Metabolic Engineering of Indole Alkaloid Biosynthesis: Case Studies in Plants and Yeast

[Music] [Music] hello welcome to nptl online

certification course on pharmacognosy and metabolic engineering so this is lecture 22 where I will discuss the

metabolic engineering uh case studies of early stepes of indor alkaloid

biosynthesis so let's go to the concept to be covered so where we'll see overexpression of TDC and S strr genes

in tobacco OV expression of TDC and Ester genes in perwinkle

and then expression of pernal genes in heterologous system and

then here root culture as a system for Indo Alid production and if time permits we'll talk about ere expression of

transcription Factor gen in pinkle so let's go to the board so first one is this that let me

very briefly work out the pathway what we have just studied so important point is

this that tryptophan we start with tryptophan so I will write

indol alkaloid biosynthesis

and pathway manipulation so what what we said that

tryptophan makes triamine and then this joins

with seanin and and make stto sidin and

strictosidine in the next step it makes strict toyin a

glycon and from there ref products form s such as

camine stenin asalin and so on so this root comes

from uh tpid and this is from

the Sate corat that means so the enzyme which is responsible

for this is one one is stopen DEC carboxilate which I have mentioned and the joining enzyme

is St Str that means titoy in synthes and this one is this SGD stto in

glucosides so now we will see this so first of

all uh the uh the scientist attempted to make uh cell cultures

of catharanthus Rosas the English name is pernal and what they have done that they

transformed the cell cultures with DDC and

SDR so uh that means uh

expression of TDC in cell culture of catharanthus

Rosas and expression ofer in culture

of cus so when TDC was expressed so it

produces tryptamine in higher

amount but that did not affect the or that did not uplift the synthesis

of strictosidine because uh the seanin may be a limiting factor there so that means expression of

TDC only upliftment of

tryptamine whereas when

is Str was overexpressed that leads to formation of upliftment

of stto sidin in marginal amount so what happens in this case an enhanced Str Str activity was detected

but uh stto in level was only marginally increased this is what happened with the cell culture

from there it the scientist so they uh came to a conclusion that uh that only the

overexpression of the genes of the same pathway is not enough perhaps the regulation lies in forther stream of the

pathway or there may be the transcription factors which need to be modulated so that the product can be

produced in higher amounts so these are the several questions uh which were subsequently

raised so when they have used now they have used the tobacco system so when they have

used the tobacco actually it's not the proper tobacco I

should write the dhiana rastika species of

Target so here both TDC and Ester

gen were transferred to nikosi rastika through agrobacteria mediated

transformation and transgenic plants were raised and what has been

found that transgenic plants are basically producing both

TDC as well as s Str and these activities were successfully

detected when that is so then it is important to see that what happened to the product so

tryptamine yes ramine

detected but no stricto in sorry no so then what they have

done feeding of

cyanin can now help in production of in production

of uh stricto in strict toid in production by the transgenic

plants so why because tobacco is not not meant for or or nikosi rastika is not meant for

producing uh the indol ALCO but genes can successfully be transferred and good point is that TDC

substrate is tryptophan tryptophan is universally produced aromatic amino acid so when TDC was expressed so it can take

the endogenous tryptophan available in the pathway and that leads to the upliftment

of trip in okay however the tobacco transgenic tobacco cannot make strictosidine

because uh although a tarpo pathway is operating there but it is not producing any

seanin therefore in order to produce trocin in in nikosi rastika uh one has to feed the cadin and

exactly they did and as a result of that uh the transgenic plants produced strictosidine

so this gives confidence to the scientist that indeed the this heterologous uh system can be utilized

for producing some of the hardly uh pathway alkaloid products even in yeah in the hrus system like tobacco

so that is the good thing so this actually give some confidence to the scientists so then they moved forther

to explore the system so now the next case study what I am going to cover I need to draw this pathway again but

anyway that is required that is the biot

transformation of tryptamine

and seanin into

plant tarino Indo alkaloids

by transgenic East so this is one of the very interesting

case studies which I am going to discuss now and this paper was published

by guard Lings at all so in the year of 2001 from Nether L lien the group leader

was Robert hpor

PT and this was published in applied

microbiology biotechnology and the volume number 56 page number 420 to

424 so let us see what they have done so we have to draw the same pathway again that means

the trpt toan makes

triamine and then Here Comes

sanin and these two joins and make strict toyin and strict

toyin Aon sorry and then the different products can be

produced such as camine

camine or AIS now if I join with

arrow the so the

source genes are s Str and SGD

from catharanthus Rosas and

the target organism it is a eukariotic system that is this sarom I just write

e so what they have done is basically they made a construct containing both s and SGD and

then so basically the cdna which encodes for stto in synthes and stto and glucosides so those

genes were used and the constructs were prepared under proper control of uh promotor and

Terminator and then East cells were transformed with these cells so that means what if we write here

that East cells

transformed with Str Str and is

GD okay so let us consider that transgenic easts are produced and this East Express is TR and SGD now what is

going to happen so East requires growth a medium so uh these genes are there these genes

can be detected okay even the expression can be studied that means the MRNA produced can

be measured but product formation most likely will not be there

because uh camine and cyanine are not available so what is required basically that you have to do

feeding feeding of substrates what are the

substrates one is the tamin

and cyanin now when this fitting was done then what has happened that uh

East uh successfully uh accepted tramin and seanin and produces St

idin now this strictosidine what is produced in the East cell this strictosidine

Leed out into the medium

so strictosidine maxximum amount

detected in the medium so that means it it is leing out or pariz of strictosidine uh outside the

East cell because uh to much strictosidine is not good for the East because e are not meant for producing

strictosidine but it is in the medium but but what happens that with time the stto in the

medium uh getting disappeared and as a result of that what happened that scientists they have

detected of camine

okay so and also a little bit of edicin now why it is

happening so although the genes for S Str and SGD are Express inside the cell so it appears that the stoy in

glucosides that is basically that is again secreting out from the East cell and it is uh basically localized in the

medium so what happens that when strictosidine are produced inside the cell uh so if

we make a light drawing this is the East cell so as a result

of St Str uh strictosidine is produced

and that is coming out in the medium and where this is converted

into camine that means the genes for

SGD is basically coming outside the medium so that means that SGD is basically there so what

happens that when you do the feeding with tryptamine and cyanin ultimated you are finding a good

amount of strictosidine in the medium and then with time that is getting converted into camine and that is

because of the SGD activity from there it is clear that expressing

plant pathway gen in East is visible and it is possible to produce the product uh by E cells and these products

are Leed out into the medium so which makes the downstream process easier so that uh these alkaloids can be

easily extracted out otherwise if it is located inside the cell then you have to break the cells that is energy dependent

process and get the things out so this is a very good system now this is fine now one important point which is this

the cost the cost of tryptamine and seanin so tryptamine is not that expensive but seanin is

very expensive so if you want to make a process uh at the commercial level so

this can not be visible but tryptamine is not that much expensive therefore scientists they look for alternative

sources of uh Cogan and they

finally uh found a plant which is called s snowberry snowberry the snowberry fruit

it's white in color maybe one of the subsequent classes I will show you the photograph of snowberry sberry fruit is

basically appears to be a good source for sanin the scientific name of strawberry

is syor carpus s y m p h o r i c a r p u Sor carpus albas a l b u s so soy fruits basically these are the

berry botanically so the fruit extract contains a huge amount of cyanin so this has basically two

advantage two Advantage what are these number one fruit is basically a source of

sugar so that means the sugar what normally one added in the cell as a carbon source for growth that can be

reduced and number two the fruit extract contains also

cyanin therefore growing the transgenic East in in a medium which is enriched

with uh snowb fruit extract which CS the need for sugar and cyanin only you have to do feeding

of tryptamine and that basically leads to the formation of your alkaloids that

means camine and this camine can be converted into aisin so this is a beautiful case study

where hardly uh catharanthus pathway genes are transferred into the East and as a

result of that East transgenic East can produce the early indol alkaloids which is produced in

the plant now let me go to the next slide so that

means we'll see the hay root system we'll discuss little bit about the hay root system for

okay for a root system of catharanthus Rus for Indo alcohol

production now what I have not said uh I have not discussed fully the late steps of Indo Alid biosynthesis uh

so before I discuss this it is important to know that strictosidine stto gluc ultimately

what it produces it produces different products

like catharanthine and vindin so scientist okay and then what I

have said that these two joints and produced Vin blastin the scientist they

uh they attempted to use hadot system for starting the Indo alkaloid biosynthesis and

regulation why herot because herot is a study system and uh it is genetically stable

therefore uh manipulations can be done so here there are a few aspects which need to be discussed before I come to

the H root so it is important that we should also know the uh what happens to the

normal suit cultures whether suit cultures of cus can produce indol

alol with the cell culture of cus can

produce indor alkaloids whether there is any role

of light in alkaloid

synthesis and any role of green

tissue in alkaloid synthesis syn

so this needs to be addressed so uh once we address this then I it will be better to bring the

head root system in the uh in this context so in the next class we'll talk about that so I will end this

class here thank you very much