Metabolic Engineering Strategies to Enhance Secin Production in Lithospermum Arizon

[Music] [Music] welcome to nptl online certification

course on pharmacognosy and metabolic engineering this is lecture 61 where under the broad domain of phenolics uh I

am going to discuss the metabolic engineering strategies applied toonin pathway aiming to produce more cin uh in

the plant lios Arizon so that is our main and with once I finish this class with this basically

I end the phenolic so now go to the uh concept covered so metabolic pathway of secin production although I have looked

I have discussed this in the previous class then we'll talk about the manipulation of ponin pathway by

expression of heterologous genes and finally we'll also try to see our current understanding on the secon

pathway as I have shown you in the previous slide that secin is basically a ninon pigments where the one is coming

from the Phile propanoids sikim and the another one is the penile so this is nepto quinon pigment

pigment which is red in color accumulated in the roots of rosum Arizon

and and I have also shown this one that is the secin production by

mitsui petram private limited so that is the first plant cell culture based product

produced by the industry now what I have ended the last class is basically the one root is coming from this Phile

alanin root that is the Phile propanoid which is basically producing parah hydroxybenzoic acid and the other root

is basically coming from the uh evalon pathway so which is from the acle qu and which is

producing the janile pyrophosphate or gpp so both gpp and parah hydroxybenzoic acid joins and I'll eventually the

pathway moves towards the production of secin okay now this have also I have mentioned that secon accumulated in the

root so that is why it is read in color and then under the dark condition cin accumulation happens and also the hay

root cultures are capable in producing secin or using particular Medium say for example M9 medium is very good for

induction of secin here and you see the secin accumulates in the epidermal region so

that is why the red coloration here but here there is no color so that means that when secon started accumulating it

accumulate in the epidermal cells okay okay now we will talk about the genetic engineering of secin so uh

now one uh interesting work is about a discovery of a gene which is called ubic Gene so UIC Gene basically encodes an

enzyme which is called Kismet uate

lies or we can call CPL this is basically from the bacterial source so so coris py what does it

produces it produces cormic acid acid from okay no it sorry it

produces uh no it produces for hydroxy benzoic acid from chorismic Acid so chorismic acid was used as a substrate

and which produces parah hydroxybenzoic acid as the product

for hydroxy benzoic acid as the product so the enzyme is basically the

CPL CPL or coris pyet lies and in this process ayic acid is released and this CPL is basically

encoded by the gene which is called UVic Gene so UVic Gene encodes coris pyrro Li so coris pyrro lies

converts CIS to hor hydroxybenzoic acid in a single step and this Gene once it is available so they

have tested this Gene in in tobacco so in the trans enic tobacco

expressing CPL enzyme I put enzyme here it produces huge amount

of for hydroxy benzo so now what I have shown in the previous

slide that four hydroxybenzoic acid is acts as an intermediate for this pathway so for this simate pathway so what I

have shown that for hydroxy benzoic acid for hydroxybenzoic

acid that basically

joins with gpp and produces produces janile hydroxy

benzo janile uh Hy janile

hydroxy basic acid or we can call GBA so this GBA

subsequently makes the cin now four four hydroxy benzoic acid

is basically produced

from camic acid or trans camic acid and that basically coming

from alanin and this phy alanin is coming from pranic

acid biogate and pranic acid is basically coming from chorismic acids and cormic acid is coming from

simic acid right so this is the pathway now putting

the CPL here will

convert cisic to four hydroxybenzoic acid in one step

CPL okay and the endogenous enzymes are also there in this pathway for example here the pal is there okay other things

are there so uh if the the UVic Gene which encod CPL can be

transferred into lium herizon then what will happen the existing pathway from Kismet to phot

hydroxy benzoic involve several intermediates now that can be subsequently

shortened by putting the this step here okay then what will happen that

maybe the normal roote will operate but if you express the UIC G and under the control of a strong promoter then what

will happen it will drive most of the cormic acid towards the for hydroxybenzoic acid and you will find

more for hydroxybenzoic acid is produced if more for hydroxybenzoic acid is produced then what is going to happen

that that will get the substrate to the pathway will move uh more efficiently towards the

formation of secin and eventually if with a bit of luck you may be able to get more of secin

here okay so that is basically the rational so uh then what they have done is basically this loots Heights group

from the University of Tuan so they basically made the construct so that means they put the UVic

gen under the control of a promoter and a Terminator so they have used the strong

promoter uh which is ptop promoter ptop which is a modified 35s promoter the promoter of ptop is added

here which is called PTP which is modified

CMV okay modify it 35s promoter and the putter also they put a

Transit peptide sequence so that it will drive the product inside the

organal [Music] so and The Terminator should be there

and this is the transit peptide this is the promoter and this is the coding gen and why they put the transit peptide

because the product to be uh transformed the protein to be transformed inside the organ particularly endoplasmic reticulum

uh so where the reaction will occur now uh so they have used normal CMV promoter as well as the much stronger version and

uh what is going to happen as a result of that so we we are going to see okay another thing is this that

uh okay so this is what is the pathway which I have shown in the previous one

so the cormic acid in one step makes for hydroxy benzoic acid this one this is what is this root so this is 4 HBA but

the normal pathway is basically Al through this route now what they have done so they

found that significant increase of for hydroxybenzoic acid upon expression of CPL that means when CPL is expressed or

UIC is expressed and it encor the CPL enzyme so CPL successfully converts CIS to for hydroxy benzoid and then that may

leads to more production of four hydroxy uh janile Ben acid but the end product formation was not it marginally affected

so it did not make much sense on this secin slight increase of cin so what they also try to understand here that uh

what is the contribution of the uh existing penile propon pathway so in order to check that basically

they they did basically inhibitor feeding which is AIP so AIP competitively block

the fin in ammonia lies as a result of AIP blockage what is going to happen if the AIP is blocked here then this

pathway is blocked so if CPL contributes to this pathway then by blocking AIP you will see is some formation of for

hydroxy benzoic acid otherwise if the more four hydroxy benzoic acid is not because of the CPS but maybe because of

uh certain other epigenetic modification then it will not really make any sense so that is why they have done feeding of

AIP so upon feeding of AIP what is going to happen if it blocks here then it blocks here then there will be some

production of secon if the pathway moves in this direction and if the pathway is not moving in Direction then the total

uh secing pathway will be blocked so here they have uh nicely put the outcome of such experiment so you see here that

this is control root line where no UIC Gene is here and this is basically the transgenic root line where UIC Gene is

there so one is the plus a plus AIP that means here in the control loot line they did feding with thep and the minus is

that no feding so when they have done fitting with uip in the control root line what is going to happen if you see

the control pathway that means if I draw this one so think about that this is

nonexistent think about this is not non-existent so here in the control here you have done AIP feeding so it so what

will happen eventually the cin content will be less so the four hydroxybenzoic acid will be less okay in the wild type

so what is happening here you see that when AIP they have fed they fed four hydroxy benzoic acid glucoside content

is very less secin content is very less as compared to no AIP feeding control where you see some amount of cin

formation along with for hydroxy benzoic acid okay and now what happen to the transgenic line where UVic is expressed

so there what happens normally without AIP you will find uh formation of cin in in pretty good amount and for hydroxy

benzoic acid glucoside is also formed but when the the AIP was fed in the transgenic line what

will happen the secin content is now less if you compare with this one and this one this is less not only that the

four hydroxy uh benzoic acid glucoside content is also less that means what

that means the both the pathways are contributing to secon information both the pathways

contribute to secon inform formation that means this pathway contributing to secon information because of the

transgenic and this PA is also contributing to secon information is the while type so in the transgenic line

both the things are active so even if you block a IP the secin content is reduced because this route is not coming

only this route is active and that is why you see the content is getting suppressed both in this one and this is

what the har root line without AIP it is producing red color SEC accumulation but

with plus AIP in the control no coloration similarly here also that uh the transgenic line without AIP it's

more coloration whereas with with the IP the color has been reduced so this paper was published in the plan molecular

biology by the somar atol so SAR was a post do and uh someone probably was a PhD student so it's luds

height was the group leader from University of tubing in Germany so they published this paper so I I think I am I

have explained the things in a simplified way no further discussion I'll go rather we'll go to the another

aspect the L height group then next once they have checked the Ubi C now they have uh utilized another uh enzyme which

is uvia a now what is UIA we need to see Ubi a gene basically encodes a plant for hydroxy benzoid janile trans

okay no no no no no in plants in Plants four hydroxy benzo

HBA or 4 HB we write 4 HB janile

transfer is so 4 HB janile transfer is what it does basically it basically joins 4 HB and

gpp so it joins a gpp

and 4 HBA and it produces janile benzoic acid okay so this is the this gen is

called 4 HB janile transfer or we can say 4hb

janile transference okay now 4 General transfer the properties of this is that this is

basically a membrane boundary enzyme

and uh and this is located in the

ER endoplasm reticulum and it is basically regulated by

light light hormones elicitors and media

composition now enzyme

activity correlates with secin formation that is more enzyme

activity more secin and his enzyme

was partially purified

however no structural gene was

cloned and and the last point is this that this is highly

specific to gpp but not

fpp so therefore the for hydroxy Jal transfer is there in the pathway but Gene is not available in the hand but it

properties known therefore the scientist they decided that to hire a similar enzyme

from eoli that enzyme is basically from the eoli so a

bacterial having a bacterial Ubi aene which encodes

four hydroxy benzoid poly penile transference which encodes four hydroxy

benzo poly penile transfer

is and although this is also a membrane bound enzyme

membrane bound enzyme and and it has broad size specificity it

accepts both gpp

and f p therefore they decided that to use the UIA Gene because there is no plant Gene

available and it is more or less similar but it is a more broader subst specificity and it is also membrane

bound so to transfer this gene into Laos spum will may do the work so the UIA

Gene was taken from the eoli the Ubi a which is for hydroxy benzoid genile transfer which is basically the this is

the plant Gene for HB genile transfer is the plant Gene and this is basically the uh equal right the bacterial Gene

and they have expressed in again in the he root using Co transform he roots and the construct is very interesting so

that is why i' would like to tell a little bit about this about the Gene construct so because they want to put it

inside the endoplasmic reticulum and to stay there so they have used uh

uh a fusion of UV a

with h d so this is basically a fusion of UV HD so how we write UV

a HD and then they put a signal

peptide and they put the promoter

so uh this is very strong promoter who used which is called uh o3m promoter which is uh octopine synthes 3

with a manop synthes promoter that is a very strong promoter and then they put

a Terminator here NOS now the purpose of putting is that

when they put the H Del so h d is basically is is basically a tetrapeptide they have take and that

actually function as signal retention uh inside the endoplasm reticulum so it function as a signal for

retention of soluble and membrane bround protein inside the endoplasmic reticulum so this

leads to the retention of soluble and

membrane bound protein inside the ear okay one small

correction I would like to say in the in the previous one now when I talked about uh this one so the construct which is

the transit peptide so this Transit peptide was used basically to transfer the protein UIC that is the CPL

protein to move inside the not endoplasm return but mostly inside the plastid please make this correction

okay okay so this this will return inside the endoplasm reticulum because the pathway late pathway are in the

endoplasmic reticulum so that means when this Gene is active so then what is going to happen that this UV a will make

uh this GBA which is three genile 4 hydroxybenzoic acid this subsequently converted into janile hydroxy hydroxy

genile hydrone and then one pathway moves towards the formation of secin and the other path moves towards the

formation of uran and uh so the outcome is this that there is only marginal

increase although the gene has worked well and as a result of that this product uh for hydroxy janile three

janile 4 hydroxy benzoic acid this product produced in huge amount the enzyme activity was very consistent but

eventually the secon in uh formation was not that much affected so what it indicates that perhaps you need to know

more about the more Downstream pathway so the downstream pathway that is the downstream pathway is

important exerts more control okay now uh the third one is the by the

same group what they have done another paper they published in 2002 this time they have taken HMG qu

reductors which converts HMG qu to mevalonic acid because that is the root which contributes to the gpp formation

and they have taken the UIC so UVic converts cores to four hydroxy benzoid so that you get more of 4 HBA and

myalon is basically HMG qu deduct is if it work it converts HMG qu to meon so more of gpp so it is expected that more

gpp uh will be produced more FOH hydroxy benoic acid will be produced and as a result of that the pathway will move in

this direction and eventually it produces more of three janile for hydroxy benzoic acid and event ually

more of secin uh so again the margonin increase was marginal although uh the three

janile for hydroxy benoic acid is produced in substantial amount but uh uh the final product formation was not that

much but the metabolic engineering attemps of the on the design strategies what they have adopted that is uh uh

scientifically that was very well designed but however uh scientists could not see the expected result because more

complex regulation exist in the downstream process now in addition to this what the what scientists they're

also attempting they try to see if any transcription factors like uh e one so E1 is a transcription

Factor so that uh perhaps plays an important role in the regulation of the pathway so they made an attempt to

express E1 as uh different way C is one is the control this is the control while type and EV is basically the uh

expression Vector that is only the expression Vector to check it parked or not then the third one is basically the

EI where basically uh e L1 they try to block it so it is basically the EI construct you see that there are RNA

approach okay and the fourth one is basically the OV expression construct so they put ere expression construct this

is O EO okay so EV is basically the vector simply the expression Vector the EI is

basically the uh blocking of the E uh and the fourth one is basically the over expression so what you see from the

root coloration itself that this leads to more coloration the right one so that indicates that over expression

strategies worked well and what we see here also that when you put it in specific

medium so first one is the normal grow growth medium and second one is the same line medium so what you see here that uh

the content of this product is much much higher in the OV expression construct where you see more

of secon formation there then and EI of course it is very less uh EV is empty Vector so it's unaffected just the it

contains a gas gene or GP Gene so that function as a while type okay so what scientist they predicted that

this strategy worked well and uh like ere expressing a transcription Factor like eil1 indeed makes sense in the

production of secin in lios Arizon yot so and this is the PGT so this ISS that

basically that is an upliftment of this en this step this is this what is that Ubi a

what we call when you use the bacterial Gene but the plant Gene was this uh janile for hydroxy Beno janile

transference which makes janile parah hydroxy benoic acid and eventually it makes the secon so uh so what they they

check the expression of these enzymes and they found that that expression got uplifted so uh so basically this is an

another update of this secin biosynthetic pathway what you see that there's a cell

wall and then outside the cell wall there is the apoplastic space and then cins are

basically coming out and when it's a cell cultur it secretes in the medium it's basically

coming out so it's not known whether any transporter involved in that uh and scientists made different attempts like

they try to block here they try to block here and they see that how the regulation happens so what we have

learned is this that uh the pathway is this for hydroxybenzoic acid and gpp which makes this uh janile hydroxy

benzoic acid then it makes janile hydroquinon and then from there the cytochrome p450 will make three hydroxy

janile hydrone and from there maybe the pathway produces intermediate leading to the deoxy secin and then ultimately the

secin where the other pathway moves towards the dehydro syono furan and which makes the

uran so this is the in general the pathway and uh so our current understanding is this that we know this

much that this one we know we know this pathway contributes we know this pathway contributes we know janile

hydroxybenzoic acid then janile hydroquinone and hydroxylated genile hydroquinon and in the latest

information is this that these this ayto 450 which converts gerany hydrocone to hydroxy gerany hydrocone has been

characterized and but what converts gener hydroxy gener hydron towards the secin and towards the benzo quinon this

remains elusive yet okay so uh so this is basically the uh hydroxy this is cytochrome p50 450 p450 hydroxy which

converts this uh hydroxy gerany hydroquinon uh into the other intermediates like uh two oxyer and

hydrone and then uh one of these pathway contributes to the formation of deoxy curan and other moves towards the

formation of cin so we'll not go into that details simply what you uh should know that the hydroxy enzyme has been

characterized but beyond that which is not known and uh much later another paper

also published where this is that this is uh uh secon atile

transference cin atile transference has been characterized from the pathway if means

lioas Arizon which basically converts secin to other secin derivatives so it is not only the secin but other secin

derivatives that also plays important role and in order to produce more of secin it may be required in the future

to block this activity possibly in the future by RNA I or anything still do not know and if it if

that is possible then it it may be possible to make more ofon accumulation in future so with this I end now the

class so this is in a nutshell that one cytochrome p450 has been characterized and there may be the oxyon toonin here

is another cyto p43 which is only partially characterized

and so what you need to know is basically details of here so then only it is possible to make

more secin as per we desire in lithospermum Arizon and years to come when we'll be able to achieve this so

this is about uh in brief about the metabolic engineering strategies so far being explored for the production of

ponin in lithospermum Arizon using herot culture as a model because it is the organ culture so it will be more stable

than the cell culture so with this I end this class thank you