Metabolic Engineering of Monolignol Pathways: Case Studies in Lignin Manipulation

[Music] [Music] welcome to nptl online certification

course on pharmacognosy and metabolic engineering this is now lecture 5 one where I will talk about few interesting

case studies on manipulating the metabolic pathway of ligin so that is why metabolic engineering of monolignol

Pathways so in the previous class I have uh discussed in detail about the classical and contemporary views of

monolignol biosynthesis I have mentioned the role of different enzymes which basically

uh makes uh this monolignol formation possible also I have discussed about the possibility of new uh ligin components

which come which may come from Keo M as well as five hydroxy and so I hope it is clear to you

so now with this I go to the case studies so an outline of monal biosynthesis I am not discussing here at

the end I'll again bring the slide so let us specifically go to the uh specific case studies so this one is

basically manipulation of ligin quality by down regulation of cinile alcohol dehydrogen so so you see that camil

alcohol dehydrogen is where CAD will function so CAD is basically

converts the uh Conifer alide

or copile alide into Conifer

alcohol and or copile alcohol

so this is the enzyme which is called C A which is called Camile alide de

hydrogenous uh so this is a generic name so that makes both the

reaction uh so c a d and I have recently mentioned that now there is an Evidence of

acdd and uh if the ligin is dominated by this this eventually makes the guile g u a i a c y l and this makes the

serile component so it's a g and this is the S so S is to G ratio is important for

the liin quality and also for the delignification issues okay so what they have done here

that they have down regulated the the plant model plant was used tobacco and they have down regulated the gene by

using this anti sense expression of this CAD so so manipulation of ligin quality by

down regulation of Camile alcohol dehydrogenase so what is going to happen that when the cad activity is blocked or

suppressed then uh this leads to less accumulation of piperal alcohol or copile alcohol as

appropriate or or it is also possible uh because if you think about the new views so new views what I have said although

uh we will explain this from the new views although the paper published in 1998 that time it was not clear at all

so what we know that from Conifer aldhy copile alide is formed right so it is possible

that uh by uh by blocking CAD here the pathway May

flow in this direction okay leading to the formation

of more of sying liin it is possible okay so uh what is going to

happen that although the ligin content will be reduced that is the most important point

because you are blocking the cad but it will affect the liin quality that is why this terminology was used and what has

happened that these two are the two antisense line so antisense line 50 is more stronger so more suppression was

work and if you see this is basically the control okay and if you if you compare this with

this what you see here uh clear changes in the coloration of the lignified portion okay so this was further studied

with different staining and what has been found here that uh this looks more intense as compared to other and also

here so and when they check for the cad activity you see in the anti sens line the cad activity is minimum when they

when they compared with the control so that means indeed CAD suppression occurred and as a result of that there

there happened to be or it have there happened changes in the liin quality which is reflected from the

cross-section viewed on the microscope with different staining so uh and in this paper what they said basically this

is the way the liin quality can be manipulated and particularly you can think ahead about the wood quality so

the wood coloration wood coloration this this discovery can really make contribution to the wood

coloration or novel wood colors can also be uh created by transferring this Gene in an appropriate PP paper producing

plant such as Aspen or the next one which I'm am going to

discuss this is another very interesting paper which published in nature biotechnology in

1999 so the title says that repression of ligin biosynthesis promotes cellulose

accumulation and growth in transgenic trees so what they have done as as as I have shown you that very briefly

I'll tell you the pathway so uh camic acid makes parakum aric acid and paric acid

converted into paroil qu and then paracom oil qu either it

makes par uh

karile alide or it

may mix C qu and eventually that contributes to

uh G residue s residue and this is H

residue okay the enzyme here is 4 CL uh so what they have done they here

they made antisense separation of this foral so the plant was used is uh the Target and plant or Gene source is same

the popular or no Aspen Aspen is the English name which is called populus tremuloides this is a plant

which is used for making Pulp and Paper so they

created transgenic Aspen

with anti sense expression

of PT for PT stands for prop terides so so what they thought this may

be the best step to block the pathway okay now one thing I mentioned that the interconnections between the

pathway that is metah Hydrox ilation converting Cil Conifer converting Kumar to Cil so this was discovered in 2004 56

onwards but this paper was published in 1999 even then the uh these scientists from Michigan State University led by

Vincent Shang who was the leader in discovering this Pathways so they thought such

a nice possibility that blocking Force at the beginning will make impact into the end product now

question which came to our mind that there are multiple

fors now in fact the foral what they have chosen that is specific for paracom KO so this foral is actually

for hydroxy uh syo qu lias but specific for paric acid so that it accepts paric acid

as a substrate and make parar Co not the fural heric acid as a substrate or synoptic

acid as a substrate that you must remember so this is the beauty and then what we see that what has happened as a

result of these what has happened the outcome significant reduction

in ligin contents let's see so this is the

pathway so this is the foral here this for they have blocked using anti sense PT

forel so when this is blocked then what will happen the flow and I have mentioned uh by now it is it is clear to

you that this pathway as such is not contributing towards

monals okay I have mentioned that so what is going to happen that here basically the flow when you block

here will be reduced and if it is reduced then definitely the one flow which goes to h that will be also

reduced the flow which goes to G that will also

reduced and the flow which goes to s that will also reduce so as a result of that what they have found

repression of liin biosynthesis but interesting point is

that this promotes cellular cellulose accumulation and the growth of the transgenic trees so I'll show you uh

some of their results so here this is the control plant so where no normal force is

functioning no expression so these three these four are different uh transgenic Aspen expressing

uh antisense for what you see here the growth you see is much more in all the transgenic Aspen with antisense

expression that is surprising but that is what happened so it did not really affect the

growth but it reduced the liin content but compensated with cellulose accumulation and that is why that is

reflected in the growth now as I said the rep of ligin biosynthesis that is beautifully shown here in this left side

so what they have done basically fluoresence microscope of the cross-sections of a nodal portion of the

transgenic and the control line so this is basically the control and this is basically the anti

sense Express transgenic line so they have used agrobacterium tence mediated transformation but I am

not uh discussing that because that is not within the perview of this course and I have mentioned about how

Transformations are being done so we are interested only to see the consequence of genetic manipulation so

here you see in the control with this uh ultraviolet excitation at 365 nanometer this shows autof fluoresence so autof

fluoresence is the characteristics of the phenolic compounds so the autof fluoresence you see

here xylm but here the autof Florence level is

low why it is so because it suppress the LI formation this is very clear and next

point is that it it promotes cellulous accumulation so you see that this is how what this is a diagram I put it

here where ligin or cellul actually they form the complex in the cell wall okay so what happens that when

cellular and hemic cellular are also there uh so when uh ligins are suppressed that somehow compensated by

cellulose enhancement how it is happened they are unable to explain but it happened and as a result of that whether

it is good or bad it is indeed good because more cellulose accumulation as a result of

compensatory mechanism it is good because that makes the

plant Aspen transgenic Aspen plant more suitable

for PP and Pier making why you know the pap quality is dominated or determined by

the cellulose quality if the cellulose content is more the paper quality will be more and also another important point

is that in order to make the pulp and paper one need to delignify so delignify means the wood has to be treated with uh

heart's corrosive chemicals which are not environmentally friendly like costic soda and so so that will delignify the

wood so now once the ligin content is less so the treatment delignification treatment will also be reduced so that

will be environmental friendly and second point is that it is producing more cellulose so actually it is

basically a gift gift of the scientific Venture so which can be indeed used for making palpant paper and that is why

this paper published in nature biotechnology so nature biotechnology Journal outstanding papers with with uh

with uh possibility of practical utility such papers are published and this is indeed outstanding although it's pretty

old but in in a class even in my uh theoretic in my classes uh where I do the board work I always bring this paper

instead of uh discussing some of the latest papers uh because it is not the year of Discovery important or the

latest paper it is important the scientific content so this is what it is so and uh the other aspect what is this

this the same group this is the group basically LED at Michigan State University by Vincent Chang so their

group also published another paper in 2003 in proceedings of National Academy of Science pns where they have done

three things number one uh

pt4 anti sense

suppression number two uh number two is this CD 5H over expression and number three

simultaneous expression of

both PT foral anti sense plus

PT c a l d5h sense

so pt4 CL if you again use a strong promoter the level of Lin reduction will be more so that led to the formation of

desired results that is a significant significant

reduction L contents and ptld 5 expression what happens that that

basically leads to this one this leads to

more accumulation of actually this is a online class had

it been a uh offline class then I could have asked the same questions to the student and student seeing the board

they can guess and give the answer but here you whatever I tell you have to listen so more accumulation

of s units isn't it because this contributes to the serile unit and this contributes

to the gunit units now when you make over expression

so that means the pathway oh sorry I I have made a wrong marking so it's it's not this one it

should be the this one so uh that means the pathway moves this

direction and eventually makes more s so more s has some Advantage because that will be easier to

delignify if the S component is more than the G so that is the intention so that has been achieved and the third one

what they have done so there the the block this uh uh

for so and

and then they overexpress this one and as a result what is going to happen I will try to tell

you so the result is this that plants expressing both trans gen resulted in 52% less

ligin and 64% higher SG ratio s is first so the S is

dominated and 30% more cellulose it's a very ideal situation so that is what happened and HG ratio increased also

accelerated cell maturation in stem secondary xylm pointing to a role of sying liin Mo

in coordinating secondary wall biosynthesis so what happens when this happen that is more SG ratio that

actually leads to accelerated maturation that is wall thickening and lignification and that happened because

of this C5 AB regulation and what you see here this is basically the control and this is basically the

transgenic so here uh what they said that this leading to accelerated maturity

here then compared to this one and also what you see see that this is basically the

control control the uh uh the Lin content was more but in the transform lines where this for was suppressed you

see that the uh the liin the the the foral activity is getting

suppressed and not only that the liin content which is represented here the contents sorry the digin contents are

also getting suppressed and liin is to SG ratio SG ratio is also getting is getting

enhanced and the top one the middle one is basically the enzyme activities enzyme activity for when it is

suppression then the activity of forcl is less as denoted by the red bars and the activity of

the CL 5 H enhanced as a result of op expression so what has happened that this basically makes accelerated

maturation with more of s component that is useful for the industrial uh exploitation of these

plants for making Pulp and Paper with less effort for making uh delignification and with a

more s component and that is also more of cellulose accumulation so that is all good so this is the paper which

published in uh PNA so here they have used three different ways either singly or both

together so the last one now I will discuss that is a case study where down regulation of uh hydroxy Camile coate

hydroxy Camile transfer this is what is the HCT so the

HCT uh down regulation and that affect lfic

development and forage quality so HCT what it does it basically converts Kumar oil KO to CFO Kumar O

simate So Kar KO to Kar sikim okay let me uh use in that way

and Kumar converted into cilat and then is converted

into CFO qu so what is clear here so down regulation of

HCT uh in the transgenic alpha alpha FX this so what they have done is basically they

have used the antisense antisense suppression or antisense expression

of HCT and what will happen if that is reduced

then this flow this flow will be affected so this flow if this flow is

affected that means uh commod koi will accumulate okay but uh the flow from this towards this will be less

and eventually the other L path will be less so then what is going to happen one thing is that if you make antien seption

of HCT it is very important because that will leads to what okay spaces less I put it here that

will leads to less uh G

residue even less s residue because you are controlling at this point HCT because

HCT is the point where uh which forms Karo simate from Kar qu Kar simate converted into C Sate by c3h that again

converted into C qu then cile Co converted into feral Co then feral Co subsequently utilized

for pushing the pathway towards either Conifer alcohol or uh V Conifer alide to Conifer alide

five hydroxy to eventually the copile alcohol so all root goes through this and if all rot goes to this and if you

make changes here then obviously this will be affected so the ligin content is

significantly suppressed that is the outcome but one more thing you have to

understand that you are allowing in this way to accumulate more of paracom quay so paracom qu level will enhanced so if

paracom coil level will enhance there may be a possibility that that the pathway

may move uh that is which contributes for the formation of what which contributes

for the formation of Kumar alide and eventually

par karile alcohol so once is accumulated so it is possible

that this pathway flow moves in this direction and this direction eventually what will happen that paracom alcohol

will accumulate more and that will contributes towards the formation of H Lin in

higher amount whereas this will be lower amount

so the plant which was used uh which is uh uh legume

trifolium so that plant okay so alpha alpha yeah alpha alpha

trium is the scientific name uh so the alphaalpha plant is basically it is a dcot it is supposed to

produce more of S and G but now because of antisense suppression it suppressed the total Lin content but the

composition got changed so it is now accumulating more of H liin so when more of H lignine is

accumulated whether it is good or bad that is the question we need to understand because 4 quality so this is

used as a forage crop forage crop means cattle feed so normally cattles and ships they prefer to eat grass but they

less prefer to eat duts okay whereas the alpha alpha is used as a forage crop now what happen that if the problem is this

that uh for if the forage crop contains more of S and G residue that is the the leifi

portion that actually eventually helps that eventually makes difficulty by the this grazing animals to digest as a

result of that they are facing indigestibility leading to a disease called blood disease and that eventually

kills the animal itself because of more ligning content in their feed so that is why there is an effort

to make less lignified uh forage crops so that the cattles or grazing animals can safely

eat this now this is something very interesting here what happened in case of this alpha alpha now the G and S

component are reduced because you are blocking at H C but that in turn leads to more Kumar KO accumulation and that

Kumar qu channelized towards this H Lin pathway so where you you know you have this uh CCR and Cad and eventually it

makes paracom alcohol and it makes more amount of paracom alcohol and that is now channelized towards the AG liin So

eventually the transgenic alphaalpha accumulating H ligin in higher amount that is actually very good and people

have checked the scientist they checked that in digestibility they found a positive results so it is basically

promoted as a very appropriate forage crops with less signification through this transgenic

approach and this paper published in phytochemistry uh in the year of 2007 again I must say there are more latest

papers but I prefer to discuss this because it scientific implication is much much higher than only the

technological papers and this is finally the uh the picture so CK stands for the control these are all control and these

are the trans genenic although what happen that the the plants appear to be suppressed it is not like the Aspen

Aspen what happens that the transic plants are more healthy but here no they are with

less healthy less suppressed but the good point is that whatever they are the uh the the presence of

this uh Sile units which is dominated which is shown here by this purple red coloration you see that that is now

getting changed so that means it is not accumulating thein so this is basically

the transgenic and this is the control so the relative levels of a are

strongly reduced although G units are reduced by similar however more striking

compositional changes more striking composion increase in the H unit and I said the implication of that so with

this and this is basically the the content of the ligin acety bromide digestion these are the control

and these are the s antisense transgenic lines it is suppressed and and uh and monar yield

also they have shown as you see here that in the transgenic line the yield of

H is more than that of the control

black one it is more whereas the others are suppressed both both G and S are suppressed are

suppressed but H is enhanced so it's a wonderful scientific achievement and that is why I have covered and with this

with three three or four different case studies I have given you some flavor of metabolic engineering of the ligin

pathway so with this I end the class so basically before I end just to tell you that this HCT was once was targeted C4 H

was targeted and the results are really outstanding and these are the different units so this is basically the

biosynthetic pathway what we have also studied that the complexity but complexity uh gives new tools for man

population with this I end this class thank you