Comprehensive Biosynthesis and Metabolic Engineering of Lignans, Rosmarinic and Chlorogenic Acids

[Music] [Music] welcome to

nptl online certification course on pharmacognosy and metabolic engineering this is lecture number 52

where I will discuss uh biosynthesis of ligant including pyot toxins and then

we'll talk about the cfic acid rests particularly uh rosar acids and chlorogenic acids and we'll also uh

discuss very briefly about a few metabolic engineering applications with this

Pathways now let's go to the next slide so the concepts that will be covered in this lectures are uh first the

biosynthesis of ligant and then we'll talk about the rosmarinic acid pathway and once we finish the rosmarinic acid

discussion we will briefly talk about one specific metabolic engineering applications of rosmarinic acid and then

we will talk about the biosynthesis of chlorogenic acid and then finally we'll end up with several uh metabolic

engineering case studies with the chlorogenic acid pathway so if you remember in in the

earlier classes I used to show this scheme so again I am bringing this particular schematic diagram here what

is showing here that the Phile propanoid pH we are disc as we are discussing phenolics for the last few classes and

we'll continue to do so until the end of the course about the phenolic so phenolic basically originate through

simate coris pathway so what is showing here simate pathway contributing to the formation of Phile alanin and this Phile

alanin subsequently converted into trinamic acid and pal is one of the most important enzymes of the red limiting

enzymes of the pathway and then this camic acid subsequently converted into other hydroxycinnamic acids which I have

discussed and then from there uh different Roots originate uh like ligin ligant so lignin pathway I have

discussed I have discussed about the biosynthesis of monol liol which basically act as the precursor of the

ligin polymer and uh so what we are going to do here is basically we will talk now about

the ligant so the ligant are you see C6 C3 2 that is two mole

molecules of monol liol joins together and form the ligant so while we are studying ligant ligant are very

important compounds so they are produced by the plant uh themselves to defend from various iotic and biotic stresses

but uh ligant are very important pharmacologically active compounds particularly as antiviral as well as

anti-cancer agents so that is the practical application of studying these compounds and then uh once I finish

liance then we'll talk about other uh other cic acid EST so cfic acids is

like C6 C3 and then it joins and then it forms different molecules okay so Phile propanoids monol

liol and ligant Phile propanoids are C6 C3 units as you know that this is the C6 and this is the

C3 units okay sorry this is C6 right and so this is called Phile Pro proile and then then what happens

then basically uh these are the monol

LOLs which are again C6 C3 compounds and these basically two molecules are monol signals joins

together and form liant so this is a characteristic uh diagram structural diagram of the

ligan so what you see here if you look carefully that two monolignol molecules are joining here one

is this one and another is this one that is why it is is

called uh C6 C3 2 yeah this is a

slight this 63 right okay so uh this is artifical

structure of a lan molec and ligant undergo several modifications leading to the formation of new liant no

ligant we will not go into that details but just you should know that new liance and no liance are basically derivatives

of different ligant so bio synthesis of liance basically as I said that these are two monol liol as you see this is C6

C3 and this is again C63 and they are the monol lials these two joints and

form the ligant basic ligant and then it undergo sepal modification and leading to the formation of different complex

ligant one such example is this one pinol so as you see here that these two

with two different colors it is it is made clear that this is coming from one and this is coming from

another same is true for malool so what I have said is this that

it's all about the Phile propanoid pathway and this Phile propanoid pathway basically what we have we have studied

in in details about the monolignol pathway like 4 CL HCT C3 Prime H HCT all these enzymes we have studied and

finally we also uh discuss the formation of Conifer

alcohols or copile alcohols or paracom alcohols and these Conifer alcohols what is this that this

basically uh or copile alcohol this joints in N numbers and form the polymeric

structure which is the ligin but only two such molecules will join and form the liance

this is the basic difference I I hope with this it is clear so what you see here that this Conifer alcohol by some

some entic reactions it makes this intermediate joining and then ultimately it forms different structures as I have

mentioned pinol is one and uh and even it it can also joins with the ligin molecules as well ligant can also

join with the ligin molecules as well now what is important point to tell here that uh kif perhaps I have mentioned if

not then I'm mentioning now that Conifer alcohol is a monol liol so that basically by the action of a glucos ale

transference it this this should be a GT it makes coniferin coniferin is nothing but

a glucose okay I just this is you see the glucose is here so this

coniferin from the cytoplasm it moves into the cell one W this is the cell wall and

then again by the action of glucosidase it is back to ciferal alcohol and then it makes different

functions so for transportation it requires a sugar molecule addition and then again will be

Detachment now here what is showing here that the early stages of Lin formation includes this okay coniferin is called

for glucoside of this and that takes part in liin formation and this is basically the LI liance I am I'm showing

this one here these are the two type plus and minus so what we will see that when two monol liol joins it always

forms plus not the minus so that means that is very uh specificity which exist among the

enzymes which joins these things earlier what was thought that it was a random reaction uh so Random

coupling cc8 random coupling but much later it this was identified this coupling was catalyzed by an enzyme

which is called derent protein please remember Deen protein so

it's a Deen transform basically to guide is a Greek word g so it's a guide protein and which basically oxid is in

nature and derion protein basically it joins two monolignol and form the first step of the ligan biosynthesis so this

is a is a a and then next comes to b b is

basically pinoresinol synthes penor resinol synthes and uh okay what I can do this a I can

make it in this way and then the next

reaction is c c is basically C and D are

pinol linol reduct

is Lis resinol linol yeah linol there is a mistake here so it

should be a l is C I will be added

here l c the so uh pinol converted into lisin and then that is subsequently converted

into SEO ISO linol so the enzymes are called pinol linol

reducts so this is this one and then

e which converts SEO isolid resinol

to mati resinol so matol e so it is called SEO isol linol dehydrogen so dehydrogen

enzymes is basically working here and then finally we see F which actually a methy transfer is which converted into

other products and here you see that deoxy protoy toxin is also there that is is also a part of this so uh point is

this the pathway first it makes pinol which is a stable compound pinol converted into

linol linol converted into SEO isol resinol and then that is converted into mati resinol these are the different

Roots you don't have to remember that but for the shake of knowledge you should study uh and uh also you see that

here that cisem in in cism Lum so that there also this pathway is

operating next so the myos synthesis of major ligant so this is basically a a uh Lum a lum species

so scientists they explored cell and organ culture in order to make more of this ligant there in order to study the

digant biosynthesis so it is always better to have a culture system that is why I'm showing here so again just a

revision cor two molecules of Conifer alcohol makes pinoresinol plus pinoresinol and that one root May

contributes toward the formation of camine okay say the cism is the English name of the plant that converted

into pinol linol reductors which converted into linol then again plr the same another

reaction catalyzed with the same enzyme which makes SEO isol linol and then there is a de hydrogenous which make

maol and now this through multiple steps it forms phoyo Toxin and I mention photophy toxin

as one of the potent anti-cancer drug so that is why we are studying lignan and this pathway has well stud

has well studed by German groups including the cloning of the genes and the OV expression and the antisense

oparation but that metabolic engineering applications I'm not basically discussing here

now apart from this this uh Conifer alcohol can also converted into dehydro dioner alcohol and which will be

subsequently converted into ISO dihydro dehydro chroner Al it's a big name but the enzyme is called Phile kumaran benzy

ether reductase okay this is a new information uh that means the Conifer alcohol can

also uh utilize for producing more complex Conifer alcohols as showing here but the main root what I have discussed

is this this we have now started so uh okay this we have now start it fine okay so and finally why I bring this issue

because hinin is a molecule of interest and hinin is produced by a species of Lum Lum Corum

bosam bosam so this is H hinin so hinin is one such ligan so hinokinin is a very pharmacologically active compound

against particularly Hepatitis B uh so virus infection so it's it it has potentiality to make into antiviral

drugs uh so this is hinokinin so hinokinin biosynthesis has been studied by the German group and this is

outstanding paper is it published in plan Journal J's journal and so so what is showing here that this is

a synthes so and then there is a hinin synthes and hinin synthes makes hinin or it can also uh this can also that is the

Matti resinol through multiple root it produces hinin one is through this root another is

through this root anyway we you need not have to worry about this things uh but just to tell you that how complex is the

pathway however the camine camine is as I said camine is also formed through this pathway camine

so camine is formed in the lam species that is well known and these are the different enzymes which are showing here

like PS is pinol synthes uh like PS is porol synthes and so what

is and then plr I have mentioned sdh I have also mentioned then PLS is new one

so this is the PLS and then PSS is there then uh HS stands for hocin synthes so this is important HS stands

for hinin synthes so which actually finally the ultimate enzyme for the formation of hinin which is a bioactive

compound so uh few genes of this pathway has been characterized uh but not the whole

pathway yet so now come to the phoyo toxin as I have shown that photophy toxin originates basically again from

this ainol so phoyo toxin is basically here and for and this is basically

matol so matol makes this deoxypodophyllotoxin DPT and that subsequently make PPT phoyo

toxin now the pathway which is shown inside this box that is particularly operating in

this tuja but not in the others so that is why is showed in this way otherwise this

matol can be converted into deoxy poopy toxin okay and then uh this phot toxin uh will

subsequently uh methylated and then it forms C6 methoxy prototoxin

and so on and then also at some Stage IT joins with the sugar so you see that the glucoside is also there so this is six

myoxy protoy Toxin and then a glucose is added so it is now glucoside

so here is the glucose molecule added and similarly phoyo toxin uh can directly converted into

phoyo toxin 7 glucoside because it is very toxic so it has to accumulate it in the cell and the the molecule must moved

into the vacle and so that and it it retained as a glucoside so there will be induction of glucos transparence which

joins the glucose and safely stored in the vacol so here is this that this is of more of

practical application so extraction and semisynthesis leading to etoposide so pyot toxin

molecule as you have uh seen here uh this

molecule so this phoyo toxin molecule uh

uh is used as a basic

uh basic molecule For the synthesis of etoposide so what is etoposide etoposid are nothing but the modified pyot toxin

as you see here that uh any R1 and R2 position this is R1 position this is R2 position so in case of aopo side the

methy and hydrogen's added in case of eoos one is methy and other is this hydroxy and in case of teniposide

here one is this this ring added and another hydrogen added so these are different

modified version uh semisynthetic version of phoyo toxin all of these have potential uh to inhibit the cancer

growth so among this etoposide is the most promising one and that is synthesized basically semi synthesized

from the naturally occurring phoyo toxin so that is should be your text home message but normally phoyo toxin

accumulates in the romes of phylum species so like phylum peltatum this is an endanger plant you

will hardly with a thorough search in few pockets of Himalayan uh high altitude you will find this phylum buil

atum and uh this is a flowering and uh there is another plant called copham hexandrum earlier it was

called poopum hexandrum so that is mostly confined to the China China region so these are the plants are good

source of Pho phot toxin apart from that Lum flavum common one Lum album then hip

sence common lamasi plant is also uh accumulating phoyo toxin apart from that

juniperus and other gyos sperms also accumulate phoyo toxins so some natural alternative sources of phoyo toxins

are uh shown here like deoxy poopy toxin this is three and these are the plants from where this photox s

derivatives can be isolated but uh large scale isolation leads to uh extension of the plant so that is why

Pym is is is a highly endangered plant so ATMs were made to make cell and organ culture for making pyot toxin partial

success has been obtained not fully yet so with this now I move into the another aspect of this today's lecture

which is basically the cafeic acid esters so there I am going to cover Ros Maric acid and chlorogenic acid both of

them are cfic acid Ester so what is showing here in this is basically the metah Hydrox silation so metah Hydrox

silation is very important Oro meta par so and so what we see here that Kumar qu by

the H C it makes Kumar simate or Kumar quinate and then subsequently uh by the action of C3

Prime H uh which add the hydroxy group at three position

so that makes uh that makes parakum Esters like CFO Esters

like Kumar uh so that means that uh Cil simate C quinate so these are different CFO acid

estas also the Cil hydroxy Phile lactic acid that is also a cic acid estas which subsequently converted into rosin acid

so all the CAPIC acid ests are basically shown here so we have discussed about capil simic acid now if the C MO is

joined with uh quinic acid not simic acid then it forms Cil quinic acid which is called

chlorogenic acid so chlorogenic acid is nothing but the capile quinic acid so uh this is what is because of this metah

Hydrox silation and then the proposed biosynthetic pathway of rosic acid a scientist from Marburg University

Germany Professor Mikey Peterson she actually is worked for decades for ranic acid bios synthesis using Lamas plant

like colus blumi that is was a model and she has decifer the biosynthetic pathway of

uh rosmarinic acid using mostly using the cell cultures so what you see here as I said that rosic acid is also the

caic acid Ester so how rosic acid is formed basically from for Kumar oil KO and from the tyrosin the tat enzyme it

makes four hydroxy Phile pyic acid so that hydroxy Phile pyate reductase R means reductase which makes four hydroxy

Phile lactic acid so four hydroxy fenile lactic acid and paracom aric acid they joins together uh by the action of the

enzyme called rosmarinic acid synthes ra stands for rosmarinic acid synthes and which makes this intermediate compound

uh which is four Kumar four hydroxy Phile lactic acid and this subsequently by the action of cytochrome p450

enzyme makes either Cil for hydroxy uh Phile lactic acid or for karile 3 Prime 4 Prime dihydroxy Phile

lactic acid and eventually this by the action of again another cytochrome p450 it makes the

rosmarinic acid and this rosmarinic acid can further converted into lithospermic acid and salanic acid so these are the

sometime marker compounds of the lamas species so this is the biosynthetic pathway and uh this is the structure so

as you see the structure this is the structure of kumaro ko and which which is formed from paric acid and this is

the structure of for hydroxy lactic acid which is formed from for hydroxy Phile pyic acid by the action of this enzyme H

PPR reduct is and then uh rosic acid synthes joins this together and which makes this four Kumar for hydroxy lactic

acid and then again there will be cytochrome p450 that means hydroxy that is it is called 4 cphl 3H and this is 3

Prime H so when it is 3H it makes Capo when it is 3 Prime H then it forar 3 Prime 4 Prime dihydroxy and eventually

there will be another hydroxy so this is I have shown another

cytochrome p450 and these are all membrane bound enzyme and eventually that makes the complex rosic acid so to

basically mopat group they have purified this uh membrane bound enzymes and they obtain the inter terminal

sequence information from their basically the design degenerate primer Bas and then they clone the genes so it

is a very very tedious process and they published these papers in very good journals so like planta

or JBC probably so for planta probably I have seen that one yeah so so this is basically an

later there's an overview published in the famous Journal phytochemistry by Michael Peterson's

group so in brief the biosynthesis of rosic acid is basically through paror oil Co and for hydroxy Phile lactate and

eventually rosmarinic acid synthes and then two cytochrome 4 450 it makes rosmarinic acid I think it is clear so

now the next attempt was to make if to make rosic acid in the microbal system so East was chosen as a model system

because it is a uh eukariotic system uh so what is showing in the Orange is basically the chimic pathway so the

orange the orange root are non-existent in the plant so this orange root so these are the climatic pathway and

through which rosic acid is formed in the East system whereas the black arrows are basically the pathway which is

operating in the plant so so they have hired uh fug from the plants and then fug from the microbes and

eventually they they are able to synthesize the rosic acid which I should put it in

the uh R yeah so so the native genes are indicated in Black so these are the native

genes OV Express genes uh East gen je in green so a few e genes are there and then over Express heterologous genes in

purple so these are from different sources heterologous genes and then Arrow orange arrow SW basically it is

the chimic pathway it is non-existent imp plants but once they put these jeans together finally it makes rosic acid and

this paper was published in synthetic biology in the year of 2020 so basically this is an East cell this is East cell

and where uh the this is again a denovo synthesis of rosic acid because glucose was used as a substrate as you see and

they have basically tailored few ples few East genes so they have also subsidized or they have also blocked few

East genes and they also use other genes from different sources and eventually uh the East cells were able to produce the

rosem Maric acid so that is interesting and this is the typical plant which is called

rosemarinus uh so it's also a Lamas plant and here basically the leaves are very aromatic and if you touch you will

get the smell of uh this uh aromatic compound so ranic acid is not indeed aromatic it is a phenolic compound it

accumulates but it also produces a lot of volatile phenolic compounds uh so but if you analyze this plant this

rosemarinus by hlc you'll be able to detect rosmarinic acid now from here we will move into another cic acid EST

which is chlorogenic acid what I said that chlorogenic acid is formed by joining Cil qu along with the uh by

joining Sorry by joining kumaro qu with the quinic acid which makes comaro quinic acid and then that subsequently

forms Cil quinic acid or so the c quinic acid is called chlorogenic acid so and then the capil M can be detached so the

hqt is basically this it is a transference hydroxy Camile queet transference and HCT we have shown that

is again a transference so what is showing here that this is how the chlorogenic acid so chlorogenic acid

this is 5 capy chlorogenic acid and chlorogenic acid several chlorogenic acid can join together so and as a

result of that it produces uh 35 Dapo quinic acid so these are the complex chlorogenic acid

substitutes uh so and it can also make four five Dy quinic acids so that means the plants where chlorogenic acids are

dominating so proper hlc analysis can lead to the formation of different uh cafeic acid EST including

chlorogenic acid four five dapy coic acids which is again a derivative of chlorogenic acid or this five capile

quinic acids so now uh so so let us talk before I end this class let us talk

to metabolic engineering studies so this is the structure of chlorogenic acid chlorogenic acid also found in solarus

plants like potato and when you cut potato tuber and leave it you will find that it forms uh it

forms a layer on the cart surface which is basically a subin layer and that layer if you peel off and analyze you

will find that is basically accumulating lot of chlorogenic acid there so chlorogenic acid is basically forming

sometimes a cross linking in the wounding site that that stop the integration of

pathogen or other thing inside the plant cell so what we see here that this parar

acid parakum qu converted into paracom sikic acid by HCT so we have studied HCT in details in

previous classes and which makes parakum sikic acid this by the action of c3h which makes Capo simic acid

similarly uh HCT can also catalyze another sort of reaction where it takes quinic acid instead of sikic acid and it

makes paracom quinic acid so paracom quinic acid by the action of again c3h it makes Cil quinic

acid so Cil quinic acids are nothing but this is called chlorogenic acid so it's a parallel pathway exist uh and then uh

this cap chlorogen IC acid plays important role it is a strong antioxidant it has a defensive role uh

and also there is an alternative root that is that from camic Acid Camile glucoside can be formed and that that

leads to the formation of karile glucoside Cil glucose and then it makes Cy quinic acid but this pathway is

not uh that much well estblished so the established pathway is basically the

this one so what scientists they have done is basically they uh overexpress

this uh HCT which converts this uh uh paracom KO which joins paracom KO and

quinic acid into paracom Que quinic acid and then it it subsequently converted into cinic acid so

as so the uh hqt enzyme was targeted so this enzyme what you call HCT is basically it's basically hqt basically

is a queet transfer so here what is showing HCT is basically the H

QT hydroxy camil queet transfer so when they overexpress this in tomato what

they have found that leading to enhancement of chlorogenic acid so what is wild type means the wild type so this

is basically the chlorogenic acid in the wild tape and you see here if you particularly see the scale so here it's

less than6 but here it is reaching one so almost the content doubled uh and not only that they have also used

uh suppression of this uh Gene and that leads to suppression of the chlorogenic acid that I'm not showing but this

result was very interesting because chlorogenic acid producing more chlorogenic acid tomato in tomato makes

tomato more strong antioxidant Source because tomato contains lopine which is a strong antioxidant so that is a

carotin now you are adding a phenolic antioxidant there which is chlorogenic acid so that makes tomato even richer

source of antioxidant and this paper was published long ago in nature biotechnology by a

group led by Kathy Martin Professor Kathy Martin from JN Center nor so she actually worked on this Phile propanoid

metabolic engineering uh for a long time so uh the same group also uh they made another attempt this time they have used

arabidopsis taliana M uh 12 gen so basically it's basically a transcription Factor Gene so they have transferred

this gene into tomato and with an aim to enhance the both flavonol as well as cfile quinic acid that means both

flavonol pathway as well as the chlorogenic acid pathway in tomato okay now flavonol pathway I will discuss in

the next class but this is in brief that uh the Kumar koi joins with malony koi and

then eventually it forms different uh molecules including dihydro cerol uh and then cerol and then cerol rutinoside

similarly quatin quatin routin oide so routin is a very common this is a dominating uh flavonol which can be

detected in the Tomato very well so these are basically the flavonols so qu in cerol these are the flavonols whereas

the other pathway is this one uh so which we have studied that is the uh chlorogenic acid CFO quinic acid so the

CFO quinic acid is there C which is the typical chlorogenic acid that again by joining with another molecule of eil Co

which makes the Dapo quinic acid and that F the joining of another molecule of Cy COI it makes triail

quinic acid so these are all Cil quinet derivatives and what they have done actually by overexpressing this m

transcription factor that leads to huge synthesis of both flavonol as well as uh this C quinet so S2 and S12 so the here

the black the black is almost uh invisible so black is there under this red background which is the

control okay it's a control so that means when you when they did not transfer the gene whereas the red one is

basically the transcription Factor Gene which was transferred so as a result of that you see particularly I marked S2 so

one is the pill tomato pill and one is the Tomato flesh okay now uh the S2 is basically the five Cil

quinic acid so five Cil quinic acid that is an enhancement in both as compared to control huge enhancement similarly S12

S12 is trapy quinic acid that is also huge enhancement of course S8 S10 so these

are flavonols I purposefully not mentioning here because flavonol I have not covered yet but we cannot really

totally detach it because this paper discuss both now so that means that a transcription Factor uh expression of a

transcription factor in tomato which is hi from arabidopsis that actually push the pathway very well and it enhances

both the uh chlorogenic acid root at as the flavonol root leading to the formation of uh these metabolites in

huge amount and as a result of that what you see here is this that

the this is the control and this is the

transgenic now the fruit color also changed the fruit color also Chang that is what it is that is very

interesting because of the accumulation of this compounds and this is all about this so

I have covered the ligant I have covered the rosic acid and I have covered the chlorogenic acid so with this I end this

class thank you