Monoterpenoids in Floral Scents and Metabolic Engineering Insights

[Music] [Music] welcome to nptl online certification

course on pharmacognosy and metabolic engineering with this is lecture number 41 where first I will discuss the

monotaro as component of florals and volatiles and then I will briefly uh discuss one me metabolic

engineering case studies where a gene of monotoring synthes was expressed in a non in a

model plant with an name to produce floral scent and we'll see what is going to happen upon expression of s gen so so

let's go to the uh concept covered so the concept covered is monoterpenoids dominate

florals and boket that is our first part of this discussion and then we will bring the issue of metabolic

engineering so uh when you talk about the floral scent although in this class I will only emphasiz on the monotaro

component of Flor elent but uh to give the proper introduction about FAL sent is

this that Falin consist of uh multiple group of compounds okay so monot Tarpin or monotaro are one such group apart

from monotaro the cisoid are also present in the floral scent and also benzenoids constitute a major portion of

floral scent in addition to that aliphatic alides fatty acid esters that also constitute the floral scent uh so

floral and basically a mixture of different components so that means that different metabolic pathways are

operating in the flower in order to produce different send components and uh but the emphasis now I

will only give on the monotoring components and when I will talk about benzenoids pile propanoids then there I

will bring the issue of benzenoids and components so just to fulfill your curiosity that

how we study floral and biology as I said that floral scent when we get the smell through our alory nervous system

that it's basically a mixture of multiple compounds uh which could be as many as 50 or as low as 10 to 15 so that

means that when we uh we it is important that we need to analyze the scent component so so how to analyze that so

gcms is the major technique which is uh usually employed for analysis of s volatile

compounds okay and uh next slide I will talk about how s volatiles are trapped but now so let us go to this concept so

upon gcms analysis we have uh we now we have identified that are multiple uh multiple uh volatile

compounds present and based on that for example uh we get say

B we get C we get D so these are the different volatile compounds so uh so once we know this once we know

the chemistry of this and we need to find out the logic or any relationship exist if any between

these compounds so and based on our chemical knowledge if we can uh if we can theoretically predict that such

relationship exist for example C can be formed from B simple by a Hydrox relation

then there must be uh hydroxy

enzyme which is carrying out the reaction okay and then for example the

in The Next Step so that means C2 d d compound is basically a methylated compound so we can think of that there

must be a methylation reaction or methy transfer reaction is going on which relates to methylation so methy

transparence reaction so like that way and then once we know know the structure of B then based on our knowledge of the

pathway we can also uh make a prediction that b probably is formed from a and a by another

enzyme which perhaps uh chin shortening enzyme orain cleage enzyme is functional and that leads to the of B that means by

analyzing the compounds it is possible to find out the relationship between this and we can predict what sort of

enzyme activities are there so once we know that then what we need to do you can get these

compounds synthetic compounds and then you can carry out invitro

enzy so invitro enzy that means you take a test tube add the compound a which is the substrate for the cpage enzyme and

you make a protein preparation for from the floral tissue and then carry out the reaction in the test tube and see

whether uh the crude enzyme which consist of the chain cage enzyme as well makes B from a if it is successful then

it's fine so the next step will be you add in the test tube B and carry out a similar reaction and see whether it

makes C and accordingly the reaction which constitute uh say methy transference

reaction uh so whether that exist or not so that way to you have to add the C in as a substrate in the test tube you may

have to add a methy donor Sam and as a result of that maybe the D will be if D is produced then you can be certain that

such enzyme indeed exist once we know that then it is possible to uh look for the genes

so once we know the enzyme so we can also already find out the what are the genes so the genes to be isolated so the

earlier uh days not earlier days even uh even decade back or less people are using uh degenerate primer based Gene

isolation because no sequence was available uh and then then you have to get the partial uh fragment and then

that to be cloned and then full length fragment to be synthesized uh and then subsequently it

can be used for functional analysis or those who are not interested in that simply to study the expression then uh

from the core cdna fragment primers can be designed and expression can be analyzed but now nowadays with the

advancement or with the uh cheap availability of the transcriptomic data so the transcriptomics approaches are

usually followed uh and from the transcriptome data the genes are isolated and

characterized so this is in general so that means first compound enzyme Gene so that is a classical concept and the

concept is still valid now what I'm showing here this is basically called a floral head space so

of a live plant flower is inside so that is emitting volatiles and this is a glass vessel so which is called Head

space so and where uh sents are emitted by the flower and this head space is filled up with that and then it is

connected to a vacuum uh through a vacuum pump and in between uh one has to keep a filter what they have used so

sometimes we call this as a column uh which will absorb the volatile so if you uh if you attach it with a vacuum pump

and in between if you put this column then what will happen the all the volatiles will be trapped in the column

which what is written filter here and then later this can be detached and the the volatiles can be uh the mixture of

volatiles can be collected in organic solvent and later analyzed through GC or gcms and eventually you will get this

sort of chromatogram and and with the use of uh library nist library mostly the

compounds can be uh identified and Confirmation can be done by running some standards this is in general and this is

one real example in the basically in the laboratory this is what is the head space and this is a live flow inside and

here this is what I call the column which consist of polymeric adsorbent which will absorb the volatile

compounds uh and then later it can be detached those compounds through organic solvent by passing through organic

solvents and then it can be concentrated and analyzed uh using gcms to identify the

compounds so as our point of discussion is on monot Tarpin monotaro so I have mentioned in the previous class that l

monotoring alcohol so lineol constitute one of the major uh

component of Jasmine floral sense so this is basically a Jasmine uh this is Jasmine

flower and the scientific name is jasam sambuk and so this is the map

pathway which is which is constituting the ultimately the monot tarpine so the dxr DXs are the

two R limiting enzymes and here is the tarpine synthes so tarpine synthes is a big class of enzymes so where there are

multiple tarpine synthes ex enzymes exist such as El stands for LOLOL synthes okay uh b stands for oin synthes

uh this is both are tarpine synthes so bet synthes b stands for so uh these are the major major tarpo monotaro

components of Jasmine so that this was studied and in order to know more about the biology of the scent emission it is

important to study the physiology of this and um just I'd like to show you uh one uh time course analysis so this is

basically the dxr which which converts deoxy dulos phosph dxp into M so this is the enzyme and the activity was measured

throughout the floral lifespan and you see the activity follows a

typical rhythmic pattern and similarly different part of the floral tissue was also

analyzed uh uh and also the petal layers are also analyzed okay and this is tarpine

synthes activity Tarpin synthes also activity also follows a typical pattern so this is from the throughout the

floral lifespan and what has been found that petal tissu is actually is the place where maximum tarpine synthes

activity exist and the all their content Tarpin synthes activity outer layer is little bit more and this is basically as

you see CMS uh uh analysis where different components are uh detected so that means uh when tarpine synthes assay

is done so the gpp is to be taken as the substrate and the uh flower

petal protein extract was used as a source of enzyme and this

gpp if it is able to make the products for example linear L and other components so

that can be detected through gcms analysis okay now what I will show you very interesting thing as the flower uh

remain alive for several days so what is shown here that this portion is basically the night

time what is shown under the shed condition and the the portion unshaded are basically the

daytime what you see particularly for the LOL the LOL uh maximum LOL uh concentration you

find during the daytime that mean during the daytime compared to the night time for

example the Lal that as in case of harnessing although that is not our point of

discussing the C it follows a different pattern but if you again look into the geranial linalol so they are also

maximum during the daytime simply similarly another monoid which is uh neurol that is also uh maximum

accumulation was found in the daytime later when we will study the benzenoids there you will see that benzenoids

accumulation uh actually reach this peak during the night time because there is a

relationship between the benzenoid emission and the pollinator phit particularly The Moth so but the Tarpin

particularly monot Tarpin are emitted more in the daytime so such such import such

information is important because if one want to study the gene expression or isolate the gene so it is important that

which time point the petals to be used for such analysis so this is an

overview that is the sub cellular storage and emission so this is the plastid where map pathway is operating

and of course in the cytool MV pathway is operating okay so our point is only to see this now right now so

uh it makes gpp and then the gpp constitute the gpp basically serve as a substrate

for monot tarpine synthes so like tarpine synthes and it makes the monot Tarpin including LOL and this may

directly emit okay or this can be stored in the vacle so this can be stored in the vacu

either as free endogenous volatile or we can call free internal pool or this can be

uh and this can undergo glycosilation that is it linked with the glucose and then it's safely stored there and as an

when required then there will be de glycosilation or glucosides activity that leads to the uh release of

the aglycone monot Tarpin and that eventually will be emitted so that means that there are three ways one one the

monotor produced directly emits second is the monotor pins can temporarily stored as a free monotor pins in the

vacu OR for a long-term storage they can link with the sugar and stored there so this is basically an overview uh of the

different monotoring components and how they are stored in the in a plant cell and they are always low molecular

weight compounds liphophilic liquid and the emission white hems because it's a high vapor pressure exists now influence

of temperature on biosynthesis and emission so one study was done with another jasminium species which is

called jasminium oriculum which is called ji or ji uh so for these plants were grown in the

plant growth chamber which is basically the picture of growth chamber uh where the four different

temperatures were used so in one instance the plants were incubated at 20° another instance 25° another

instance 30° another instance 35° okay and uh the plants were kept just at the time of bir formation so the

birds developed and the and it bloom and then emission happened and accordingly the volatiles were collected this is

basically the head space uh and then it analyzed not only the not only the emitted volatile also the internal pool

of volatiles what is present that was also analyzed so what you see here that 30° is Optimum for the maximum emission

of linalol as compared to the other temperatures so even uh 30° temperature

is is slightly better than 25 for lineol accumulation inside the cell so from this

knowledge uh what is the practical application is this that okay growing the plant is growth chamber for

commercial purpose is not visible but such study will throw you light how can you grow ji or

julum so so you can use a shed net you can use a uh plastic house and you can maintain the temperature 30° by putting

fan and cooling pad and then you can grow there and if you grow the plants outside where temperature variation is

large then you'll be able to find out the difference in the uh concentration of the volatile components which

accumulated when you grow at 30° and when you grow in the outside the field so it has a large impact because uh

these flowers are used for different cosmetic application not only that even for the as a food ingredient

particularly it's mixed up with the black tea or green tea to make herbal to make jasmine tea so it is important that

the concentration of this compound should be at the highest side and not only that suppose if you want to make

other products like a and other uh so from there maybe different uh it's uses in different uh cosmetic applications so

there are also so it is always better to have a plant material with higher concentration of the desired metabolite

so this is one way one can really make impact in the Horticulture industry uh by enhancing the uh

volatile tarpine contents in the flower simply growing at a particular temperature and which is

possible so and so then there are some fundamental studies like as this Tarpin since sy so tarpine synthes expression

was studied at four different temperature so like 30 here particularly TPS there is not much difference uh so

the gene expression level is more or less same but what happens the beyond the expression level that is of course

the enzyme activity so that perhaps makes difference in the final production of the volatile components this is what

it is so it's not all that gene expression uh Hy gene expression makes the final product it may not be as you

see that okay 20° tarpine synthesis is slightly uh less expression but whereas 25 30 35 there is not much difference

similarly it is true for both the MV pathway gen also so that means the post gene expression period is important or

ultimately for which will determine the concentration of the volatile metabolites accumulation in the floral

tissue so so this is basically uh in brief about some studies done with Jasmine uh with an name to give some

clue to the Horticulture industry how can uh how can the cultiv or the farmers can grow justment uh in a more

scientific way that can leads to more product accumulation and which will give a better profit to the farmers so that

is an applied aspect now we'll go to more of the fundamental aspect that is now we talk about the metabolic

engineering application so so

the metabolic engineering of

[Music] monoid pathway I'll try to explain in a very simplified

way uh first of all what is the target what scientists wanted to achieve so they wanted the first of all the source

Gene Source gen is LOLOL synthes l i n a l lol synthes l i s

now one uh one information I need to tell you that linalol synthesis basically the gene was isolated from a

plant called claria Briar it is a plant of native to South

America on aggressive family it produces big flower and the flower emit volatiles Which con which is basically

dominated by monot Tarpin as well as by benzenoids so among monotes among monot noids rather the the

linalol is one of the major dominating compound so actually that was the driving force behind isolating the genes

of linalol synthes so before Gene isolation they have also studied the pattern of linalol synthes enzyme

expression throughout the floral lifespan as I have shown in case of Jasmine so here I would like to

show you this in the form of a uh graph so like the level of activities uh so

level of laral synthes activity

uh in in uh

flower tissue throughout their

lifespan so for example okay this is -2 this is

-1 okay and this is one this is

uh two and then this is three and so on so what is this basically this is called

time post enthesis post anthesis means that when

the when the B matures it opens andjust it it coincides with the bursting of the anthers that means

the this is the anther uh so this is anther uh mature so when the anther will burst that is

called deance of the anther so that leads to so and what you will see

that Poland are released these are nothing but the

so this happens uh the phenomenon called anthesis so

time post anthesis means that that at 1 it is the time where when the anther bursts and it releases the poen and that

is the time also The Petal opens so that means the B matures and it it fully blooms and at this are the points where

the laral synthes activity was measured so laral synthes activity was measured so like I'll put the scale y AIS is for

the activity so the Lis

activity so and that may be a unit called uh picoc cat or nanocat flower tissue so that anyway I'm not

talking in that details uh so what I'm going to show here is this that how the Lal synthes

activity behaves so this is two and then this is uh

one and then one goes down and it goes further

down two and then now if you join this point then you

will find that uh so that means the maximum linal

synthes activity will find at minus one that means just the day before the opening of

The Petal okay so that means the petals are more or less matured but the B is still

closed and that is the point maximum Lal synthes activity was detected and one is basically Bally when the uh one day Zero

means at the time just opening one is the day uh just the anthesis after the anthesis so it is basically getting down

so this is typically the pattern of linalol synthes activity so this is very important so to know that how this

reg tarpine regulation happens because it will be also interesting uh to uh to see that uh how this differs

okay uh in the different uh plants how this differs in the different plants okay I just made a

small leave it like that okay now this is what it is uh and uh next point is this that this Source

what is the the source of this is linear synthes from the claria so if you go to the

next slide so the source Lis

from claria r and now the

target target plant they have chosen arabidopsis taliana so arabidopsis is basically a

model plant used in plant biology okay and uh so they transfer the linalol synthes gene from claria to arabidopsis

so the method what was used is simply agrobacteria mediated transformation so that means the LOL synthes

Gene is here so it should be under the control of a promoter and a Terminator so

Terminator is added here and promoter is here but in between the promoter and Terminator uh what needs to be added is

basically a Transit peptide sequence and why because this is the sorry this is the

promoter right okay I will so why Transit peptide because what I have said in one in one of the

previous classes that the monotoring pathway is operating inside the plastid and this is basically we are

transforming the gene into the nucleus so the the through agrobacteria mediated transformation so that

means using a [Music] tumes the gene was transformed in in

arabidopsis and it is integrated into the nuclear genome from there uh transcription will happen and it makes

the Nal synthes messenger RNA and then finally Nal synthes protein but if you do not put the transit peptide then that

protein will be there in the cytosol and if it is there in the cytosol it will not get the substrate because remember

the gpp is the substrate and gpp is basically localized in the plastid so therefore the transit

peptide sequence was added because that once the protein is produced after translation and it should contain

the transit peptide at the inter terminal region so it will drive that protein inside the

chloroplast and then it will be able to function so Transit peptide adding basically driving

the uh protein into the chloroplast what is the site for the enzyme activity okay so that is what has been done now

question is that what is the result result is this that they have successfully

produced transgenic plant arabidopsis plant expressing Eli

Gene okay gene expression was confirmed everything but what happened that when they try to measure the

LOLOL uh from the floral tissue unfortunately

no lar L was detected in

transgenic aidis flower where Eli was expressed so in fact Eli was

expressed constitutively so so the constitutive promoter was used that means throughout the plant linalol

synthes was expressed but anyway it is expected that linalol is mostly is a botar volatile as the plastid is the

component so they check first from the flower or later also they check from the different parts but no linalol was

detected so then why no linalol although the gene expression worked very well uh so that means the gene was functional

and the substrate gpps should be available uh so why it is not that is what is the question is so then they try

to find it out what happened so then they did a very careful analysis uh they have made extract from the floral tissue

as well as the vegetative tissue and what they found that it is not linalol but basically they have the this time

they detected detection of

s linol beta D

glucopyranoside so this is what they have found in the tissue so as the name indicates it has some relationship with

the linog so what sort of L so it appears that linalol perhaps produced and then

it under goes glucosylation leading to formation of this compound which is nonvolatile and which is stored in the

back wall okay then what they did basically they uh basic they try to get the LOLOL out Simply they

they treated this with beta glucosides so beta glucos is what it

will do it will clip the glucose out so now what you see that

linalol plus bet glucose okay I put bet glucose for for better writing

Plus so glucoside is treatment in the tissue now leading

to detection of linalol plus beta D glucose okay so that means it is clear that

Lis that means LOL synthes gene or successfully expressed the Gen was function it it took all the all the gpp

available in the tissue and converted into

linalol but the plant found LOL to be a strange molecule because it's not meant to produce LOLOL so immediately what

happens that glucos ale transfer is induced and as a result of that linalol is stor as LOL gluco

Panos site okay so what happens that this is because of

this induced glucos ale transfer is endogenous and this is

the transgenic Elias or Elias Gene we can say transgenic elas

Gene means that the gene was basically transferred from a different plant so I okay better we use the

term Gene from this uh what is the claria claria species El gen so this is what is the outcome so

that means plants are successfully producing linalol but converted into linalol glucoside and that is maybe a

sort of detoxification mechanism or some s of adaptation so that means the message is that that whatever you want

to do with the plant plant may not do they also equally clever they will do whatever they feel that which is to be

which is necessary for their proper survival so this is a nice work and this paper basically published

in a famous Journal which is called which is plant journal the pl

Journal full name is the plan Journal uh in the year pretty old 2001 volume 27 page number 315 to

324 so the what authors have written that linalol produced by the plant in various tissue is not present as free

linalol but efficiently converted to nonvolatile linol glucopyranoside by the action of endogenous glucos ale transfer

so what I have said that this is induced so the result present demonstrated that yes monotoring production can be altered

by genetic modification and that the compound produced can be converted by endogenous

enzyme activity so there there is the challenge how can you really make the plants to produce pre linalol it should

not get converted into a glucoside that is the challenge and that is the difficulty with the plant system so with

this I end this class thank you