Metabolic Engineering Enhances Alkaloid Production in Catharanthus Roseus Hairy Roots

[Music] [Music] welcome to nptl online certification

course on pharmacognosy and metabolic engineering so this is lecture number 27

where I will discuss several case studies on metabolic engineering applications in catharanthus Rosas herot

system so the a of course was to enhance the alkaloid content and Har root system was chosen so I will come to that so let

me briefly tell the concepts what I'm going to cover so first one is the expression of a meib

transcription Factor Gene MK myc in har root culture uh we will see what happened

when uh attempts were made to express this transcription factor in har root next is the expression of

orca3 and sdd gen in har root the third one will be the expression of D gen in harot culture the fourth one will be the

expression of gtnh either in singly or gtnh works here three

together in hary root culture and finally we will see the expression of dxx DXs and G10

H in one instance in another instance DXs with Anon synthes ASA gen in hery root culture let us now go to the

board so this is

metabolic Engineering in

catharanthus Rosas here root system and of course the

a basically was to enhance

the alkaloid to enhance the contents of alkal if I

that will be better to enhance the alkaloids well and the system used

was here root Cure by now it is clear to you that how genes can be inserted into hay Roots so

basically this is what I used to call earlier the same terminology I'm going to use now so this

is co transformed car root

cultures so if you remember that c transform hary root cultures

are raised by

transferring uh without uh without making any changes in the

original R plasmid which is there for uh

which is there in agrobacterium rogenes which contains

the tdna for R and of course it has a uh virulence

region so here uh recombo and plasmid has to be

incorporated so which contains the synthetic

uh uh tdna construct having the Border sequence and here you insert the

gene or Gene of Interest so this is Gene one this is

Gene 2 of course this will be under the control of promoter and Terminator which I am not showing here and

what what should happen is basically this V will work both in CIS as well as in

trans this work in trans t r NS and this is s so it allows this tdna to go out and this tdna

to go out so this is basically the tdna of the synthetic plasm

so well and then what will happen so both uh tdna

will integrate in the uh PL genome

and eventually this will be selected based on

the based on a marker which is an antibiotic selction marker so

maybe a canamy and resistance marker is there and that leads to selection of and so these two will integrate and as a

result of that what we are going to see is co transformed

here root culture and this here root

culture uh we'll Express the genes like G1 and Gene G2 and here it contains the ra

genes which is required for here root phenotype formation okay so this is in brief the

mechan me ISM of hary root for transform hary root culture and scientist whoever did this work

they they prepared the synthetic plasmid which come under binary vector and then they have inserted the Gen there and

then they put the plasmid the combinant plasmid inside the agrobacterium rogenes and then they did

transformation in catharanthus Rosas young seedlings and as a result of that herot emerges and the her Roots were

checked uh using antibiotic selection pressure and then finally they look for the uh the look for the growth

performance and then finally the product so with this let me first uh talk about case study

one where uh EXP

expression of CR M C1

gen in here root culture I use this small terminology so that means c h myc is

basically MC MC1 is a transcription Factor MC1 mc2 that plays important role so these are the positive regulator so

the gene was was put under the control of a promoter and a terminator and then Cenas Rosia Co

transform her root cultures were raised so we are not going to those details we will see only in a

nutshell what uh this Co transform hary root culture showed us or what this her

culture produced so what we are going to see we need to see that what happens to the

content of the alkaloid so what they have decided they have decided to measure measure several alkaloids among

which they particularly focused on aalin and catharanthine content so uh I don't think that I have to

again mention about this so one root goes to

aalis and other root goes to C and thin so this form by joining two substrate one is

SE ganin and

cryptomine uh so what happened the outcome the outcome is this that uh [Music]

aalin content enhanced by 13 to 14

fold as compared to the while type headout so this is pretty enormous and

the cathar Anin content also enance but that is not that

much that is only 3 to 4 fold so to sum up expression of catharus

Rosas MC1 which is a transcription Factor Gene when this Gene was was uh transferred or overexpress in

her culture of catanas Rosas this leads to enhanced production of uh Indo ALCS and particularly the impact was

seen with the asalin content as well as in the catharanthine content so this is a metabolic

engineering uh case study where positive result has come so next we go to the next case study

so so this is the heading ke

study two so here what we see okay uh one thing I forgot to tell

that uh this work uh was reported

very recently in a journal called Horticulture environment and

biotechnology in the year of 2022 uh volume 63 page number 709 to 7117 so case study 2 case study

2 we will see that sorry o area

okay over expression of o r c A3

and SGD in

cus culture so

when so they have used the inducible promoter which is called this uh

gluc corticoid inducible promoter though I will mention here promoter used so normally the promoter normally what is

used is cauliflower Mosaic viers 35s promoter one is constitutive example is cauliflower

mosaic virus 35s promoter but here this was not used here they have

used uh gluco cortic

inducible promoter so when uh I will not go into this uh promoter details here uh because

otherwise we will not be able to finish the major outcome what we are going to discuss so here uh but of course I'll

give you the reference and uh uh the details can be studied by the students themselves so here using this promoter

when they attempted to express orca3

alone that did not make

so significant changes happen in the uh total amount of alkaloid contents

however when what C3

plus SGD both genes were put in the construct under the control of a glucocorticoid

inducing promoter that leads to changes detectable changes in the

alkaloid content so

significant change changes detected so that

includes increase in serpentin

content and by 44% and

as micin contain by

32% catharin content by

38% and tonin content by 40% not only

that it also showed increase in lock nine and hard Hammer so which I will write this

side and hard hammerin uh this is

by uh hard hammerin by 56% and lo nine by

60% such significant changes detected when uh both orc3 and SGD was expressed under

the control of a glucocorticoid inducible promoter now this is very interesting because this is root and

what I have said uh yesterday that if you briefly look into the pathway that is this taronin

in root tonin is not uh converted into

vindoline instead different pathway operates in roots that leads to the formation

of Lo nine and hardam medicine so what has been found that tabarin content

enhanced okay catharin content also enhanced

and the pathway leads to aimation content enhanced not only that serpentin

content also enhanced so this is a very positive

result and this paper published uh in a a journal called

protoplasma in the year 2016 volume 253 page number 1

2 5521 1264 and there are two authors one is Son and

PS and they are from Colorado State University USA so this is another uh positive

result with metabolic engineering application let us go to the next case study EXC study let me go to the first

slide D gen so so case study

three so this is expression of De

atile vindoline for atile transference that means expression of d

g a in cus here root culture so

here what they found so the same C transform head root they have used uh system and the D Gene was expressed so

dat you know that this is the uh more or less the terminal step leading to the formation of vindoline so if I put the

pathway so that means I think that is required to explain uh say this way if we write

tonin tonin one

2 3 4

uh and this is4 H uh L

T 16 H then is T6 omt third one is T3

t3r then comes to nmt and

then uh d4h and then last one is

the D which makes

vindoline and tonin in the root system it produces

lock rock nuris

or from there it can produce

or or medicine okay

now what happened as a result of that so the first of all how they have studied they have studied the they have

checked the enzyme activity of dat enzyme activity they have

checked uh and next what they have checked they have checked the metabolite content so what they

found dat expression leads to the accumulation of hor

hammerin so what happens when D expressed that leads to accumulation of hor

hammerin that is uh pretty interesting uh what is h hammerin it is here why it is

so because d8 is basically situated here in the pathway okay

but uh what is also known to some extent that there are an enzyme like

t67 e like m T and then uh there are enzyme like uh

T 19h so here

so the mat and uh this is t67 e t 67 e now this

mat Matt the full form of mat is minov venin 190 atile transpar so that means this m and that is more or

less similar type of enzyme so when you overexpress that in the he root of catharanthus what

happens that that which is meant for vindoline it is not working because in order to have

vindoline accumulation you need to have the light so that is not happening but the that enzyme is produced in higher

amount but what happens in the dark the pathway from taronin moves in different direction like Lo renin and

hor hammerin so this is operating in the root constitutively operated so so the extra copy of that is rather now working

together with mat and that leads to the increase of flow in this direction and leading to more hard hammerin

accumulation so this is an interesting paper so this is what is the explanation I try to

provide so I just put reference

phytochemistry uh 207 volume number 68 page number 19222 1

931 so this is a paper from Canada uh led by Vincent DEA so the next we go to the case

study four where I'll use here only

uh okay uh is study 4 so where basically the

over expression of G1

H and or C3 leading

to leading to improved cathan in

production I already told you the outcome okay so G10 is one of the early enzyme I

have not covered in that details so which basically converts the geranial to 10

hydroxygen anal and this subsequently uh takes part in the formation

of SEO loganin

okay and or C3 we know that that is basically a transcription Factor

it affect the pathway so uh what happens that when they have used only

G10 GT H expression that also leads to

changes in alkaloid levels but when

both G and what C3 were expressed

that that made subsequent changes uh and produced more catharanthine which is

very prominent more cther Anin compared to the other alcal more catharanthine was

detected and interesting point is this in this herot line when uh they fed absc acid that means

feeding of absc acid

in coexpressed a hay root that leads to uh

the catharanthine accumulation up to the level of

1. 96 which is almost 2 mg per G dry

mass of G root this is also very interesting outcome and this result published in the

journ called uh

plant sale reports in the year of 2010 volume 29 page number

887 to 894 and the last case study which I will cover very

briefly uh so which is uh case study this will be case study

five where uh DX is

plus G10 H in one instance and another instance what they have

used uh DXs plus alanate synthes so this they have

said a uh

AA so so DXs is the pathway which is basically uh the first enzyme of the M

pathway and GT is basically uh it is towards

the end enzyme of the map pathway that means when you are expressing both DXs and G1 that leads to

more seanin formation and

similarly uh in other case use DXs so DXs contribute to this is the first enzyme of the map pathway that leads to

formation of cyanin eventually whereas the

ASA which is anate which is product of the uh simate uh Phile propanoid pathway and

eventually this leads to the formation of more

tryptophan so that means more triamine and more triamine more

seanin that leads to the formation of strictosidine and here this

seanin joins with the normal damine level and

makes strictosidine so let me beefly tell what is the outcome of

this so outcome in general that it increases the indal alkaloid accumulation

uh so DXs over expression uh resulted in significant increase in asalin serpentin and lo

nine so aalis I

serpentin and what I say the Lo nine that all enhanced not uh by Lo by

49% upliftment uh aalis by 67% upliftment okay and serpentin by 26%

upliftment this happened and when both DXs and g10h was used uh so that leads to cause significant increase in asalin

Lo n and tab so I'm not showing the data likewise when DXs and ASA over Express that

displayed increase of hor hammerin Lock n

Lo medicine and also ton so that

means content in sense or Hammer content content enhanced as well as Lock N content enhanced so here some data was

provided tab enhanced by 34% horam medicine enhanced

by 30% and do n enhanced by

27% that means this is also very uh positive outcome as a result of that because you know that it is basically

enhancing the stto sidin and that eventually leads to formation of

all Downstream products so this paper published in the Journal called metabolic

engineering engineer ing in the year of 2011 volume number 13 page number 34 to

240 uh it's from United States that's the same group so these are all uh the case studies which I have discussed in

this class all showed positive impact uh on the levels of Indo alkaloid accumulation in catharanthus

Rosas so this is what is the take-home message and with this I end this class

thank you