Metabolic Engineering of Vanillin Production Using HCHL Gene in Plants

[Music] [Music] welcome to nptl online certification

course on pharmacognosy and metabolic engineering this is lecture 59 where again under the broad domain

phenolics now I am going to discuss metabolic engineering for vanilline production so in the previous class we

have seen the vanine biosynthesis vanine localization and different views of vanine

synthesis uh argument counterarguments uh so that all really contributed to the knowledge of vanilin

but now if you think of vanilline production so vanilla planifolia plant is a slow growing plant

so even it's a monocord transformation is difficult agrobacterium still not yet uh

been standardized so one has to use the particle gun or gin gun even then uh the growth of the tissue is slow so

therefore to hardness transgenic vanilla planifolia for more vanilline it Still Remains a dream so

therefore uh alternative system should be explored for the production of vanilline so the

concept to be covered now is basically discovery of a microbial degradation pathway of ferulic acid and then I will

introduce hchl enzyme then we'll talk about opportunity of expression of HCL in plants and then we'll see what is

going to happen when HCL Gene is transfer into the plant and what is what happens to the vanilline level so this

is all our topic so uh coming back to my uh discussion that ferulic acid so ferulic acid here will be exposed as an

alternative or as the starting material for vanilin and ferulic acids are basically C6 C3 component so it is a

abundant component present in the cell wall of dcot as well as in the monocot as well so ferulic acid was used

uh by different groups who are working with the microbal transformation and success uh success were

achieved so this is one such work which I'm going to dis tell about the degradation of a ferulic acid

degradation or sorry ferulic acid degradation pathway by a microorganism so I said feric acid are present at

abundantly in the cell wall so therefore ferulic acid can be harnessed from Agro Wass so aggro was particularly the cwall

Wass are a good source of fic acid and such an aggro generally they are dumped into the soil so the soil reached with

Agro West can be checked for isolation of microorganisms uh

that are capable

of utilizing ferulic acid for

their growth so in other word using ferulic

acid enrichment technique taking soil from uh reach in Agro

West uh so one group in United Kingdom they isolated uh

bacterium which is a sorus fluoresence

so 103 is the name of the strain uh particularly the person who isolated his name was Dr Arjun nard so

it's it's name as that so these ponus floresent were capable of utilizing capable

of utilizing ferulic acid

as soul carbon

source that means when you grow this microorganism in a medium you don't have to put in an enrich medium

simply uh minimal medium so that means where you have only the TR Sals

Plus I acid as so carbon

Source okay and and the microorganism can be inoculated and that mean this sud

Florence and it will grow when it is growing that means it has to get all its nutrition from the fic acid so during

the process what will happen this ferulic acid will be utilized isn't it yes it will be utilized and that means

utilization means the f g is structur to be broken down so utilization means f a structure must

be broken down on a Time course basis so that is what is called the feric acid

must undergo degradation so which is called feric acid

degradation so narbad

actually uh check the fuic acid degradation so they have grown the cous fluoresence is

in 103 in the minimal medium and then they did time course analysis that is on hourly basis and how the amount what the

feric okay uh so the feric acid was given at zero time and see with different time points how the feric acid

content is getting reduced and what are the different

products formed as a result of that so someone is interested can uh see this publication I think this is available

free in the net so one can study that so that that describes about this novel pathway so while doing so finally what

uh they have come across that ferulic acid under goes degradation through this root that is ferulic

acid first converted into fural qu then feral Co will be converted into hmp KO and then it forms vanill

then it vanilic acid and then it forms protocatechuic acid and then what happens there will be

the ring cleage and total used

up so this is the process so on a Time course basis narbad group they have found

vanine velic acid protocatechuic acid PCA and then rink cpage and then again they try to look this is where when this

is happening is it a entic route or non entic roote while doing so they found that it was indeed an enzymatic root So

based on the available literature uh they try to predict several Pathways and then this particular hypothesis they

tested and it worked well and so and accordingly they frame this so what we see here is that first it converted into

fural qu so and and when it is converted into fural qu so there must be an enzyme so we know we have read the plant for C

so which converts feric acid to fumaric acid to Kumar qu so here also the bacteria induced a forcl which converts

fic acid to FAL qu and then there is another enzyme induced by the bacteria which subsequently identified as hchl

which converts FAL qu to vanilin and then there will be U uh vanilline

dehydrogenase which converts vanilin to vanelc acid and then protocatechuic acid and so on so uh Nar birs they

characterize these enzymes so and and then when the particular look into these issues from ferulic acid to vanine they

found that it is not that simple it's likely that involve a qu thester so it's qu so that is why qu is formed that

means FAL qu so uh then they try to see what is happening is it if you remember that when we talked about in one of the

previous classes we talk about the benzoic acid biosynthesis so there one we call non beta oxidative root and

another one is one is qu independent root what we have mentioned in the

previous class about HBS which convert paric acid to parah hydroxy Benz alide which is coind dependent root and

another one is that qu dependent

and beta oxidative root and the third one may be

Co dependent yet non

beta oxidative root now this particular root is basically belongs to third one that

is co dependent but non bet oxidative root this root and what is showing here in this side is

basically the qu dependent beta oxidative root that is showing here and the one in the left side is co dependent

nonb oxidative root so major difference is that the end product is again a qu but here end product is an aldhy but

Co is involved that is it is called so that is why this is called as a novel root and this novel root they have also

isolated this novel enzyme which is hchl so which under goes hydration remember I talked about hydration and cleavage but

that was that paper published much later and this was a 1998 paper so this is first concept they have given so first

hydration then second one is chain cleavage okay so

now uh so what is the message message is this that hchl and and the hchl enzyme

converts fural Co by an intermediate

into vanine and in this process it releases atile

Co and in this process it requires addition of water so this is basically a two-step

process first one is the hydration and the second one is

the chain clivage I think I need not have to show but if you want I can show the

intermediate structure and and this is

okay I think it is clear so the C3 chin is now becoming

C1 so this is the uh HC HL enzyme what is hchl hchl stands for four

hydroxy C Mo

qu hydrates lies it was described like this so much later now these people are

calling van vanine synthes so okay now other question is this this hchl enzyme not only accepts peral KO but

also so the substrate specific cities so what are the

substrate we have written okay only one perile qu so apart from per

qu uh Forum qu was tested file Co was

tested Copo KO was tested and camil KO was also

tested and what happens as a result of this test

so the first one it produces vanilline second one it should produce

four hydroxy balide and the third one it will make it

will produce Proto alide however it did not work with Copo

KO neither C KO so so at least the enzyme worked with

FAL qu Kumar and cfal KO and they have also checked the C values for this okay now all so far what I have discussed

this is all about the micrell pathway now if you remember the Phile propanoid path one thing is now clear to you that

all this file karile or

Capo these are the uh these are

the substrate or intermediates

of Phile propanoid pathway operating

universally in plants so if it is so then this hchl Gene so it it's basically it an

opportunity so hchl Gene offers

opportunity opportunity of hchl Gene to

manipulate plant metabolism

[Music] so this is basically [Music]

metabolic engineering for

vanilline production in Plants

using hchl gene it's a bacterial Gene so two

systems were used number one is basically uh he root system of daturas stramonium

so he root culture so we call them now as Co transformed or

transform y root culture and second one they have used

tobacco plant so in both the cases agrobacteria mediat transformation

was used so in the first one a rogenes was used so recombinant exacum rogenes

uh harboring the HL Gene was used and second cases the combinant agrobacterium tumans was

used Harding the HL PLM so that was typically the binary

Vector approach was used so in case of agrobacterium isogenous so where the while type uh RNA will be there along

with that the binary Vector containing hchl was introduced in case of tobacco so there are disarmed agrobacterium

tupaan containing only the V was used and the HL binary Vector was introduced there and uh so both strategies for

different purpose for Co transformation we need to have the heot phenotype but for the tobacco we do not

want the uh the uh Crown G and therewi the disarm strain

was used so first I will describe the uh work what happens in the hay root so the construct basically they have uh uh

slightly modified the construct because it is originally a bacterial Gene so they have done certain side directed

mutagenesis so that it it it makes the starting point uh as the plantbased so it's a changes in the KAC sequence so

that it it when it started the translation the it's the first amino acid should comes methon in there so

that minor changes were made and then it was put under control of a 35s promotor which is constitutive and not Terminator

and then it was put under b19 so and then it was transformed both the toaca LI tissue and tobacco Li tissue so

now now this is basically the hay root culture of luras stramonium

So Co transform this is the co transformed

oh containing the hchl and this is simply transform that is control only only

the wild type agrobacterium rogenes was used so if you notice one thing uh you will see here that the coloration so the

here is slightly reddish in color as compared to this one and uh this is basically the southern block so what is

showing here that there is no band here also in one line it's almost F and that was also reflected in the northern so

these are the co transform lines whereas the control line no band was there so this actually

uh proves that yeah so the indeed code transformed hay roots are created and so and and they are showing Northern

signals that means the hchl gene are expressed so in those days Northern was used rather than the uh PCR and Northern

is a much more uh foolproof technique because Northern signal confirms that your plants are transformed just simply

artificial you may get lot of problem with agrobacterium sometimes but northern will not show that so that is

why Northern but nowadays Northern people are not doing even we are also not doing in the life so uh next next

what is this that okay the gene is Express it produce the protein uh it produce

yeah the protein now whether whether it produce the protein or not so that means whether hchl protein was there or not so

the antibody was earlier antibody was raised against the purified hchl poal antibod so that was used and basically

what is showing here these are the hchl protein which is single band which was detected in the transform

lines so you see here that H6 h10 h11 whereas C5 or H4 uh uh C6 h8 it was not there that was also reflected in there

so that means the protein was successfully produced that was proven next is the protein means the en whether

it is catalytically active or not that has to be checked so so FAL qu was synthesized and the root extract was

taken uh from the combinant from Co transform route as well as the control route so

when control route was taken control har root with fural coincubation it did not produce veline whereas when Co transform

rout was used you see this is the substrate Falco and velin formation is detectable that means the the the enzyme

the transgenic enzyme is functional and then in order to be confirm that indeed the enzyme is working well so this is is

some sort of uh checking enzyme activity time course checking so this has also been done so

this is the initial uh so at 0o minute that that Turkish blue color you see the peak

height is more so this is the substrate now with time it is this is going down you see the blue you see the red

you see the uh yeah purple and accordingly you see the peak height initially no product

but now product formation enhanced with time that means there is a linearity so that means the enzyme

activity uh is pretty stable so that has been proven so next is that it it is important to see what happened to the

metabolite whether vanine is produced or not so what is expected that hchl will produce vanilline so when the hlc

analysis was made so two major paks were detected one is this uh these two first one is four

hydroxy benzoic acid GL glucoside and second one is F four hydroxy benzoic acid glucose Ester and this was

subsequently confirmed by uh obtaining standards from different groups who who synthesize

these things uh and apart from this uh so apart from this van pic acid

glucoside was also detected along with some uh

van v n i l l y l vanil alcohol okay along with some vanil alcohol so that's all detected in the co transform Roots

but it is almost nil in the sorry is almost nil in the uh control line you see you cannot see anything

as compared to this so that means that is a tremendous changes in the

metabolism of the co transform Roots upon expression of HL Gene and what again I'm showing here here is basically

the cell wall material isolated from the co transform so the H 10 is the co transform line and this is the control

you see color differences there the co transform are red in color and which is reflected that for hydroxy benic acid

content is also more here compared to this is control and other interesting point what

has been found the ligin monol liol the AC this was basically acylated Conifer alcohol that in the transform line it is

the peak height is this much whereas in the control line it is more that means what happens that upon HL expression the

pathway towards Li monol Lal formation got reduced and as a result of that when analysis were done with monolignol the

content was less so what is basically happening let us see very quickly in in a line diagram

[Music] so uh

okay so uh for Kumar Royal KO I start from here and then maybe

uh KO is there so for Kumar KO normal the pathway moves towards the formation of uh H liin right so this is

four uh Hydrox for uh karal and then this makes towards the formation of

uh con file

alcohol now foric acid is also forar Alco is also very important molecule because it

also produces flavonoids

including antoin right now what we have seen the HL has multi subate specificity so the enzyme

here the natural enzyme here is the plant enzyme so far first one is that CCR and then C A this is

CHS right and if the new pathway if the hchl is working then what will happen it

basically uh competes with

CCR and CHS and then it makes what it makes it makes four

hydroxy Benzel deide but no four hydroxy benzal deide was detected what has been

detected is this uh

four hydroxy benoic acid for

hydroxy benile alcohol not only that the next step it forms four Hy hydroxy benzoic

acid glucoside four hydroxy benile

alcohol glucoside right similarly for ferulic

acid so it also competes

with CCR and HL is here so ideally it should make

vanilline but no vanilline was detected instead what has been detected is basically

the vanelc acid glucoside so this vanilic acid glucoside must have formed from

velic acid and uh then it forms this so then what is happening here is that as a

result of this actually vanilline is perhaps produced but then vanilline immediately the vanilin de

hydrogenous so I I rather write simply DH not any specific uh plant

induced a lot of dehydrogenases are available and then it produces the bandic acid and then there will be

induction of glucos Sile transfer is these are all

non-specific and then it makes this one similar thing happens here GT means glucos ale transference and

then there may be this this be de hydrogenous

row g e n and then this may be an oxidase present here okay so

okay so what we see then we see finally these products the question is that whether HL

has job done his job yes HL has done it job otherwise in the control you have not seen this but it it produces huge

amount of for hydroxy benzal deide so plant found them as a strange compound immediately it induces dehydrogen it

converted into acid which is relatively safer and then that is also produced in huge amount so it produces it induces

glucos ale transference a glucose molecule is added with the for hydroxy menic acid and so finally what you see

here basically the products which are produced as a result of this are for

hydroxy benzoic acid glucoside

or for hydroxy Bic acid glucos Ester or vanic acid glucoside so this is basically the product formed as a result

of hchl because dehydrogen and glucos Ale transfer is is functional so that means okay and now let us see what

happens in the tobacco the tobacco some interesting thing happens so the tobacco plants they have grown for two

generations it produces flower and what they found that the flower color was paler in the transgenic as compared to

control now pal flower color means basically there is a competition for the pathway of antoin so why because the

anthos pathway Works in this direction now if the hchl is very strong so it is pulling up all the substrate towards its

activity or the uh the km values is lower in case of hchl than CHS so that actually allows more Kumar qu to get

access towards the hchl than CHS so whatever may be so if the pathway moves in this direction then

obviously there will be there will be uh

reduction in this pathway and as a result of that antoin content are less now the ligin

content also they found significant changes so this these bottom three are basically the

control these These are the control and upper one is all the hchl expressed

transgenic you see first then unusual coloration was developed as you seen in case of

harot also slightly redish so that that develops this not only that if if the if you see the gym Vel the fenol gym

usually Flores under UV light and they have used perhaps a specific UV light is where it is showing green so if you see

the control the fluoresence is much stronger than that of the transgenic that means what the content of the ligin

is reduced as also I have shown in this slide that the AC Conifer acidil Conifer or ciferal alcohol content is reduced so

because these ultimately form the Lin structure so if it is reduced then obviously it makes impact and similarly

the fol staining also reduced in case of transgenic than the control so these all basically speaks in of hchl expression

but these are more of pleotropic effects but the opportunity is this that this HC HCL can also be used to reduce the

ligdin content also to manipulate the quality of the ligdin content which has not yet been exploded so this is about

the metabolic engineering of veline so in principle it is possible but there are lot of hardles so plants are Mar

more cleverer than us so whatever we wanted to do plant may not always agree to that that is why they they induce a

certain Pathways and make and try to keep themselves safe uh from this unusual metabolites

accumulation so that is the message so there's a long way to go and with this and this is basically in a

nutshell what what happened as a result of that so this is hchl as a result of that basically there is a diversion of

pathway and this is a new pathway not not existed in Plants this pathway non existed in plants and as a result of

when there is a diversion obviously the existing pathways are getting affected as we have

seen with this I end this class thank you