 Thanks for the opportunity and I guess the responsibility of this giving a talk in this session. I Hope I do a good job So We heard this morning from won't borion who laid a great foundation for me I'm gonna have to go over some of the same material, but I hope to to Minimize that overlap and and talk about some other issues That I think are important and some other Findings that we've made that I think are really new and important Components of say that that what we might call the toolbox for manipulating bioenergy species so There are many entire conferences That are held about biomass improvement, and I've got 25 minutes or 20 minutes to talk about what I think are some of the targets that we've been able to have an impact on and What are those what are the topics that come up when one considers what? How we might improve biomass because it's not just about the topic. I'm gonna be focusing which is on which is Elicit the bottom there biomass quality. How do we manipulate lignin? There are many other issues that are gonna that we need to consider If we're going to be successful and in founding a bioeconomy to address at least some components of the problems that we're facing Amongst those amongst those topics is yield if you ask an agronomist or a plant breeder What are the three most important things when breeding? Breeding crops they say yield yield and yield Other components that are of course are very important for sustainability include water and nutrient use efficiency and pathogen and pest resistance and of course those have an impact on on that yield equation But what I'm going to talk about today is how we can manipulate quality of biomass to hopefully enhance its value and The as you will have noticed the figures on the left is not a figure of about plants because you most people I Or the most of the people in this room probably don't have a particularly great affinity for plant breeding but you all know about dogs and Those two dogs. I think it's a what is it a great day in a Chihuahua or something like that all derived from a common ancestor that the wolf About it's debatable, but about 15,000 years ago So thanks and some of these species like these miniature dogs these lap dogs are only a few hundred years old so they are the product of domestication and They I think Clearly convey that the genetic diversity that's present in an individual organism in this case the wolf can be exploited and Lead to the sort of diversity that we see in in these species Of course time gone by these were very important attributes to have for the use of dogs now where there's more recreational if you will so When it comes to plant breeding if I've brought you along with that introduction I'd like you to introduce you to The cob if you will of the the species that was the progenitor of maize or corn That plant is called T. Ocente. You've got a ruler there to see How big that cob was you can see it's a single row of kernels and yet from that species humans were able to tap into the genetic diversity To of found in that in those plants and mutations that arose to develop a crop that feeds a huge amount of the world Now that process took about 5,000 years it continues today but the the genes that Contributed the bulk to the of the characteristics that allowed us allows us to domestic allowed us to domesticate maize Number in about half a dozen these are genes that affect Cobb size row number as well as the overall morphology of the plant and in fact the the The the increases in productivity over the last century in corn can largely be attributed to Changes in the body plan if you will of a corn plant that allows us to plant many many plants closer together in narrower rows And of course the fact that we don't have to have horses going down the rows. We have more sophisticated machinery So we've done a lot to improve maize but that most of that of course there's been there's been improvements in various traits including GMO herbicide tolerance and pest and pest tolerance But most of the effort there has been focused on yield. It's been focused on the cob and the point that I want to make is that the the actual quality of the biomass that's generated by corn or I Think looking forward at more reasonably Dedicated biomass crops the opportunity to improve that that biomass has really been almost entirely not exploited So as as won't pointed out in his talk we need to have access to genes In order to improve these crops we need we now have available to us CRISPR-Cas technology You know as I mentioned this was corn is five thousand years in the making Sally threw down the challenge of Fixing the problem in the next 24 so we have a big challenge ahead of us but we need to have access to the genes and understanding the regulatory machinery that affects the expression of those genes in order to move forward so Our work as what's does we are collaborators on a single g-sept grant focuses on lignin biosynthesis This is a very important polymer to the plant. It rigidifies the stem And and the water conducting tissue to allow these trees to stand upright But as as won't mentioned earlier. It has a very important impact on biomass Processing particularly for securification because it it slows down or reduces the efficiency by which we can extract useful fermentable sugars from that biomass To the plant. It's an enormous commitment of fixed carbon Something like 20 or so percent of the biomass is is lignin So that represents a lot of this amino acid phenylalanine being shunted being generated to begin with and then shunted into this pathway and and a point that I want to get to at the end of my talk though is That it does have compared to the polysaccharides of the wall a high energy density And that is something that looking forward and I think that's what partly what I'm supposed to be addressing today It's something that we need to be Looking for ways to exploit So here's another depiction of the lignin biosynthetic pathway in the top left You can see the processes that go on inside the inside the cell where the amino acid phenylalanine Is used to make three so-called mono lignals the building blocks of lignin The upper one is as won't mention is used in very small quantities that the G and S monomers Caniferal alcohol and cynical alcohol are much more abundantly used these monomers are synthesized in the cell and Then exported into the cell wall where they're activated to free radicals by peroxidases and lacases and then chemically combined to form a polymer now What's into what we have learned over the past 20 years and have furthered our understanding of through G sub funding is that? the type of lignin that is made is dependent upon the balance of those mono lignals that are being made and won't Mention that his CSE mutant develop deposits an H lignin and that is really quite remarkable in terms of our understanding of our typical biopolymers And it's something that again we can take advantage of so if we think about Biological polymers that we're familiar with nucleic acids proteins polysaccharides The synthesis of of nucleic acids DNA and RNA this this is template driven So to make a strand of DNA molecule you copy a strand of the others the opposing strand in order to make a molecule of RNA you read the template of of Excuse me of the DNA In order to make proteins you read the read the information off of the mRNA strand Polysaccharides are a little bit different, but still they're Architecture their chemical composition is strictly dictated by the enzymes that assemble them now if we compare that to lignin We find that Its composition is dictated solely By the amounts of the various monolignals that are being made and that provides us That's why lignin modification we consider to be the low-hanging fruit of biomass improvement It is the most it is the easiest thing to manipulate and in doing so we change the chemical Characteristics of the polymer and we change the architecture of the polymer and all of those both of those things We think play into the impact that we have on biomass Processing so there's there's I think a number of opportunities. We've seen that have come out in the literature Some of them like won't show to with CSE we can and I'll show another example of that include blocking lignin synthesis That would seem to be a pretty straightforward thing to do, but it is not without its consequences Particularly if we block it to a very very significant degree We can change lignin composition from We've we have made lignins that have only s units only g units and as I'll show you today only h units as well as other types And again those have consequences on the chemical of the chemistry and architecture of the polymer We can redirect lignin monomers into other other molecules, so Cj Luz group at Brookhaven National Lab Took advantage of or evolved basically an enzyme that would cap the hydroxyl on the aromatic ring That was required for activation to free radicals and in doing so they divert those molecules out of lignin synthesis So it's a different a different way to reduce lignin content and then as well presented We can we've developed control over where lignin should be deposited and not only do we know that in a sort of a directed strategy But we know that there is tremendous genetic diversity in in crops maize and sorghum that range from plants That are very very soft to plants that are more like a hard bamboo So there's all sorts of opportunities not only to to use our modern strategies including CRISPR-Cas or genetics to Sort biotechnological approaches to modify lignin, but also take advantages of inherent genetic variability Okay, so I know we've we've had some people talking about mutants, but I still think this this whole concept bears a little bit of Explanation because this is a broad audience So I just wanted a two-minute primer on on using mutants So if we take a normal plant we can mutagenize it with very radiation or chemicals to and Then Following a round of reproduction identify some mutant with a trait of interest and that could be a plant with low lignin for example that allows us to not only identify the gene Eventually that was mutated that led to this effect and thereby give us a gene that whose expression We can manipulate in order to Modify the traits of the plant so thereby understanding gene function But it also lets it's also informative with regard to What type of variation a plant will tolerate will a plant tolerate a reduction in lignin of this much if we mutate this gene Will it tolerate a production of only G subunits instead of a GS copolymer? So we learn a lot about the actual function of these molecules in planta Okay, so again, this is a way that we can get at these genes that will hopefully allow us to Improve biomass crops not in the 5,000 years that it took us to improve maize, but in in a 20-year time frame All right, so the first example I have To go back to this this slide Deals with basically one of those first arrows in that pathway a gene that is an enzyme That's required for the deposition of all lignin We were able to identify a mutant in this step and learn what that what that gene encoded But also what effect blocking that gene had on the plant viability Our lab also uses a rabbit-opsis and so what you see here is what's called an allelic series three mutant plants all of all of which have Increasingly severe mutations in a particular gene involved in lignin synthesis and you can see how The more severe that the mutation is and the lower the lignin is I haven't shown you that data But take my word for it. The plant on the right has the least lignin of all That you see this increasingly severe effect that we've called Lignin modification induced dwarfing. I don't want to leave I want to make sure I start start by saying This does not always happen via the same process in all plants So there are some dwarf lignin deficient plants that are dwarf for a particular reason others that are dwarf for another reason And we really have to have a comprehensive understanding of that in order to Really Be able to manipulate plants in the future The next example is I think an example a little bit further down the pathway that Where we were we eliminated the deposition of the production of these G and S monomers And in that case these the lignins and these plants were only made of H subunits But the plants didn't like that at all that you can see the plants on the left or what we were left with and Those plants are severely dwarfed and we thought well, it's probably due to a lack of ability to transport water normally But it turns out that the answer is a little bit more complicated than that and I'll get back to that in a moment the third example I want to just briefly mention is Not doesn't isn't represented by one of those arrows, but by all of them So what we identified was a protein that was required for the regulation of the entire lignin biosynthetic pathway And so when we mutated it this the the resulting plants had lower levels of lignin and we're also Dwarfed like the plant that this is the plant that's shown on the right This is a and I don't want to get too deep into the weeds if you'll pardon the pun on genetics, but This this plant called ref 4 3 on the far right was a very special mutation in that it didn't Inactivate the protein it actually if you will activated it it turned it into a Constitutive break on the pathway so To go back one of the students show used traffic lights earlier This this this mutation changed the protein. So it constitutively shut down the pathway when we knocked out that gene and It's paralog so there were two genes that perform the same function but in a rabidopsis What we found is that the we found the opposite the pathway was enhanced. We got more phenylpropanides So what we deduce from that is that the normal function of this protein is to modulate the pathway both up and down And so it's required for a pathway homeostasis So what is this protein? It wasn't as I said an enzyme of the pathway But is an instead a component of a large molecular machine that Directs gene expression. So what this? shows is this the line that runs around is DNA and This this molecular machine is called mediator It's made up of about 30 proteins and it makes contacts with DNA bound transcription factors shown at the top and the bottom the smaller proteins and When it does so it interprets the information of what where when how much? gene gene should be expressed and recruits the transcriptional machinery and directs a coordinated output of gene expression And I'll get back to those transcription factors a little bit later So we we started to get an Unexpected view of the of how lignin synthesis is regulated and then we got a real surprise when we generated some double mutants, so we crossed that Very sad sick lignin deficient mutant that we call ref 8. It's the plant more or less in the center there that very much dwarfed one with these mediator knockouts and We found that these plants grew normally again Which was not at all what we expected and told us that it perhaps wasn't just an architectural problem And a low lignin problem, but in fact a modification of gene expression that That occurred in the plant as a result of the interruption of this biosynthetic pathway so what we did was an User an approach called RNA seek which allows us it's basically It's an amazing time to be a biologist really it's it gives you a window into the nucleus it tells you what genes are being expressed and How they how they're being expressed differently as a result of a particular stimulus or a developmental stage or what have you in our case it was in response to a block and lignin biosynthesis and That so the gene the Venn diagrams on the left show up regulated genes the ones on the right show down regulated genes And what you can see is the gene that the mutant that low lignin mutant those numbers are shown are in that yellow section huge number of genes are misregulated, but when we bring in that Mediator knockout into the same background all that gene expression is restored to normal so The way I look at that or I tend to explain it Am I really running out of that out of time? Have a five minutes. Yeah, that's what I thought. Okay So the way we think of this is that so this this mediator complex and this one particular subunit that we're working on med 5 Is normally again a homeostatic mechanism. It's not there To rescue the growth of lignin deficient mutants. No, that's just something we've discovered What it what it does is that it's we think is measuring Metabolites that are accumulating in this lignin deficient mutant as a result of this metabolic block And when it is faced with this abundance because of this block it goes haywire So I think I like to explain it like Anaphylaxis so there's probably somebody in this room who has a peanut allergy And we but We all know that our immune system is there to protect us But in some individuals when that system is stimulated inappropriately They go into anaphylaxis that can kill them So what we think of here is that the this knocking out this mediator subunit is like the epi pen for the for the Lignin deficient mutants we can block that response and allow those plants to grow again okay, so What did we find when we looked at the lignin of these mutants like the plant that won't Describe well, we were actually even able to push it a little bit further We we found that these plants basically made a lignin that was only only in Derived from each subunits This is a lignin type that wasn't really even expected to polymerize So it was very exciting to see that with this genetic modification. We could we could achieve that Um, we also found that the secarification efficiency was dramatically increased Um Again without Pretreatment the figure on the left the dotted line is the this triple mutant that we've been working on Um, okay So that's and given the the expense of pretreatment We think this is a very interesting strategy and also want to to then transfer this into a real crop to see whether we can Get the same advantages So I want to just wrap up very quickly by talking taking this one step further and talking about Uh What are called suppressor screens? So if we've identified a mutant that's of interest Uh, and it has a particular trait Can we learn even more about this system by doing By trying to mutagenize those plants and asking can we convert the plant back to more like wild type? Can we suppress this mutant phenotype? So in this example The top left plant is a wild type plant The one to its right is this mutant of mediator that causes the dwarfism this plant That's there were the that protein is constitutionally putting the brakes on the pathway And then all the rest are plants that we where we had remutagenized the plants and looked for ones that regained the ability to grow like wild type What did we find we were able to use whole genome sequence analysis? Thanks to support from gsep to to very rapidly identify the mutations in all of these plants and identify What genes were responsible for restoring growth? And what we found was shown in the colored bars the black bars are a number of what are called intergenic mutations The colored bars are other mediator subunits. So we're using this approach to now tear apart How um how mediator functions? So I mentioned that we have these transcription factors that mediator makes contact with I really wanted to mention the work of my former postdoc and gsep funder fundee In our in our project who did a suppressor screen on that original lignin deficient mutant and found the plants in the middle Now he's got a plant that Grows normally that plant is Defective in a gene that is responsible for Bringing a transcription factor from its site of synthesis in the cytoplasm into the nucleus where it can act And so by doing so it rescues the growth somehow that transcription factor is really important And the plant on the right is one where he's knocked out that transcription factor And corroborated his results. So now we know that another player in this Dwarfism these transcription factors that interact with mediator So I want to conclude just by by by saying Giving a couple of examples where we need to think about lignin. Maybe not being all that bad It's a significant portion of biomass It is more highly reduced and it's then then the polysaccharides And we've now determined that it's amenable to catalytic degradation and conversion So through our I would say thanks to gsep funding We were able to leverage that into Funding from the DOE in the form of an EFRC our EFRC focuses on lignin modification and utilization And so that group the group of Madi Abu Omar has developed catalytic strategies for Move for converting lignin in biomass Into these substituted Propylphenols that could be used for downstream processing into Your choice of a fuel or other value-added Compound while leaving a carbohydrate residue that can still be secured and used for fermentation And if any of you had Looked in science a couple of weeks ago. You may have seen this this study which featured Contributions by John Ralph again funded by gsep and myself on another way of Converting lignin into value-added products With a pretreatment that involves formaldehyde stabilization and subsequent hydrogen allysis So i'll i'll i'll just leave it at that by saying that i think that we're Developing a better understanding of not only the catalyst but the regulatory machinery that's involved in the deposition of lignin And that mediators involved in that process We know that we have now a better understanding of how dwarfing comes about in at least some of these plants and I think that's going to be essential as we Move some of these technologies into real biomass crops We have this new type of lignin that could be valuable in a bio refinery context if you're going to be Catalogically converting lignin into products. Maybe you just want one product rather than two or three And that this may allow us to make more efficient use of biomass And with that I'll just acknowledge the people that did the work and candy now is here and some people thought she did a nice job on her poster Which I was very proud of And the other people that with whom we've worked including a lot My former postdoc sirius and john ralph and others So thank you very much for your attention so A little bit too long. Sorry Is Yes Clint you did a very good job of explaining the analogy between polymers of different composition of clay gases proteins and templates and We know a lot about secondary tertiary structure of those macromolecules Maybe i'm naive, but I always thought of lignin as lacking secondary and tertiary structure And I guess I don't fully understand Is that a known problem and is it the secondary structure of that? Polymer that's important for stability or is its association With cellulose the dictator of this mechanical advantage So how does that help us explain what we need to solve? So it's closer to the second We still really desperately need a better understanding of of lignin structure What referred to it is sort of filling in the gaps and that's really in in the wall and that's really our level of understanding at this point With regard to secondary structure, you know, we don't believe that there's any chirality to the molecule and we don't believe I don't think there's any evidence that points to a secondary structure per se when I was referring to architecture What I was referring to Although it didn't have time to explain is some of these some of these lignins like the pure s lignins Can only be beta o4 linked So they they nucleate and then they grow out in a linear structure And yet those lignins as despite Being not being cross linked in the way that normal lignins are thought to be Are perfectly functional Which is which has, you know left us scratching our head in in terms of what what the function or how the structure and function are related Good afternoon. My name is Kurt. I had a question in at the inception of your research. Are there Pure reviewed or agreed upon assessment of risk for extra genomic transfer of your work. I mean obviously there There are precautions, but could could you speak to that? So are you talking about the genetic modification in arabidopsis where we're Bound by you know, good live practice or are you talking about moving to Crop species Both Well, we certainly have to contain all of our transgenics Mutants are not governed by the are not regulated in the same way And But when we move to other species, so we've made transgenic Tobacco for example and certainly it has to be disposed of properly and when we move to poplar Uh, we have to follow those same protocols, but when they go out to the field then we fall under the jurisdiction of aphis So under Those regulations which are Extensive And which we take very seriously my colleague rick myelin Renews that permit every three to five years. I'm not sure which And under those circumstances we have to go to painstaking efforts to make sure that the The the plants that we're putting in the field do not flower so Poplar has a juvenile period of about five or six years perhaps seven and so we do we we have to prevent dispersion of Of the trans gene in the pollen And we do so by compassing the plants you can cut them to the ground and that resets their clock So they do not they will not flower for again and for another five years or whatever So I hope that I hope that answers your question. Thank you. Okay Evan Hughes, is there any chance you have you or others are on the track to some Form of lignin and being able to make it so it becomes a super high value product instead of a problem Well Instead of a problem. Well, yes, as they used to say or still do say you can make anything with lignin other than money If that's what you're referring to I think that these catalytic strategies are really what what are called for because we're making we can convert these into Low molecular weight building blocks in about 50 percent yield Uh, the the the work that I mentioned at the last with formaldehyde stabilization got yields with the high s lignins Which are which are characterized by the beta o4 linkages that are easiest to break over at over 70 percent Um, so we certainly see a bright future there But we've we've only really been working on that for the past few years But I would certainly hope to see that be part of a Bio refinery process Okay, I think we have to stop there. So let's go clean one more time. Thanks