Metabolic Engineering of Carotenoid Pathway to Enhance Provitamin A in Crops

[Music] [Music] welcome to online nptl certification

course on pharmacognosy and metabolic engineering well now go to lecture number 43 where I will discuss about uh

carotenoid pathway genetic manipulation that means metabolic engineering of carotenoid

pathway so here I will cover two case studies one is basically genetic engineering for gazan thin Rich

potato and second one is about improving Prov vitamin A content in tomato so let let us go to the board

now so genetic engineering for for zanin

Rich potato so how the strategy so they used the Gen which

is zantin epoxides z e which is janin epoxides I have mentioned in the last class and

they have used two strategies one is basically by over

expression and another one is by anti sense expression or anti sense suppression anti sense

inactivation well so uh and the gene s was potato itself so

the gene Source potato Z

something uh so to show you the pathway once again for a brief

revision is this that lopin lopin makes

Alpha carotin and beta

carotin and from beta carotin zanthin is produced okay and

zanin in The Next Step converted into into ananin and

then V so and th s

Thea and then y okay and then it may move to Neo zanthin so in the while type

potato while andin accumulation

is pretty prevalent and in the wild Ty potato you will also

find Litin so this is what happened in the normal wild type

potato so while type okay

so the step what uh they have decided is basically this

step which is this ZD which converts Zan 2 and th

Zin and Anan to ventin so this also I have shown in the

last class that there are two successive epoxidation is required leading to formation of Vos zanthin so

the strategy one is basically overe expression that means the Gene simply extra copy was

overexpressed and the outcome of such OV

expression led to the phenomenon which is called

cotion that means the additional copy inserted into the uh plan genome and it express that eventually leads to the

shutdown of the zanthin epoxides activity so which is called Co oppression so extra copies

hutting down so the co supression term was used uh nowadays it is not using much but the paper basically it is

published in the year 2002 so that was the time when this RNA interference mechanism was not fully

iated so this paper was published in the Journal called metabolic

engineering uh in the year of2 volume 4 page number 263 to 272 the first author is RAR r m

RAR from German laboratory okay so this leads to co operation Co

operation means the no Viola

zanthin production in the transgenic plants

whereas in case of antisense Separation that is straightforward because there it is the strategy is

clear basically you want to shut down that and as a result of that here also same thing will happen

that no banthin or in other words the V banthin

amount was diminished drastically okay

and maybe little bit of ananin accumulated but bolog total gone but as a result of that what happened in both

the cases that uh this leading to the formation of

more more Zan so that means when you are blocking this by anti sense or try to make OV

expression both leads to basically shut down of this pathway leading to more accumulation of

zanthin so the level of Zan level

increased from 4 to 130 fold depends on the uh expression level

okay another point is that when higher zanin level was found that also Al showed that increase in the total

carotinoid content so the next point is this that increase in

the total carotenoid content was

noticed and uh this increase up to 5.7 ft as compared to the

control and also what they have found that in in few lines uh reduced amount

of Litin was also found D amount of lutin was also detected this much is fine so that means

that when you try to block here what happens that anyway as a result of that what expression it it led to co

oppression as a result of antisense it's basically it blocked the uh enzyme so Zan in both the cases Gan amount

enhanced as a result of this enhancement so so what they have noticed that the total carotin and contents also enhanced

but the Litin amount was got uh reduced in in particularly in fuel lines but and the another interesting observation

although it is difficult to correlate but I'll try to uh make a correlation that there will be they also found two

two threefold increase uh concom

tant 2 to 3old increase

in Alpha too this is O

feral which is vitamin E content in the transgenic plants

so now why how can we correlate with this too feral increase so let me try to explain because Toof feral is not here

in this picture but what they found as a result of this separation the too feral content increased let us see that so uh

too feral normally produced from map pathway so I just briefly tell you the origin of too

feral so one say one is the Sate

pathway which supplies tyos and another one is

the map pathway and this pathway is basically contributing to the formation of

chlorophyll as well so here if I write briefly loril

and of FTI tail is there p h y Ty y Lyle pyrophosphate is produced so from tyrosin what happens

that it produces the homo gentisic acid homogentisic acid

ho what is called hga

so homogenic acid and fle tail they joins together and forms the tocoferol which is vitamin

E and the relationship is this that map pathway also contributes towards the formation

of kerotin prots

including Pro vitamin a which is beta

carotin so the relation is bit very distant but what happens that uh uh and the

fle pyop phosphate is basically it is produced if I show it to you that it is basically coming from

ggpp so M pathway this

is uh coming from and is a GG reduct is enzyme plays a

role and uh so so the relation is this that when the total carotin content

enhanced uh what we found as a result of this antisense separation I don't I me the point is

this that enhancement perhaps drives the map pathway the flow of the M pathway flux

more towards this pathway and it is possible that as map pathway is more active so perhaps that leads to more

formation of this uh okay I made a one small mistake so uh this gpp reductors will be here uh

more GG reductors it is here so more formation of ggpp so ggpp when there is

more formation of ggpp because ggpp takes part in the CARiD formation so some amount of ggpp is basically

channelized towards the formation of uh this too feral through this file tail so uh right

ggp like this way so we can modify the pathway a little bit more uh instead I take this

out so this is basically the origin of tocoferol and its relationship with the carotenoid pathway uh although this was

not discussed in that detail in the paper but uh as a scientist we need to understand that why this happens so this

may be the explanation for this now with this I move into the another case stud that is

uh improving Prov vitamin A content in tomato content in tomato now we all know that tomato

basically accumulates lopine and lopine is basically a very

strong antioxidant so there is always a need for accumulating more lopine in the

pathway so how like like how lopine is formed although I have discussed so I just make a simplified pathway I not

showing all the details so lopine is basically formed from uh this uh jeta

carotin and that is coming from phin and what I have also mentioned in

the earlier class that this is phyto in De saturas and this is called gartin

desaturase so these are the two plant enzymes which involve in the formation of

lopine and so the uh the the work what I am going to discuss now

basically uh there are two group which contributed in this uh Improvement one group is from UK

group and the second group was from the Italy group so you UK group originally aimed

for making more lopine in tomato and what they have done actually they have

used a bacterial Gene which basically makes lopine from phyto in one

step this is called

CRT I uh carotin isaris

okay so the this is basically from The heterologous Source uh I will not talk more about this here when I'll talk

another case studies I'll will talk there so this is basically the bacterial Gene so

the hetus source so the constructs what they have

made is B basically they made a simple construct that means

the C and in between they put

a Transit peptide and this is of course they put the antibiotic resistance marker gen and all other

things and which I'm not putting they use the agrobacterium

[Music] Tums as a vehicle to transfer this Gene in tomato

now uh a promoter was used this promoter was an constitutive promoter so throughout the plant the Gen is

expressed and Terminator is there so that the transcription will stop there and why TP because uh this is okay

a heterologous gene source and you are putting inserting this gene into the genome but eventually as I have

mentioned that the pathway carotin pathway is basically operating in the plasti chromoplast eventually it forms a

chromoplast so the transit peptide is required to drive the translation product into the plastid where it is

expected that this enzyme will get the appropriate substrate and run the pathway so that is why this they have

put it now what happened as a result of that what they expected that when they have put this CTI under the control of a

very strong promoter uh uh so it should work and Def yes it has worked and it should make lopine but what

happened here that when they try to analyze they found that it is not the lopin but the the

pathway moves ahead and produces beta carotin

okay so that is unexpected result and beta as beta carotin is basically the uh the precursor

of vitamin A so that is unexpected but in interesting result but why it happened

that is the question the the gene should the gene function led to the formation of lopine

yes it it achieved but lopine subsequently converted into beta carotin this is part of happens because of the

presence of endogenous genes which is called beta lcy which I have also discussed in the next class so more

lopine in the cell allows the endo enous beta cyclist to function and it converts this to this so this this was unexpected

result and this paper was published in uh nature biotechnology in the year uh 2000 it's phical in the June

issue okay anyway the result was unexpected but the result was interesting and it has direct bearing on

the health issues and that is why the paper goes to uh nature biotechnology now let us see what Italian group

attempted to do Italian group is the job was uh pretty straightforward what the uh attempted they have made

basically two constructs one is the over expression construct or OE

construct so the over EXT expression construct is basically they have taken again uh a

heterologous gene but it is from the plant Source only so they have taken arabidopsis uh beta Cycles gen Arab of

CIS LC y this gen they have taken and they express it under the control of a promoter and

Terminator so here they need to they also need to add a Transit peptide because eventually it

should be driven into the uh plased and but the promoter what they have used

they have used a uh organ specific promoter or which is called tomato PDS

promoter tomato PDS fatures promoter it is tomato PL specific

promoter and whereas here it is basically the c m 35s promoter which is a constitutive promoter in the other

case okay and then uh and they have transformed uh tomato plant using this construct and

the the other construct which was basically an anti sense construct or as construct so the antisense construct

here they have used the Tomato Gene itself so the Gen

was Tom beta LC y lopin beta Cycles

whatever and then uh Terminator to be added here and then a Transit pate

and the promoter promoter was same Tom PDS promoter and the transit peptide also I

need to show it here which is TP and you have to put this but one important thing uh what we

need to show in both the cases in OV expression construct you have to put the direction

this this direction in this way whereas here you have to put it in this direction so that is why it is anti

sense so in a reverse way so that means here what happens that the three

prime portion of the Tomato beta Cycles DNA was cloned in anti sense orientation so anti

sense orientation under the control of the same promoter now what what you expect

that they all have done the manipulation uh with this step particularly so in case of OV expression

what is going to happen that uh so the Tom eventually we should see the fruit so the Tomato when if it is lopine

Rich then it should be red in color

but uh when you put the arabidopsis beta cyes gen so it leads to the formation of beta

carotin along with lopine so a substantial amount of lopine will get converted

into beta carotin so accordingly the color of tomato should change

into orange if it accumulates more and that is a indication that beta carotin

accumulates whereas uh whereas in case of antisense suppression where basically you are

blocking the step then what will happen that leads to more lopine so then how do you distinguish so if you take a o type

and the antisense Express Plant so the intensity of the red color will differ and from there uh it is possible

visually to check the consequence of this gene expression on tomato plants so Italian group was very successful uh in

in achieving their result and in fact the OV expression construct actually it produces orange or bright red

coloration of the fruit color okay and in case of antisense construction what happens that

slightly darker Hue H

in red fruit was noticed okay and the total koton content also enhanced so

I will I am basically not uh going to give you much of this content but uh yeah okay I can also produce for your

uh information see from the antisense construct uh from the OV expression construct so over expression construct

when we use so there what they found that the total carotenoid level reached up to 112

microgram per gam fresh weight uh whereas in the control was

only 66 whereas when antisense construct fruits

were measured so there the total carotenoid level is basically uh lower than that of the

control so it is it's about uh 97 microgram per gam uh as compared to the control

control is around 55 or so so and the work of the Italian group was also published in a very good

Journal that is called plan Journal so the Italian group work was published in the plant Journal again this is in

the same year 2000 volume 24 page number

413 to 419 so this is what is the Improvement of pro vitamin content by metabolic

engineering uh the first group actually they targeted uh the

bacterial carotin isomer which converts phyto into lopin but eventually they found more beta carotin but the second

group they played with the like beta Cycles one atemp they tried to block it and another one they try to overexpress

it and as a result they got the desired result so this is basically the outcome from the overe expression construct if

you see the color this is orange type and this is basically from the anti

sense okay so this is the real picture which was what was provided in the Jal so I

hope this is clear to you and with this I end this class thank you