Secin Biosynthesis and Industrial Production Using Plant Cell Cultures

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course on pharmacognosy and metabolic engineering this is lecture number 60 where under the broad domain of phenolic

I will now talk about the secin and its biosynthesis perhaps you remember that one of the very early classes I have

mentioned about different plant cell culture based products so where I mentioned about secon it's basically a

red dye uh and and this is basically the first cell culture based product which has gone to Industry produced by mitsu

petram limited so cin is basically a nap on pigments so we will see that so let us

see the concepts to be covered what is sorin why there is a need for secin production plant cell and organ culture

based syonan production and metabolic pathway for cian production so these are the topics which we'll cover in lecture

number 60 this will be followed by lecture number 61 where I will talk about the metabolic engineering for the

secin pathway okay let's go to the slide so secin as I said that this is basically a

red pigment so red pigment so it's a napen on pigments so if I draw the structure it's like

this this double bundle and

then okay so this is basically the secon in pigment you have o here

and then there is this is a napon pigments

and this is red in color

now how what are the different uh uh okay uh how this pigments is

synthesized number one is an aromatic

ring the problem is biosynthesized from sment pathway

then number two a prile side

chain is attached to

the aromatic ring and number

three is basically CC Bond formation

occurs between the aromatic

ring and the rile side

gen resulting in the formation

of of the last ring of the quenon

skeleton so it is quite clear this is the rile side

chin and then this is the aromatic ring and then third thing so as I said this is a pigment which is red in color

and this is produced mostly in the members of the family boragen so the boragen is the botanical

name of the family Bor borin under this the name of the plants

the most important plant name I will put it which is uh lithos spum

Arizon so this is the plant which is producing the secin now apart from this there

are a few other plants which are also producing secin these plants

are I I I write it here these plants are

aravia aravia species then anusa

species then Unos species then eum

species ium ium species H ch

so I will mention about other this the species name of this gener later so but the main uh plant which is used as a

system for exploring the codin production is lithos spero later uh people are using arnia anusa as well so

which what we'll see now why we study secon next question we study secin because it's it's it is basically used

as a constituent for the Japanese national flag so

Japanese national flag is basically red in color and that

red color comes from secin now apart from

this uh the secin is proven as antibacterial compound so it is used in different cos cosmetic products

Healthcare products but most important secin is nowadays uh

tasted as anti- cancer Regents particularly different lung cancers cin has proven good results and if you make

a search in the Google secon in you will find more than 70 80 papers appeared on these different uh applications of secon

in particularly in the in treating the cancer cell lines and how it it basically inhibit the cell line growth

all these things but but that we are not going to cover because that is not on the domain so we our main domain so our

main interest is that biotechnological production of cin and it's used apart from the non- pharmaceutical say normal

Healthcare products like uh um cosmetic application like uh other other uses as I mentioned for this color FR so now we

go to the next slide so this is basically the lios per Arizone plant so and this is The

Twig and and the this is the root so you see the root is basically red in color and and uh once it accumulates lot

of ponin and then it turns towards this brown color so Brown means it is a very concentrated amount of cin is present

there and that cin is basically accumulated in the uh bark so it's it's it's secreted out from the cell

uh and then it accumulates there in the bark and then it is possible to extract the cin out of it okay and next is this

that uh demand of secin was very high so therefore earlier uh secin the lios

speron plants are harvested indiscriminately from the nature and particularly Japan and Korea and that

leads to a situation where the plant is slowly moving towards endangered species so at that point of time the Japanese

scientist they decided that they should explore the alternative way of secin production so the first approach was

cell culture if Cell cultures can produce cin or not so and uh for your information this attempt was very

successful and what you see here is basically the cell culture of cin lithos spum growing here so the uh

the one red this is basically grown under the dark condition and the one uh yellowish and white in color this is

the cell suspension culture grown under the light so what is clear that when the cell cultures grown under light it is

not synthesizing the red pigment however when you transfer this culture in the dark that

synthesize the pigment this is exactly opposite to antoin because antoin requires light for its synthesis but cin

is basically the dark condition so next what we see is this the biosynthesis of ponin so biosynthesis of ponin we will

uh discuss later so this is basically the structure which I have already mentioned uh okay and uh biosynthesis

what we are going to study that one root basically comes from sikic Acid pathway this is the simate Phile propan pathway

and another root is coming from the Tarin pathway from gpp and this is phy alanin and then ement and then these two

joints and then the pathway moves and ultimately it produces the secon so that we are going to see later okay now this

is another species Arabia eukroma so Arabia eukroma was explored as an another promising source of sein and

arnia grows actually plants are available in India so the scientists from csir Institute of Himalayan bi

resource technology so they basically were very successful in producing the cell culture of Arabia and which can

synthesize the secin so what is showing here is basically the gous culture of cin K culture of Arabia here and here

basically it started synthesizing the pigments okay and this is basically a microscopic picture but most important

here that if you concentrate here the bottom one so this is basically when it's

grown uh in Ms medium Arabia it is not really synthesizing cin or even if it is synthesized cin it synthesize very low

amount but when it transferred to AP medium that is Aria production medium so and that leads toonin

biosynthesis so basically they have designed a production medium and that with that production medium they are

able to synthesize to to make the cell cultures accumulate uh cin now again the cells cell suspension culture of Arabia

eukroma under light and dark condition so again under light condition so that is almost no

coloration but again under the dark condition you see that it started synthesizing

secon so that's very interesting so next is the once they have uh standardized this so they have actually scale up in a

laboratory scale bioreactor 7.5 L bioreactor what you see here that is the red colored pigments accumulated so this

is basically the secin

pigments okay now apart from Arabia eukroma there's another species

Arabia hispa so that is also another source of opsonin and this paper published very

recently so they also the these scientists what they found that normally

hispa is basically uh producing secon in its root and first they have developed callus culture and then you

see kalus cultures were capable of producing uh secin is red in color and later they also established a yoot

culture this is hary root culture and Har root culture synthesizing secin and again here from this scous culture they

are able to make the suspension culture and this suspension culture produces

secon so next we go back to the lithospermum work so lithospermum is the main species so which is exploited for

secing production at the industrial scale so here one important important point is this that normally the whites

medium was used and but whites medium the problem is that white medium when they

supplemented with uh indol latic acid it produces cin but the amount is only 120 mg per liter

okay however uh what they thought that maybe the secin production requires some manipulation of the medium this is

called empirical manipulation that is by manipulating the maccro and micro elements for example you see that sodium

sulfate so in VI medium it is only 200 but it is, 1480 such a huge amount and they have also enhanced the content of

calcium nitrate okay along with uh along with some other manipulations like uh boric acid and uh uh so Crose

concentration also slightly enhanced and so this medium they call M9 medium so M9 medium is

basically uh is standardized as the production medium so that Medium when when lithos eison cell cultures are

grown in dark in the M9 medium they start synthesizing the secin normally the lithospermum cell cultures are grown

in either white or LS medium so there it ly produces any secin so the take-home message is the M9 medium now taking this

into account uh the and this paper was published in 1981 in PL cell reports hujja so they're from Japan and this is

basically the mitsu petrm so where uh they have I have shown this picture one of the earlier slides when I talked

about elicit or S culture based metabolite production this one I have shown and also I have shown that this is

growth dissociated product so first there is a growth of the cell cultures and then the

product formation uh so that is why the cell growth is

basically favored by LSR whites medium and this is by M9

medium now if you see here right side so here basically this is the production Medium as it's mentioned here that

uh this is M9 medium M9 medium and this is basically the

medium for the growth so this color indicates this color indicates that this is basically non production of pigment

but the cell growth and then once the cell has grow sufficiently then uh the medium were removed and the cells were

transferred to the another bioreactor which is basically the production and

here they have supplied the M9 medium into it so that is that is the M9 medium first to supplied in this in medium

vessel so where M9 medium is mixed up and then from here it moves here and then uh you

allow to grow the cells in in M9 medium then what you will see with time you will see the formation of the

secin secin accumulation

here so this I have taken from this ctin biotechnology Journal so this is basically the method which is followed

by mitsu petram so mitu petrm m i t s u i so this is basically an overview first

one is the plant lithos Perma merizon plant and then from there you grow the cell culture that is

under light and dark okay and then or you have used here the the production medium maybe here

also you you have used M9 medium and then next stag is this is number one this is number two the next

three is that laboratory scale uh scale up using bioreactor you see the red coloration is nothing but the secon in

accumulation and from there it goes to the industrial scale that is number four while this is also a two stage

bioreactor where the first stage was basically for the growth medium uh and the second one

is for the production stage first one is the growth stage and second one is the production stage and eventually the

secin are produced now what has been found that cell

culture is a very good system but over time cell culture tends to become instable therefore there is always need

for selecting uh proper cell lines or new cell lines which are capable of

producing cin so that requires a significant R investment which is also costly but that process need to be

continued if C culture based products are being produced in the Biotech Industry so the R&D should be active so

alternative way is basically to root why root because roots are basically the organ whereon synthesized which I have

shown in one of the earlier slides so now here cultures were used and you know by this time what is hary root culture

that simply the lios Arizone plant can be infected with wild type agrobacterium rogenes and as a result of that if the r

plasmid tdna of the r plasmid transferred into the plant genome and then it leads to the formation of he

root phenotype and this he root phenotype uh upon slight manipulation of the medium so it is possible to make uh

secin so uh like the media manipulation I have made it I have told that is you have to

increase the concentration of calcium you have to increase the concentration of sodium sulfate so and that leads to

cin and this is basically um and based on that they developed a root culture uh medium so root the that means the normal

Ms or LS medium here it can support growth but but in order to have the secin synthesized you have to modify the

medium so they have that is why they have used the term root culture medium root culture solid and liquid

medium where you will see that it is producing secin so one thing is happening that the

root cultures are producing cin and these cines are secreted into the medium so that is indeed a good point and then

uh and then uh they have used a column uh this X2 column basically for uh for adoption of secin uh so that this

ionin can be later harvested so this is all about the cell culture and organ culture based production of secin so now

what about the biosynthetic pathway I have shown in one of the slides so one thing is that if this is your

secin so it is a neptoon pigments and this secon is synthesized by joining two Roots one come from

the Phile propanoid and another comes from the tarpo and then these two molecule for hydroxy benzoic acid and

janile pyrophosphate joints and it makes janile hydroxy benzoid and this janile hydroxy benzoid is considered as an

intermediate and that subsequently through multiple step process within with the formation of J hydroquinon as

intermediate it eventually forms the secin so this we are going to see a little bit more details and I will end

this class perhaps the current status of this is this is based on the on a review report published in

2022 where is this that there are uh there are different enzymes uh because there are lot of things which

remain undefined yet however uh C cytochrome p450 enzyme perhaps this is a hydroxy this has been characterized and

recently this has also been characterized this is also an hydroxy as you see o addition is there so these are

the new enzymes which have been characterized and that actually

advances uh a little bit ahead of the secon pathway what we knew this earlier 10 years before and with time maybe the

whole pathway will be elated but this is very difficult because some of these are membrane bound enzymes and then not only

that the secon is secreting out so basically it involves different organal is like G bodies endic reticulum and

finally it comes to the apoplastic space so that part is still also not clear but uh so with this I end this class and in

the next class I will talk about the metabolic engineering of secin pathway there we'll see the pathway and what are

the attempts made by the scientist and also I will tell you the current status of the secin pathway in the next class

with this I end this class here thank you