Comprehensive Guide to Anthocyanin Biosynthesis and Metabolic Engineering

[Music] [Music] welcome to nptl online certification

course on pharmacognosy and metabolic engineering this is lecture number 15 4

where under the broad group of phenolics I going to cover biosynthesis of Anin and metabolic pathway engineering for

enhanced anthocyanin production so let's go to the concept to be covered so we'll talk about the

biosynthesis of anthocyanins and then we will discuss the issue of Antoine and

antoin next we will talk about the structural diversity of anthocyanins and then finally we will

see how anthocyanins are once they produced in the cytool how they are transported into the

vules okay so let's go to this previous slide this slide I have shown several times

but this is the basic one basically the H of phite propanoid metabolism so the antoin is basically coming under broadly

the flavonoids so as you know flavonoids are C6 C3 C6 compounds and anthocyanins

are just one of these so and this basically originates from the Phile propanoid pathway by joining melano KO

with the paracom qu leading to the formation of chalone so the chalone Sy is the first enzyme so I have spoken

about chalon synthes several times uh so this is also no exception chalon synthesis a starting point core pathway

for anthos in biosynthesis and basically showing the relationship between flavonol and flavon

so these aspect I have covered in little bit details particularly on the flavon and flavonol in the previous class so

let me make a brief overview about this that uh as I mentioned just now Phile propanoid start originating with Phile

alanin converting into camic acid then cam for hydroxy then for cl which makes the activated hydroxycinnamate which is

parakum KO because the KO molecule is joined there so that is more active and that joins with three molecules of

malony qu and makes the chalone or which is also called naringenin alone uh because it next step it makes

through isomerization it makes Nar enging okay so the enzymes responsible for this is chalon

synthes and next is chalon isaris so the three molecular manoline KO joins with paracom KO and forms the

chalone so that is called claen condensation whereas for stillin it is called alol

con condensation steel B synthesis also recurs three molecu manol qu but the the mechanism of

condensation was different than that of this one so once naringenin is produced then a part of ninenine may be utilized

for the production of flavon flavon

synthes and then but the main pool of naringenin May further converted into dihydroflavonol by the action of F3 H

f3h is Bally flavonone 3 hydroxy then F3 Prime H which is flavonoid 3 Prime hydroxy and F3 Prime 5 Prime H which is

flavonoid 3 Prime 5 Prime hydroxy we will see this in due course and that makes

dihydroflavonol and dihydroflavonol one root May produce flavonol whereas the root which is meant

to produce anthoine in specific plants so that pathway Moves In This Direction Where dihydroflavonol reductors will

come and Then followed by anthos in synthes okay so it's basically anthos in synthes

and then once anthos in is form then there will be a sugar molecule addition that leads to the formation of anthoine

so the anthoine and anthos differs is that anthos first comes to

anthocyanidin which is basically aglycone whereas when anthocyanin joins with

sugar then it makes antoin and then it is basically stored

in the vle so this is from the structural point

of view as I said that chalone synthes makes chalone which is tetrahydroxy chalone so this is the structure of

tetrahydroxy chalone and so don't see CHR here CHR has no

relevance particularly here so we can simply cut it STS also we not discussing here but

what we will be discussing the next step is this this is converted into by this uh chalon isomar is forming

naringenin so the naringenin is basically coming under flavanone so naringenin is flavanone so

naringenin can be converted into IOD diol okay and the pathway May utilize in producing dihydro quatin whereas

uh dihydro quatin can also be produced from dihydro cerol directly through this root as

well okay and similarly what is showing here that

Penta hydroxy flavonone can also produced and then f3h but this is also possible so here if you see the

difference between these three structures so here particularly what you see here

is this position oh is there so this is four position o is there but here there are additional

o here in addition to this one so 2 H so one Hydrox silation happens so here the enzyme is

called f 3 Prime H that means flavonoid 3 Prime hydroxy now if you compare this

one this one with this one so here there are two more so this is called F3 Prime 5 Prime

H flavonoid 3 Prime 5 Prime hydroxy which makes dihydro mtin

and uh then these three Pathways contribute to different antoin

structures for example the one sinin which is basically magenta towards red's color so the

cadin is this one again the common enzyme is dfr but this dfr are specific so one dfr which

is specific for dihydro quatin will not work for dihydro kemperol or dihydro mtin so these are specific but the

generic name is dfr dihydroflavonol reductors because all these are dihydroflavonols so all these three are

basically dihydro flavo n plural

I okay so that is why dihydroflavonol reductase is there so which converts dihydro flavonol into Lico anthoine so

Lico so that means that is colorless so Lucine Luco means colorless licop ponine or luod

delphinine and next step is this by the action of anthocyanidin synthes which converts liyin to cadine

or uh licop ponin to ponin so ponin are generally yellow or orange in color and the third one is this the

delphinine delphinidin are blue in color so delphinidin requires two Hydrox

silation to happen that is F3 Prime 5 Prime H whereas sinin is only one whereas for Pon no substitution is

required only this position H will remain so this is uh in brief about the biosynthesis of

ano uh sinins okay and and what I have said in the previous

class that this dihydro cotin or dihydro cerol or dihydro myin can be converted into the either uh to myin

which is flavonol so that is called flavonol synthes which I have shown so in the previous class so for example

this one by the action of flavonol synthes f LS will be converted into this is L

capital will be converted into dihydro quatin will be converted

into quatin so qu setting means if we draw the structure it will be like

this so this structure is

dihydro mtin so the when it will become mtin there will be only one Bond formation

here so sorry so this becomes qu setting

or myin sorry this is myetin which is basically a flavonol so that is the side pathway

but uh but main pathway moves in this three Direction so let's now move into the next slide so ponin is the basic

structure as you see that in these two positions are open so no hydroxy added yet but cadine what I have shown that

this position one hydroxy is added and in case of delphinine two Hydrox is added so ponin is responsible for yellow

orange color so here there is a picture of uh geranium which is sp aronium there are

multiple species and uh and the sinine is a common example is Rose and uh delphinine you find it the plant which

is commonly grown in this winter season which is called Larkspur which is the English

name but the scientific name is Delphinium so that produces blue flowers okay so now what we will see how

this structural changes occur and how this different

uh different anthocyanins are created so now let's let us go to the

Whiteboard so here what we will see first is we'll draw the pillar gin once again

so this is the structure of parar

[Music] gin so which is a

Anin now if here if this o is replaced here the hydrogen is replaced and uh

sugar is added then basically it forms

pillar going in sometimes they use this term which is

Anto sinin now with this structure so what we see

now this structure is still not correct we because I have not added uh o here and two more things to be telling

here that this will be the three prime position and this will be the five Prime position so now let us see the

uh antoin name of

ano then we'll put

substit and then we'll put the color I use the British spelling color c l o Ur So first one is

ponin [Music] gin substituents at three prime and five

Prime as such that is not it's only H and what we see as a result the color is orange

or you or or red sometimes they call uh so or it next one is

sin sign it in 3 Prime o and the color will be red p

in three prime will be now oc3 color will be

pink red next next is

delphinidin here 3 Prime o and five Prime will also be o and this will

be color will be do purple next

is p in it in 3 Prime will

be O3 and P Prime will be

oh the color will be purple and

then malvidin MLV d i n 3

Prime oc3 I prime also O3 three and the color will be reddish

purple so you see that this substituents at these positions that is at 3 Prime and at five Prime

really makes difference in the color of the antoin so one thing to be noticed that

as I have said that cyanidin formation requires from dihydro cherol cyanidin formation

requires F3 Prime H that is uh flavonoid 3 Prime hydroxy which converts which adds the at three prime position hydroxy

group okay so that is why this is three prime o and then next what happens the ponin that three prime

position hydroxy will be replaced by methoxy athy donor will be there so that converted into ponin so that means

ponine cannot be formed without the formation of cyanine first cyanine is formed then it will be converted into

ponine so because I have said that is a thumb rule first there will be Hydrox silation Then followed by

methoxy uh similarly for delphinine what I said is basically it's F3 Prime 5 Prime H so dihydro cerol converted into

dihydro uh quatin dihydro mtin by this um a 3 Prime 5 Prime H so that

is why the uh 3 Prime and 5 Prime o is added and delin is produced then in the next step what happens the three prime o

will be replaced by oc3 it converted into ponin so that means from delphinidin ponin will be produced and

finally from ponin malbin is produced as you see from here this o now become O3 okay similarly like here also this

oh now become O3 like this way so it follows the straightforward

chemistry okay so uh now what I have discussed so far is this

that we have discussed chalon synthes chalon isomer is flavonone 3 hydroxy which converts nen into di

hydrol so so these are all dihydro flavonols so these are dihydro

flavonol so nenin are flavon and this step is chalones okay and in The Next Step what

we see that this will be converted into a DI hydr

either by F3 Prime H will be converted into dihydro quatin and which eventually uh makes the

formation of Codine but uh cyanine what happens actually this this is shown in

three uh in one single step actually this is three different steps are involved so first one is dfr so dfr

converts uh dihydro quatin to Lu anthoine which I have shown in the previous slide and then lucose anthoine

by antoin synthes as or ANS is same ASN or ANS is same which makes the cadin so that is still a glycone and then GT GT

stands for glucos ale transfer why 3 GT because at three position this will be added so this will be uh glucos ale

transfer and then it makes in three glucoside so what you see here this thin 3 glucoside so this thin 3 glucoside

subsequently converted into Pine as I have shown you so this is O becoming O3 here then this is O and then what

happens that different sugar molecule can join uh and it makes like uh so I will tell you the different

sugars involved in this uh decorating the anthos sinin in some of the in in later later slide okay and the

delphinine purple okay similarly this these are the finally these are the anthoine and then this basically these

are finally the anthocyanins because the sugar is attached to here so this is a brief

overview already I have discussed now coming to the decoration so uh this is the

beaing which is important so here actually these positions changes occur okay and uh this

basically B ring is coming from the Phile propanoid group and the A- ring is basically coming from the melon so what

we see that there is a Hydrox silation so Hydrox silation ponine as this is four it's fine sin in what we see

addition one more in in delin in what we see two more so this is all Hydrox silation then

methylation so this one under goes first the this one under go methylation uh sinin in case of uh

yeah okay so it makes pin this is a methy group added and in case of uh this one uh there is methylation here as well

which forms ponin in malvidin there are two methylation so like this way and not

only that what are the sugar glycos units added one is arabinose can be added galactose can be added glucose is

common ramose is also common xylos can be added and Ros can be added so similarly that rosile transfer as rosile

transfer as gluc gluc coile transfer is all these enzymes are functioning and that leads to different patterns of uh

anthocyanins with variation in the color now apart from this uh common asile units can be joined with the

anthocyanin like acetic acid can be joined cafeic acid can be joined which makes complex anthocyanins even karic

acid ferulic acid malonic acid can be joined or even GIC acid Copic acid suic acid can also be joined and leading to

the formation of complex anthocyanins for example pin three parakum rutinoside five glucoside you see how big is the

molecule so so many at least you see there are three sugar components attached here 1 2 3 okay

similarly Geno Delphin which is Delphin in 35 Dapo glucoside three

glucoside so so maybe one this is one C Unit coming this is one C because C contains

34 dihydroxy so this is c 1 this is Cil 2 uh and this 53 means that the two position either three position five

position like that way and then it makes the things so this is how the antoin in the nature are decorated leading to the

formation of complex anthos now another important point is that antoin are basically uh stable at acidic pH so if

the pH changes there will be ionization effect so normally the ph1 or2 is the most stable pH for antoin and which is

red in color and it at that it forms the FL ium C then what happens that if you add alkal so then at ph45 it is

colorless and next what happened at pH 6 to7 it it converted into quinal base and that leads to

basically blue color okay so Point here to be noted that at and

then uh if it is beyond seven uh if it is beyond seven then it will turn into yellow and once it turned

yellow if you change the pic acidic it will not convert back so that is some experiments people have

done in pit and based on this results they work out the complex chemistry so uh so that means that ano is very much

ph sensitive so pH is very important for its stability so the sometimes you see the slight changes in the anthos

coloration in the flower petals so it is not that bluish means it's all not that delin it may be possible that it's it's

a cin slightly present in in alkaline or less acidic condition in that time in the red uh the intensity of

red will be dispersed and leading to formation of bluish tint so that is also possible uh this is again a much more

complex one I will leave it so this is overview I have already uh discussed this structure so the last part of this

lecture is this once these antoin are produced so these antoin are in the in the

cytool okay methy transference glucos transference then rosile transference whatever so these

antoin cannot stay in the cytool uh so they must be stored in the vacle so how they move into the vacle

they move there are two hypothesis one is through the vesicule meical mediated

Transportation so this is vesicle in the next slide I'll show that or or they are entering into the ER

and there they basically their GST is attached to this glutose uh glutathiones transfer which plays

important role in the transportation of antoin into the vacle by uh through several Transporters so two things

happen that one either it it moves through a vesicle where GST is mediating that or

uh without the involvement of vesicle uh the anthocyanin can directly move inside

the uh vacu so and here the mat transporter plays role and also ABC transporter

plays role in this and here the pH the protonation is important because the acidic condition of the vacu to be

maintained so that the antoin remains stable so the vacu pH is very important one important point is that if the

vacular pH changes antoin may become unstable or may changes it structural confirmation so uh this is one and this

is another way of showing that that first one is that through involvement of GST and then GST helps to transport the

anthos sinin into the vacu by the use of either ABC transporter or a m transporter or anthos

sinin are basically are enclosed in in some facular structure called

autophagosome which which finally fuse with the uh vacle or membrane and uh that then it enters and finally the

autophagosome get dispersed and antoin stood but the once antoin are entered inside the vacle then it sometimes it

antoin vacular inclusions it stays so it it joins with either a chemical molecule or a protein molecule and it's stored

there so tonoplast means the yeah the vacu vacular membrane okay and the so here what I am

showing here is that this is simply a light microscopic view so here you see that

uh this is this is One S okay uh so this is one single cell right and this is single cell what you see this is

the main vacu portion and here what you see there is another this

structure so this is basically uh anic vacular inclusions inside the

tonoplast so these are the vacular inclusions and that is inside the tonoplast like this one so it is

possible to see on under the high resolution and then in addition to that there are

other bodies such as these

things these things which are which are basically present in the periphery of the vacle so

this could be possibly the uh the uh this uh vesicular structures so this was observed in our

own laboratory under light microscope and so to before I end this course I'll briefly tell about one

metabolic engineering application but this is not with the flower so flower metabolic engineering we will talk in

the next class so what here they have done look the flavonoid path is universal it present in every plant so

what needs to be done that uh why some plant leaves are not producing anthos because it is very uh temporally and

spatially regulated now now this regulation is basically controlled by transcription factors so scientists what

they have done they hired two such transcription factors from arabidopsis which are known to enhance the antoin

and that transcription factors are are D and Rose so these transcription factors are

are uh expressed in tobacco and as a result of that what will happen these transcription

factors normally influence chalon isomar chalone synthes you see flavonone three hydroxy dihydroflavonol

reductors and glucos transfers and so on so these transcription factors plays important role what happens maybe in

tobacco flow anthos in a synthesiz but in the leaf they are not synthesized maybe in the leaf tissue these are these

genes remains in the dormant condition because that is slightly that is regulated by another set of

negative uh negative some negative control was there so now that negative control is removed by overexpressing the

uh transcription factors from oh sorry antium Mages not arabidopsis please excuse me em stands for antium Mages

with Snapdragon and as a result of this expression what happens the tobacco uh Leaf once it accumulated

antoin it basically turn into yeah blackish which is basically most cadine accumulation and from these

Leaf basically they have developed the cell culture and these cell cultures produces

antoin in huge amount and which by which and from which they are able to to purify and isolate in the form of powder

so this was this paper published in metabolic engineering 2018 and so this is the normal uh uh so

this is where the am d only expressed and this is a Ross one when both are expressed you see the changes

in the leaf color and this is basically the cross-section of the leaf these are the cross-section of the

leavs here it is very clear that the antoin are producing in huge amount as compared here little bit here little bit

is you see from the coloration here sorry slightly turn but here is huge

huge and uh from there basically they have developed the cell suspension culture so

the first one in the left is basically the uh cell culture without Antoine induction and this is basically cell

culture with an Antoine induction too much antoin turns this cell apparently towards blackish but it's not black it

is a too much concentrated red coloration and this is the suspended cells which can be checked under the

light microscope and this is a comparison they have done so what we see here that with m m d and M

row uh and with another gen F3 Prime 5 Prime H so when F3 Prime 5 Prime H is not there the anthos still produce in

huge amount but when F3 Prime 5 Prime H is there that they H from pH means Bonia hybrid the the the concentration of an

you see the intensity of cation is much more even it is much more even further more probably when they have used a

asile transfer and they compared this with vtis Venera vtis Venera is known to

produce anos cell cultures of vtis Venera with elicitation can produce anthos of this level but with tobacco

expressing this transcription Factor genes along with the Gen of the pathway it can produce huge amount of antoin and

these antoin they have purified and used in different food products as well so this is very interesting with this I end

this class thank you