involvement of Department compartments within the cells are shown uh this also discussed in detail like

there is an involvement of vacol there's involvement of chloroplast there is a involvement of cytool and also endoplasm

reticulum and where these reactions are carried out so as early as 1993 this uh paper published in Journal of plant

research and by mear so this is from the Robert bport is group from uh Netherlands lien

so they have given first the overview of involvement of different compartments and subsequently later researchers

confirmed that uh this is uh the same thing just to

show you that the how complex is the structure like stto in this is stto in

agon so and and and this this is from different alkaloid formation like camine

like aisin like serpentin Vin blastin VIN pristin similarly so this is already I have covered so I can leave this one

and this one also I have just told in one of the previous slides that this is the pathway from tabarin to vindoline

which is operating in the aial part or leaves and this is the one which operates in the roots and what I said

that the enzy times involved in the formation of hor hammerin or uh Larin or atile hor hammerin this have already

been characterized now uh here this slide what I like to emphasize

here that from stemar denin one route goes towards the formation of taronin and these steps are

already characterized and which contributes towards the vindoline other roote goes towards the

cathod Anin whereas from Starin another roote originates which goes towards the formation of codin and ultimately it

forms vindoline this is another indol alol of of course monomeric similarly from 421

dehydro sizin gasio sizin aquamine leading to the formation of perine these are also different Indo

alol which also formed in different plants including catharanthus Rosas and here it is showing the formation of

alstonine and tetrahydroxy tetrahydro alstonine so the point here to tell that it is not only the VIN Christin Vin

blastin but there are so many Indo alals which are produced in catharanthus Rosas and they

origin from the intermediate molecules are shown here so this again emphasizes the complexity of the

pathway so cellular localization of both the enzymes and intermediates in a previous one like mner uh paper I have

already discussed but uh this is 1993 so this is a more recent drawing so here the involvement of uh different

compart ments like uh vacol this all already I have discussed but here interesting point is that endoplasmic

reticulum is shown here where t16 hydroxy is located and where the reactions are carried out followed by 16

omt and then the pathway moves inside the plastid and the nmt is localized here in the thid which converts this 16

methoxy to3 dihydro 3 hydroxy tonin into D acetoxy vindoline which come comes out uh into the cytool and then d4h

converted this to dyle vindoline and finally D converted into vindoline so here basically involvement

of chloroplast uh vacol endoplasm reticulum and

cytosol all are important in running the pathway this already I discussed I have also drawn some line

diagram in few of the previous classes to explain

that aalin serpentin Alin so this already I have discussed so I can skip it this is Al stonin this is serpentin

which is coming from asalin so this is just to reminder that 421 dehydr oyin which form from stto in

aglycon that is an branching point from where azaline can be formed asalin can be formed which converted into serpentin

later catharanthine can be formed and tonin can be form which converted into pendolin and then eventually it makes

this compound so 421 dehydr gasio is an important intermediate okay uh this

is biosynthesis of adaline which I also covered in one of the early classes so like adaline is the final product we are

to find in Ria serpentina so which is formed from vodin

vinin and ultimately start actually it starts from 14 21 dehydro gisin and ultimately it forms aalin through the

formation of hodin and other intermediate alkaloids uh this I also covered so

don't get daunted with the structure just structure just to show you that how complex are the

molecules and this is again from the structural point of view Al although I have covered

that but this is a uh more uh a zoom view of the pathway you can say that 421 dehydro gasio sizin converted into uh

camine and which is subsequently as and then by the action of peroxides it converted into uh

Serpentine this also I have discussed that stemar denin originates from 14 to 21 dehydroxy Yin and then one route goes

to karantin another roote goes to taronin so purpose is just to make an overview so that things will parate and

settle in your mind that is why I'm showing this things again and again with the

structur uh and then uh this also I have covered that first 34 anhydro Vin blastin is formed then Vin blastin and

then finally Vin Christine now the difference between Vin VIN Christine and pin blastin is this that here it

is uh ch3 which is Vin blastin where is Vin Christine this is

CH okay and this also I have covered only uh thing is that that at this step this is

t3o and t3r these are discovered so leading to the formation of vindoline from

catharanthine so you see this is the diagram taken from 2011 that time t3o t3r was not

confirmed so much later diagram it is there but intentionally I put this just to tell that with the advancement of

science new discover is leading to more clarification of the pathway this I have already discussed that the elicitation

signal leading to the formation of stto and synthes gene expression uh so where involvement of K

is where the some sometimes East extracts you can use so this is the point where elicit binds it could be

anything and then that activates the calcium channel which may activate the lipas leading to more formation of

jasmonate then that will be uh received or perceived by the jasmonic acid receptor followed by a protein kyes

activity ultimately leading to the formation of different transcription factors including War C3 octadecanoid

responsive catharanthus ellus it could be three anything so that is the specific transcription factor for op

liprin stto stto in synthes gene expression uh I have discussed this with

a very uh simplified line diagram where basically the role of light in vindoline biosynthesis and particularly from the

context of CR pf1 which is a negative regulator so when it is under dark condition that binds uh in the promoter

region and that doesn't allow the expression of uh CR G gta1 or t63 H2 or D which all required for the vindoline

formation however there is another transcription factor which maintains it basal level of activity leading

to uh minute formation of these alkaloids uh so the or some basal activities maintained

but then what happens that uh and when this happens what happen because pendolin is not formed so tabarin

accumulates in high amount when light comes light degrades the crpf

crp1 catanus Rosas phytochrome interacting Factor one so it it degrades leading to activation of CR

GA and that is a positive regulator which binds at the proper place leading to transcription of the lead genes of

vindoline formation and what you see that more vindoline formation and tabarin is getting

depleted because that taronin will be converted into vindoline so this pathway is more operating so the content of

vindoline is more that is basically shown by the thickness of the arrows and here basically the th the thinness

of the arrows indicates that less vindin accumulation under dark condition so this paper published in uh

plant physiology in 2019 this is again finally I have covered so I can skip this so the

different compartments involved so we can go okay now comes to the cross ction of the leaf so you know at the top there

is epidermis then uh uh and then this is the epidermal layer then there is piset mesophile pal

then there's a spongy mesop these are and then uh these are all then along with that there are spongy mopy

idioblast is there and there are lafar is there so the lafar and and spondo blast so these are

the specialized cells which can accumulate the latex so and the latex contains the

uh late steps of uh vindoline alkaloids including vindoline and and subsequently the VIN Christine and Vin

blastin so and this is basically okay this is a three-dimensional uh representation this

left side whereas the light side right side is a cross-section where it is showing that

the epidermis plays important role in the synthesis of these alkaloids including uh the formation of 16 methoxy

Taron which now moves through the uh palisad parenchima and from there it moves to palisad idioblast which is the

specialized cells where this uh dis deetle vindoline to vindoline formation takes place similarly this will will

move to spongy mesopo blast the yellow one where this reaction is taking place or it can move to the specialized cells

like latier where this conversion takes place because these are specialized cells

having thick walls and this actually uh isolate this L step of uh vindoline by sythetic enzymes as well as the products

from the rest of the cells because these alcal may be toxic to the normal cell that is how in the evolution the

specialized cells evolved and then internal fluent parena plays important role because there the early steps of

the pathways are operating uh so basically this is showing intercellular trafficking of

monoton pinoid indid pathway and this was taken from plan Journal

2008 uh much later uh review published in phytochemistry where uh the same thing

it is showing uh that this is the ID blast where conversion of e2f takes place so e2f is the late step of this

vendol in biosynthetic enzymes ler so the yellow uh background uh cells are basically these

are the either laifer or rbl cells and internal fluent parena is showing with that red

color and then uh there are showing the upper epidermis and the lower epidermis and the different pathways are

operating are showing in the left side and right side so this is nothing but what I have shown in the previous slide

it is showing in wed line diagram and

uh it's the same thing but what is important that uh uh if t3r is not there then the pathway

produces uh instead of uh three hydroxy 16 methoxy 23 dihydro tronin it makes three hydroxy 23 dihydroxy tonin that

leads to the formation of this acetoxy vindor so This acetoxy vorin Again enters into the lers it conver into de

atile vorin and then it forms vorin that is also a a

monotoring indol alol quite similar to vindoline structure and this is only the new thing

which is here in the slide and this I have taken from new phytologist remaining things are same

other important point is the secretion what the I said that cathan thing is is basically from the epidermal cell this

epidermis it moves to the uh cutic surface and I mention because a significant amount of cathann is moves

there because that basically plays an important role in defense uh so that is again a relatively

new information discovered which already I have covered in the previous slides and also I have covered that the

specific uh Transporters genes one such transporter gene is CR npf 2.9 which

actually moves the strictosidine out from the vacle into the cytool where SGD will function and it

makes the stto in a glycone so that means that seanin and tryptamine these two enters inside the vacle and

there lies the stricty in synthes that stricty synthes joins and makes strictosidine and once strictosidine is

formed it should come out from the vacu and there is a specific transporter protein which helps to move these things

out so what scientists they have done when they have characterized the gene so they basically block the gene expression

by using vus induced gene silencing as a result of this you know what happens that

the this is basically the chromatogram showing the accumulation of strictosidine as a result of that when

uh when the the when this Gene function is disrupted then what will happen

strictosidine will not move out so strictosidine accumulation will increase so that is showing here in the blue one

this is the silence line so because the transporter was disrupted so the accumulation of

stto in enhance whereas this is the while type where transporter is active so almost no stto in is detectable

because it all converted into stto aglycon and subsequent Downstream products so this was published in nature

plant5 and then uh let us uh this is more orless I have covered I have not discussed all the metabolic engineering

before I end up this class so let me make another overview again I have drawn this in in line diagram what you see

here that the roots so root what the alkaloids accumulates in the root actually as malis

Yin in so these compounds are accumulated in the root so which along with that this Lo Narin Lo nisin and

this uh these compounds whereas in the leaves of the aial part finally what we see the accumulation of v blastin and

Vin Christin uh however in the whole plant you can see all the

intermediates present in the present in the cell so any portion but particularly Leaf specific is these

two whereas root specific are there a few which I have shown now uh I have also spoken about

the non-natural indone alkaloid production so non-natural means these alkaloids are not produced by the plant

so and why they are interested because non-natural alkalides may have more importance for pharmaceutical

application than the natural alkalides therefore one strategy was taken by silencing the tryptophan

decarbox that means tryptophan will not convert into tryptamine so that all the subsequent enzymes will not get the

substrate but uh the pathways are ready only you block the early step so then what they have done they made feding of

fluro tryptamine and then flot triamine is accepted by the root

culture RNA silence root culture TDC silence root culture because TDC is not working that is why you are putting this

one uh and and that will be subsequently taken up by the different uh through different

pathway and leading to formation of different uh Downstream Indo alkaloids so that means non-natural indol

alkaloids like flot triamine fluro fluos serpentin fluro asalin these these all it is possible to make this through this

technology where you can silence the TDC and do a and do feding of the fluto derivative and that will be subsequently

accepted by the normal enzymes present in the pathway and make the subsequent products which are all Pluto derivatized

or okay and finally I have talked about the concept of metabolic reprogramming this is basically the metabolic

reprogramming where stoyan synthes Gene where they made a single amino acid substitution valin replaced with the

methine at 214 position and when and then what they did

so this is basically a mutant Gene so mutant gene they trans transferred into herid culture that means they have

generated Co transform herot culture with a mutated s but when uh but the normal pathway is still there so the

normal s is there along with a b214 m s is there and then what they have done they made feeding of

chloro methy bromo and fluro already checked and that leads to the formation of

this derivatives either chloro or methy or bromo of the indol alkaloids whereas the as the normal pathway was not

interrupted but the normal products are also there so in the beauty of this you can get both the derivatives or

derivatized product as well as the normal products so and this is the concept called metabolic reprogramming

so where the stto in synthes v214 and as a result of that you can get different product formation like as

malis tonin serpentin centin so it basically it relax the substrate

specificity and uh enabling turnover of tryptamine analoges into different product formation so this is a

very interesting paper and this was published in nature chemical biology 2009 so and finally the the Netherlands

group they also uh explore the possibility of producing these alkaloids in

bioreactors so they have given a schematic diagram how the uh work to be undertaken so the fundamental study is

this one which we have covered

soin is important they have also covered what C3 they have covered okay and all these things but then Upstream

processing that is uh this and then mutagenesis screening so this part is already done then important

is the bioreactor design and bi bioprocess engineering and the subsequent Downstream processing these

are also important and if you want to produce the alcol in large scale and so the fundamental pathway is now worked

out now more of the bioprocess engineering needs to be the work need to be given emphasis on the bioprocess

engineering aspects and scale up and that may leads to formation of these alkaloids at Industrial

Level in BIO reactor so with this I end this class and giving you a brief overview of uh Indo alol what I have

covered and with that I end the indol alol

and we'll move into the next class with another topic thank you