Comprehensive Insights into Vanillin Biosynthesis and Localization in Vanilla Planifolia

[Music] [Music] welcome to nptl online certification

courses on pharmacognosy and metabolic engineering this is lecture 58 eight where under the broad domain of phenolic

I'm going to discuss now the biosynthesis of peline in plants so the concept to be covered is vanilline and

vanilline glucosides and then vanilline biosynthesis in Plants classical and

contemporary views and then uh histo localization of vanilline and vanine glucoside uh in the

tisue uh okay so again Pine as the structure says so so uh this is four hydroxy three methoxy

benzal deide which is venine and this is originally a C6 C1 compound so that also produces from C6 C3 so it's all started

with with this basic Phile propanoids and then there will be chain cage so we are going to see that so next let us

look into the okay now natural vanilline uh usually obtained from the vanilla pods so vanilla is basically an

orchid so I will talk about it in the next slide and from extraction natural vanilline can be obtained or even from

the corn glucose glucose can be uh utilized through fermentation for the production of veline other one is that

ferulic acid which is basically abundantly available in the cell wall material and also in bran so that can be

converted into vanine because of its structural similarity as you see here four

hydroxy three methoxy uh it's the the hydroxamic acid and it is this part is same so just this chain cleavage from C3

it becomes C1 and of course this acid and this is aldhy okay uh

eugenol ugol again was successfully used as a stter substrate for production of vanilline particularly utilizing

different microorganism both bacteria and fungi okay so uh okay I will now go

to the Natural vanilline so natural vanilline is basically obtained uh from

the ripened fruit of orchid called vanilla

planifolia and this fruit is botanically called pod so this is what is showing here is a

green or rather young

fruit so this fruit with age converted into the mature food fruit and then eventually it will die so the green

becomes black in color so this is what you call uh mature fruit which is now dead that

is why it is black in color and what happened now vanilline is there

in both the cases so now if you take this tissue and analyze if you make extract

and analyze you will find uh vanilline not in the free form but as glucose

conjugate so this is glucose okay why it is so because it's an alide alides are very toxic so

as it is synthesized in the cytool then uh the plants cannot really retain so it it it immediately try to move it inside

the vacle so there will be induction of glucos Ale transfer so the glucose molecule is added so it becomes

vanilline glucoside so what we write here this is called vanilline glucoside or glucovanillin

and then when the fruit dies then what will happen by the

time there will be induction of uh glucosides so induction

of glucosides and here we write that induction

of glucoside transfer is and as a result of that what we see it

produces this glucovanillin and when glucosidase is induced then what we'll see here the glucose molecule will be

clipped out okay and

living pre vaning this I have to dra it one

again okay this is free handline so the glucose is now

out so what is available in the market is basically this for product available in the market so you

can buy it in big supermarket black colored vanilla pod or it is also called vanilla

bean okay so uh now if you Analyze This vanilla bean you will find vanilline as

the major fragrance component apart from that you will find parah hydroxy benzal

deide vanil alcohol parah hydroxy benile alcohol and then plenty of other molecules present

there so that all together constitute the typical fruity vanilla Aroma but again the if you take the smell of this

natural vanilline and if you have the synthetic vanilline available you will find the differ synthetic vanilline is

just vanine what you get in the ice cream but the natural vanilline is more of like chocolate uh fruity flavor and

of course that is expensive so now let us see how vanine because of its demand it's it's the

world's most favorite flavor compound so because of its demand and it's high cost because one kilo cost more than $4,000

or so the natural uh vanine so that is why the scientists were interested to understand how velin synthesized in

plants with an aim that they want to do metabolic Engineering in the future so the velin biosynthesis work started as

early as 19 1970s by scientist called MH

Zen in Germany so Jen even Jen proposed that vanilline can be formed from ferulic Acid so I have

mentioned about ferulic acid in one of the previous slides so ferulic acid

through a beta oxidation root it may produce vanoil

qu and this subsequently by uh elimination of hydrogen so it

[Music] produces vanin or it can also

produce vanilic acid so vanilline and vanilic acid structure is pretty same okay I put in this way

CH o O3 this is

vanine and if it is vanelc acid then this will be replaced by Co then it will

be vanilic acid and if it is Van vanil alcohol then this will be replaced

by ch2 o so that means so all these components are present in

the natural vanill in natural veline so which is obtained from the plant sources now uh after Jin propos then

there are lot of work uh carried out with this so like uh a person from Switzerland whose name is Peter Broadus

so Funk and Broadus so they actually worked during

1990s so they have used cell culture

of vanilla planifolia in order to elucidate the

biosynthesis of vanilin and what the found using cell cultures they are unable to get vanine instead they found

the cell cultur successfully produce uh vanilic acid glucosides

from I form from isoferulic acid from eventually it produces from isop IC

acid and this vanilic acid subsequently form vanilic acid

glucoside so what happens this isop ferulic acid is it same like ferulic acid when we draw the ferulic acid

structur so the no the structure difference is not at C6 but this at C6 position not at C3 so in case of heric

acid so here is oc3 o in case of isoferulic acid the O3 will

come here and O will be come here so this is ISO

ferulic acid ring part remains same co co so this is fuic acid

this is only the difference so Frank and Broadus could not detect velin and then a group from

Japan who is Kiwa kanisawa kanisawa kanisa k a n i s a w a so kanisawa and

his group in 1994 they proposed a different view of B information

utilizing vanilla pod as the source tissue okay so

there but but this was published in a conference proceedings but it never published in any uh peer reviewed

Journal that is why scientists were not very optimistic in accepting their hypothesis so now much later uh group

led by Richard Dixon so they actually try to elucidate the veline biosynthesis uh in

using both cell culture as well as the vanilla [Music]

planifolia placental tissue as a source so what the proposed that vanilline is formed from

parakum aric Acid so all of you know the structure of paric acid I'm not drawing here so paric acid or for

hydroxy camic acid so parakum aric acid will make parah

hydroxy benzal deide and uh then parah hydroxyz deide eventually will

form 34 dihydroxy benzal deide and then so that structure I put it

here so this is 34 dihydroxy Benz deide and it is likely here

uh there's an involvement of cytochrome p450 enzyme why because parah hydroxy benzal deide

is only one oh here so another o so that is why it is

basically a hydroxy but this hydroxy has not been characterized but interestingly there's

another group from Korea that group basically Al isolated uh 3 omt from vanilla

planifolia pod and this 3 omt converts 34 dihydroxy benzal deide to to

vanine so the enzyme is three Ile transfer so me that means

it requires a Sam so here the hydroxy is replaced with the

myoxy and that is possible so this this was characterized at the gene level so the gene was basically isolated and

characterized well now this speaks in favor

of paracom acid as the starting material for the biosynthesis of velin okay so this paric acid will

make this parah hydroxy benzil deide and then it should undergo another Hydrox silation which makes 34 dihydroxy benzal

deide and then uh this will convert into veline by a Ile transference so one point here to

tell that this St particularly which converts paracom aric acid to parah hydroxy benzal deide the

enzyme was characterized and it was called HBS hydroxy benzal deide parah hydroxy alide synthes so it's it's it's

it was found to be a 16 proteas type enzyme and it requires dtt for its Activation so more dtt more

active but uh when they have isolated the Gene and use it use the de combinant Gene so it did not really

uh show very much positive result particularly from the point of this vanine pathway so that is there

so they basically did not publish this work at the molecular level rather they kept it at the biochemical level only

they suggested that this is possibly an enzyme so which is playing important role making parah hydroxy benzal deide

and which subsequently converted into vandaline so this was there so the

work I mentioned this was published in phytochemistry 2002 and this was published in most

probably in PL sell report in 2004

so that was the status after that uh we see an very interesting paper from Denmark so

where uh they claimed so let's go to the next slide so this paper appeared in uh nature

Communications in the year of 2014 and they said vanilline formation from ferulic acid

vanilline formation from

ferulic acid in

vanilla planifolia and this is catalyzed by

catalyzed a catalized

by a single enzyme and this enzyme they

called vanilline synthes so

VP van they have given this veline synthes VP van and what they

proposed that this VP van is basically converting ferulic acid to feline and

that let us see how that fuic acid is formed so that ferulic acid is basically forming from this Capo qu so let me I'll

not draw the structure because all structures we have seen in the previous classes

so Cil qu from capil qu uh it

makes cafeic acid and from cafeic Acid they say it

makes ferulic acid and from ferulic acid veline is formed and this Anin eventually converted

into veline glucoside now if you put the

arrow so Capo Co to cic acid basically it's basically the theris so theris will release

the uh what it releases the which color I will take okay it release the qu higher out

and living CAPIC acid and that CAPIC acid will be converted to feric acid by a CO

Mt cic acid othy transfer so which basically adds at three position a methyle group which makes fuic acid and

this ferulic acid will converted into veline and this is they have named van and and they also proposed that this

is likely a uh hydras lies type of enzyme so I put here Hydra is

lies and then subsequently this converted into velin glucoside so if we see the structure of peric acid

here okay let us use another so this will be converted into one

intermediate this something hydroxy fenile propic acid what we have seen in case of this

uh Beno pathway what I have discussed in the previous in one of the previous classes and then this will form

are not good at all so the the

enzyme VP van VP means vanilla planifolia vanilline synthes catalyze both the step so the first step

is basically a water molecule is added so that is why this is a hydration and this is

basically cleage chain Gage so hydration what you can also put it here uh molecule of

water and here with this AAL qu is removed or acetic acid is removed no qu is involved

so acetic acid is removed so basically this may be a qu independent way of uh peline formation where from fuic Acid uh

it first it makes an inter intermediate so whenever we put an intermediate we should put it

under uh third bracket this means intermediate and then it immediately converted into vanine so

it is basically y step enzyme catalyzed by the single enzyme and subsequently they have characterized this as the

molecular level uh so the second one is called retroaldol keep cleage and first one is basically the hydration now now

this paper published in as I said it published in the famous journal

Nature uh biotechn uh so nature Communications in the year 2014 volume Five article number 4037

this time it is basically totally online Journal so it's article number is there and this is basically the group head by

bergar Lindberg mher from Denmark so University of Copenhagen so once this one published

then again the group which led by Richard Dixon uh from this uh

USA so that is samel Robert Noel foundation so uh so and they collaborated with several people in

Italy so what they did actually so they again started relooking the vandaline biosynthetic pathway again and what we

see that they have published a paper in the year

201 17 where they say a re-evaluation re

evaluation of the final step

of vanilline biosynthesis

in vanilla planifolia and this paper published in

again a very good Journal which is called phytochemistry a traditional Journal of its kind publish

this sort of work so that was published in the year [Music]

2017 uh Vol uh volume 139 page number 33 to 46 so what they said basically

they challenge the nature communication paper and they said so let me read out you listen a recent publication

describes an enzyme from the vanilla Orchid vanilla planifolia with the ability to convert

ferulic acid directly to vanine the authors the authors of that paper propose that this represent

presents the final step in vanine biosynthesis which is then converted to its storage from glucovanillin by

glycosylation so the existence of such vanilline synthes which I said VP van in my pre previous slide could enable

biotechnological production of veline from ferulic Acid uh using a natural vanilla

enzyme but now the point is this the this proposed baline synthes has exhibited high identity to 16 proteas so

the 16 proteas I mentioned when Dixon group in 2002 in the phytochemistry they said

that this is the HBS hydroxy balide synthes which converts karic acid to parah hydroxy benzal deide and that

enzyme they said Sy proteas but here also uh so here also the the nature Communications paper also peline synthes

what they have characterized and they said that that is basically show high identity to 16

proteas okay now this current phytochemistry paper uh what they said they reassured that if it is 16 proteas

it really cannot really May cannot really work with ferulic acid as the substrate

so it only able to convert paracom acid to parah hydroxy benzal deide not the feric acid so in other word if it is a

ctin protas like protein it cannot convert feric acid to vanilline so

that really is basically they contradicting or rather challenging the other group and what they said is this

that the the path way what is operating in vanilla planifolia so so it's It Started from the simate so let me

briefly put it here so uh okay so simate which which simate it should be uh Cil

simate okay we start with CFO sment c a f f e o y l let me write it in a clear way okay we can start from a little bit

of arly also like let's start from this point parar acid or for karic acid is same for karic

acid what uh D group proposed the first author was Port stalsky so this makes

this makes four hydroxy benzal

deide and that subsequently by the action of a cytochrome p450 I put the enzyme

later uh which makes uh which makes

34 dihydroxy benzal deide and and this one by the action of a Ile transference it

makes peline this I have shown so the enzyme what they said that this is a HBS or 4

HBS type of enzyme and this is a p450 and this is basically our omt this has been

characterized okay and uh this is likely to be the root or the other root what is operating in

Plants uh maybe in this direction for karic acid will will make uh forar qu and then subsequently it

makes omaro simate which we have studied and then it

makes Cil simate then it makes cafeic

acid so if if you put the pathway this makes this this makes this this makes this so

what we know that this is 4 CL this is

HCT this is C 3H and this is CFO simate Estes which will remove

the simate leing cfic acid and this uh by the action of the enzyme CC Mt

sorry we'll make we'll make ferulic acid and this fic

acid the will be converted into converted into

uh Conifer Al deide and then by the action this is by the action of

uh CCR so and

then this will be converted into Al dehy so this will be converted

into fulc acid and then this fuic acid

2 so there is a big question here uh so this

one will be LD DH alide dehydro renous

so so the biosynthesis between feric acid to karic acid is basically uh through this root and what

they said that uh feric acid may be formed but this ferulic acid is most

unlikely to convert into uh vanine so what is possible

that uh this C Mt this means the Capo

Sate so in between it is possible that it may be also that uh there may be a foral reaction also there so in that

case from this capil simate uh from the okay from the cafeic okay

from the cafeic acid a c qu may be produced Cil acid C qu can also be

produced by the action of a traditional 4cl

and then this capile koi Maybe be converted into ferulic acid by the action of

CMT so in other word this may not be the case so and then it's converted like this and

the Capo qu can take part uh in the formation of

veline through some undefined Roots okay so which may involve a

hydras which may involve a dehydrogen and which may involve a

thol and then finally there may be a reducted so that means what this group

proposed that from fuic acid to veline is not possible and uh it it should follow a different route what they has

they proposed earlier so basically this was a scientific War uh started between the two group both of them have their

own views now let us before I end this class let me show you the tissue so this is again this is again a

paper published by this uh Danish group led by Bard Lindberg M so this paper published in plant cell

physiology again on this vanilla planifolia and this time this was in the 2018 uh volume 59 page

number 300 4 to

318 so here uh basically this is a crosssection showing different part of it so the

mocar portion is very thick and then endocarp is inside the White portion and then there are seeds inside

and then placental tissues are also there and then vascular tissues are also there

so here what they have done they have done this uh this dis option

uh uh this is uh this this is a specific mass spectrometric ionization uh mass spectrometric and that uh leads to this

picture so they have done a longitudinal section and this longitudinal section they do it by cross sectioning

and then they have uh they did directly mass spectrometry of that and then what you see uh through this new technique uh

where uh the red portion is basically the

vanine so this is where the vanine is accumulating and the this green

portion is vanilline

glucoside and this blue portion is basically the sucrose so they have studied the

distribution of this metabolite that is histo localization of these metabolites using this high resolution dsorption

Mass ometry and this is really a very nice paper with the modern tools so where you could detect the metabolites

using mass spectometry a similar Le several papers are nowadays appearing in very good journals where they localizing

the specialized metabolites in the tissue using this technique not only that they have done imunohistochemical

localization of BP van so that that is there that means the vanila pronia van that is veline synthes exists and they

have uh so they characterize the gene so they have the recombinant protein they raise antibody against that and then

they have used that antibody to localize the protein VP van protein in the tissue so that is what it is so what is

showing here is again this is a transverse section so VP van localization in the mesocarp so mesocarp

is is this the thick portion so here is the BP

B localizes and and and then that uh that is also localized in

the plastids and uh so that is in brief about the outcome

of the latest research where that you can also see the uh localization of the uh targeted enzymes so this is basically

all about the vanilline biosynthesis and the vanilline localization in the Vana planifolia so the only vanilla

planifolia plant Orchid produces vanilline in huge quantity but vanilline are produced if you if you isolate the

cell wall you will find uh and if you do the fen analysis you will find veline there and but that veline is basically

coming as a result of this uh clivage of this Conifer alcohol or Conifer Alid so it's not by the normal root of pyin

synthesis so with this I end this class