Metabolic Engineering of Golden Rice to Combat Vitamin A Deficiency

[Music] [Music] welcome to nptl online certification

course on pharmacognosy and metabolic engineering this this is lecture number 44 that is metabolic engineering of

carotenoid pathway and here we will discuss the golden rice story so it is about the rice so engineering uh Prov

vitamin pathway in pathway reach endos okay engineering provitamin a pathway in endoscope okay I correct it and

improving the nutritional value of golden rice through increased Prov vitamin A content

so it's all about the rice and why rice and why golden rice so why

rice rice basically a steple food of Southeast Asia

for and particularly in southe East Asia the people who are below the poverty line

they mostly consume rice uh as the major diet so actually they are not getting uh other balanced

diet so the Southeast Asian population those who are below poverty line they mostly

consume rice as their men Mill without a balanced

diet proper balance

diet and as a result what happened continuous consumption of rice in two times of three times of day eventually

that uh uh that that led to the formation of diseases like night

blindness which is called zero thomia Talia and

the reason for night blindness is basically the deficiency

of vitamin A that means the Southeast Asian population are mostly suffering a

majority of the or sizable population are mostly suffering from light night blindness because of their consumption

food consumption pattern which is only rice because they did not have wealth to get a balanced diet and as a result of

that with time what they develop vitamin A deficiency and that is why they are under uh this under the attack of this

zamia disease so uh so now to solve this problem if the deficiency of vitamin A

uh can be solved by adding the pathway of vitamin A in the

rice plant or in other word is it possible to create to create nutritionally

reach Rich rice containing

Pro vitamin A now I need to tell you briefly about this pathway of Prov vitamin A uh although I have discussed

the carotin pathway but this part I need to reemphasize once again so uh beta lopin uh we have discussed

lopin so lopin from here it

makes beta carotin so this beta carotin uh if we consume food containing

beta carotin so in our body system there there are enzymes which convert beta carotin to transretinal

trans retinal and that subsequently converted into trans

retinol okay and then it will convert it into

trans retinoic acid so this pathway operating inside the

human body so that means human body has the enzyme to convert beta carotin into uh vitamin A so for which one is vitamin

A then vitamin A is this one trans retinol is called vitamin A and that is why beta carotin is called

Pro vitamin A okay so this is brief about the concept

So eventually what needs to be done is basically the pathway to be incorporated so that

the the rice plant will produce and Rich antioxidants like lopine and beta carotin okay and then what will happen

that if that rice is consumed then our body system will convert the beta carotin into trans retinol trans retinol

and trans retinoic acid so and then that will be the vitamin A pathway and then vitamin A deficiency can be uh

fulfilled by this diet supplementation so keeping this in mind two

scientists they collaborated at in international level so uh first of all we need to know that

uh what is whether it is possible to transfer the pathway in rice or not that is the first question uh first of all

that whether the pathway exist in the rice or not no rice or particularly rice endos sperm

because that is the aable portion so question is whether in the

rice endos sperm the carotin pathway exist answer is

no then course next question is is what about the substrate

whether ggpp is available in the ospm the answer

is yes but it is for different purpose so ggpp is not taking part into the car pathway that means the pathway is

basically non-existent so you have to basically transport the whole pathway just to remind you that from

ggpp it makes phyto in I am only showing the main compound then it

makes G carotin then it makes lopin and then the next step it

makes beta carotin obviously uh uh trans retinol cannot be made by the plant that I made it clear

so this is the pathway so only thing is that ggpp is available uh uh in the rendos sperm but not this so then what

you need to do you need to hire the genes so you need to hire a Psy gen okay you need maybe you need to

hire PDS gen you may need to have the JDS gen you may also need to hire

the lcy Gen okay then only whole pathway can be operated now putting all these genes together technically whether it is

possible or not that is again a question so there are uh so first of all whether these genes have already been

characterized in other system or not that is important so

and for your information uh a scientist professor at

the University of fryborg okay so that is in Germany his name is Peter

bear Professor Peter bear his group was working on this carotin cartin biosynthetic pathway so Peter bear

basically characterized the genes like Psy from uh

defoil the scientific name of deoil is Narcissus pseudonarcissus we from

japil and also they have lcy

Gene also from this defoil and they isolated the gene

from so CR from a bacteria

called a inia urora so this is I have shown in in in

one of the previous classes that putting this Gene is basically making phyto to lopine in one step that

is c r t i so uh this

team collaborated with another group at uh Swiss Federal Institute of

Technology I write the full name so is federal Institute

of Technology eth yes I remember eth zurk that is the German name it zurk

so there uh Professor Ino

poas uh has developed the technology what is the technology it's

basically the [Music]

technology for uh

rice transformation using agrobacterium to

so this is very important because why rice basically a monocot all of us know rice is basically

a monocot and monocot are usually recalcitrant to agrobacterium tence infection so diots

are easily uh transformed by agrobacterium whereas monor difficult but Ingo poas

develop technology where rise genin can be transformed and not only that he has also have the technology for putting

multiple gen in rice plant so therefore these two scientist they basically made a proper

collaboration and that collaboration eventually so the collaboration between these two

scientists so why collaboration is needed because Peter beer will supply the Gen which to be put and Ingo poas

will have the technology uh for transferring the genes in the rice plant okay now let us see that what has been

achieved with this so the let us go to the next Bo so that is Rise transformation so they have used uh two

different constructs one is uh okay I rather say one is the construct for single

transformation so single

transformation and another is the co transformation so they have used

different constructs for single transformation they have used a single construct where they have these genes

for example what they have decided they have decided to take the Psy

and they have decided to take the c r t i okay and obviously when CTI is to be

taken here Transit peptide to be added and then these gen

uh are putting in a proper Vector so that means that contains

uh promoter and Terminator and the it also contains the

antibiotic resistance marker gen so all these things I'm not discussing here so along with this you have a gene for uh

canamy resistance so what I call say [Music] npt2 which

gives canamy resistance to select the transform plants from the UN transformed one so see here this construct so they

have put the psy and

CTI so so what is expected that it is expected that if

you use this single transformation construct then

the if the genes are properly expressed then ggpp which is available will be converted via

phyto to lopine and lopin should accumulate in the dendros form okay and let us see the

co transformation construct the co transformation construct they have used two construct one is the phyto

synthes and the

bacterial isomer is okay see it right

and and obviously the promoter is there anyway so the space is less so I have to delete

this and Terminator you have a promoter here and then you have the gene

for phin synthes this is construct one and in the second

construct what they have put they put a uh lcy that is

uh L see why I put it here red no I should not rather I put it in the green

only along with the antibiotic resistance marker gin

so here also both promoter Terminators are there and

uh so this construct was named as PB 19

HPC and this one the top one was named at p z

p s c and the bottom one is P stands for small SP stands for plasmid z l c y h so here

basically two constructs two constructs in two plasmids and two plasmids into two

agrobacterium okay here single construct in one plasmid in agrobacterium so here what they have

done basically they have put this two constructs into agrobacterium they grow them together

and then they mixed it up and then they inoculate so the the that for PL transformation

work they have used as a immature embryo as

the explant so this explant was basically infected

with with agrobacterium two different cases right

separately so this is event number one and this is these two are event number two and

then after that uh they put it in the proper medium containing antibiotic resistance and then from there they have

screened the putative transform plants which contains

the desired Gene of interest and then through further selection they finally uh

selected uh a few populations of both the types and then they analyzed now the question is this what they have found so

in the second case uh where is the case number two case number two what is expected

that in case number two what is expected that ggpp will make fyin fyin will make lopine lopine will

make beta carotin okay I just beta carotin okay that is basically for

the for the job two and and this

is for the job one I think it is clear okay now what they have achieved that is the question so what they have achieved

is is basically uh in the first case where uh where PSY and

crti Gene was expressed uh where uh it is expected that they should get the endos sperm uh which should be red in

color because the endosperm should accumulate the uh lopine but what

happened that they did not find any lco op instead they found that in case number

one no like copin detected

instead beta carotin was

detected and along with other carotin so why it happened that is very difficult to

interpret so in the first case no lopine beta cyclus Gene was inserted so therefore lopine should form as a result

of CTI and it will not not get any opportunity to convert to Beta carotin

but what happens no lopine was found but beta carotin along with some other carotin were detected so the question is

that whether the beta Cycles Gene was there in the rice which got induced it is still not

clear but it is something very interesting fact when they have analyzed however in case of two that is case

number two there what they have found they have Al

there they have found of course no lopine but beta

carotin as the dominating pigment along with other pigments like zanin

and so on okay so that means in both the cases

beta carotin accumulates that is the good thing in case of uh two approach two where core transformation was used

so there it is expected that it should make beta carotin okay provided uh the the genes from both the plasmids are

simultaneously transferred and integrated in the proper site so that uh the trans gen will be transcribed

properly and make the functional protein and eventually it get the ggp from the endosperm and then it makes

the product so which has happened in the fast case it was really astonishing but there somehow some induction of enzyme

which converts Lop into beta carotin as AER so in the second case beta carotin was dominating Al along with zanin

and some other carotin pigments and uh okay and

uh and from the endosperm the carotin level this I must say

that 1.6 microG per gram

of carotin was achieved per gram dry or we can say beta carotin

level so this is a pretty good amount but although not achieving the daily recommended dose

but the outcome was really uh interesting and outstanding and that is why this paper published in the famous

Journal science so I'll give you the reference uh just a

moment the paper published in the famous Journal science

uh in the year 2000 volume number

287 and page number 33 to 305 it is a short paper but details are

described in the supplementary first author is e at all probably a postdoctoral fellow who

worked it but here both the Peter beer and Ingo poas were the corresponding authors so uh he's here the the title of

the paper was engineering the Prov vitamin A bracket betaar carotin biosynthetic pathway into carotenoid

free rice endosperm very clear title so rice endosperm was carotenoid free only substrate was available the pathway was

transferred and it produces beta carotin so this is an outstanding achievement and uh uh This was done by a

collaboration between the E zurk and University of rorg and this is called the Golden rice

one and after that I just take few more minutes to finish this class I just give you a brief overview that once this has

been achieved so this golden rice what they have developed they have not patended it so the they made this

technology available freely any anywhere in the world and lot of Trials were made in developing countries but this

basically boosted the uh the private players the the industry players to join in the group so they have more resources

more money so they uh started doing Venture and as a result of that they have

achieved uh golden rice so I call them golden rice 2

and in one such construct they have achieved the carotin level up to 6 microgram per gam dry weight whereas

what I have shown with the other one is 1.2 so this was achieved by uh using this I just put the construct briefly

they put Psy and uh along with that they use

uh uh as C GI and uh they have used a synthetic

Transit peptide and the promoter was different so I'll not go to that details whereas in case

of uh public laboratory the promoter what whatever used for CTI was uh 35s promoter but here they

have used uh endos spon specific promoter that is only the

difference they have used the endospan specific promoter so this was glutelin B promoter Pro pror and the phyto synthes

was was also used under the control of a oson specific promoter

uh motor so those both are both are basically endos sperm

specific promoter was used and here this crti basically they have used the synthetic Transit peptide

fused with CTI so this this was the synthetic

TP uh with CTI okay so this is uh this construct they have named as

p o gr 011 and uh and this they have achieved

by Co transformation and because C transformation means the antibiotic

resistance Mark gen they put it entirely another PL so that in the subsequent generation it can be segregated out and

you can get the trans genic plant containing only these two genes so that is why Co transformation was used

whereas in the other plasmid only the antibiotic resistance marker was used and the finally before ending what I

will tell that another construct was made by the senta group and which leads to the achievement of 37 microgram per

gam of carotin so from 1.6 1.2 to 37 so this was achieved by again using the construct so I just put it here one

is they have use the uh fyin synthes fyin synthes and the this phyto

synthes was narcissus sudon narcissus but this phin synthes is basically from maze so this is basically GM is phyto in

synthes so that is a monocot source and otherwise the

promoters and Terminators were kept same uh so that is one along with that so that means the promoter was endospan

specific promoter and along with that what the have used they have used the uh

normal uh CRT I with

the normal Transit peptide and with the uh

the normal endon specific promoter and of course the one here the antibiotic resistance marker Gene was

there so here one more thing I would like to tell just before ending

so they used phosphor Manos isomar as a

positive selection marker where antibiotics are basically Mar are negative selection marker so

Manos was used as the medium so that phosphor Mano isomer is if the gene is it can degrade the Manos and I make the

and convert it into fructose which is available for the plant to grow so this construct was they have named as P Sy y

n 1 2 4 2 4 which is gr2 eventually this is called gr2 so this one the first

construct uh this is called gr1 so what okay so I have made the golden rice 2 because use the term at

the beginning because the aim ultimately they have achieved gr2 so they have achieved gr2 from

gr1 so gr1 led to the accumulation of 6 microgr per gam carotin per dry weight of

endosperm whereas here it accumulates up to 37 microgram per gam so that is a very outstanding achievement and this

was achieved by the uh private laborator senta Laboratories okay so this is all about the golden R story so this uh high

carotin content golden rice are basically uh exploited by the companies for profit and those who have money they

can buy those who don't have money they have to go for the uh golden rice which was created by the public

Laboratories so with this I uh I just show the last slide which is the uh picture so as you see here uh this is

the while type rise that is untransformed this is the golden rice one and this is the golden rice 2 and

here it is showing again in another way so there is indeed distinct difference between Golden rice 1 and golden rice 2

with this I end this class and this is a very nice example of metabolic engineering uh and I hope you have

enjoyed uh thank you