 I think, so our next speaker is Claudia Schmidt-Danert. She's a professor in the biochemistry molecular biology and biophysics at the University of Minnesota. She's also the director of the Biotechnology Research Institute. Studies were in Germany and after her PhD studies, after her PhD studies, she moved to Stuttgart where she was the head of a molecular biology group and that's where I met Claudia. And I can really say that Claudia is the one that taught me how to do molecular biology when I was on sabbatical there at that group and I'm always grateful for her because I use molecular biology every day now. Then she, after that she did postdoctoral research with Francis Arnold at Caltech and did some of the work on the molecular breeding of the pathways for carotenoid biosynthesis. And then she joined the University of Minnesota. Her talk is going to address the problem of how to get a whole series of enzymes to work together and she's going to talk about making nano compartments for these enzymes so that they can work more efficiently. Welcome. Okay, I think I get started here. I'm waiting that the computer restarts, it's not mine. They try this new technology of YouTube and recording the lectures and I agreed to do this. So now we see how this will work. So before I start, I'd like to thank the organizers. I only played a very minor part in the organization to get this kick started, but Romas is the key player here who initiated and brought the Biotechnology Institute into the fold to organize this fantastic first North America, Japan symposium on enzyme technology. So as Romas alluded already to, and now something I don't know, couldn't find that event. Okay, there was another screen on top of my screen. So I'm going to talk about protein-based biomaterials and specifically I'd like to give you some insights and overview of our most recent research in using actually proteins to support enzyme catalysis and biotransformation reactions. Several years ago, I became interested in how natural systems self-organize themselves and how we could use these principles to improve our own biosynthetic and bio-catalytic reactions that we would like to perform for bioprocessing or bioprocesses. And if you look at the left side, you see some of the fantastic examples on how nature patterns things and creates these patternings and organizes itself. And I became interested how can we program this and how can we redesign the same principles to help us with some of our processes. In natural systems, they are all programmable because they are genetically encoded, they're dynamic, they can self-repair, they can build these fantastic, hierarchical structures and these structures also have new types of functions. So I thought, can we use some of the building blocks and some of the principles, such as proteins to design materials that can help us to, for example, in my case, what I was interested in, organize biosynthetic reactions or enzyme cascade reactions? And particularly use these protein-based systems or proteins as building blocks to build scaffolds that can be produced in cell-free or microbial systems that then can basically use proteins to build materials and these materials you can then utilize and engineer in design to build platforms for enzyme immobilization, for cascade reactions or in vitro-biocatalytic reactions or biosynthetic reactions. Then also can potentially assemble in these hierarchical structures and build really macro-scale materials with enzymes embedded in these materials. And then at last, not only using these protein-based materials but also interfacing them with inorganic molecules. So basically build a reactor system that is kind of a division, a reactor system that you can genetically encode in a microbial organism. You basically encode the building blocks, you encode your enzyme catalysts, produces, and the vision would be that it just builds itself into this assembled materials in interfaces with an inorganic molecule to make a robust, rigid materials. So in this presentation I will give you, I will talk about two topics. One of them is programmable protein materials for biocatalysis and biosynthesis. And doing this first in kind of 2D and our most recent efforts is see if we can get this into 3D assemblies going from the nano to the macro-scale. And if I have time at my last part of the lecture, I'd like to see, I'd like to briefly introduce some of our efforts in basically bringing in inorganics or mineralization, bio-mineralization processes into these materials. And I will show you one example where we're thinking about interfacing it with cells to make these engineered living materials. So my primary motivation for building these protein-based materials is for the design of enzyme immobilization platforms. If you work with enzymes, it is relatively straightforward to immobilize one or two enzymes on a material, a matrix through chemistries. But if you want to design more complex immobilizations or do some more complex immobilization by integrating four or five or six enzymes, and it gets really more complicated because you have to think about the chemistries that you want to use. And they are often not very compatible between these different types of enzymes. So I thought, what about if I could use a protein, a much more benign immobilization method by using a protein material where I could use some tags and some other covalent, genetically encoded mechanisms to immobilize enzymes and build these immobilization platforms where I can easily immobilize multiple enzymes with finding basically methods that do not destroy the enzyme's activity. And at the same time have a material that behaves very much like a matrix that you use in immobilization. It makes actual material that you can recycle and reuse. So we worked several years ago on building these kind of micro compartments in E. coli with the idea actually, these protein-based micro compartments to build these nano reactors, nano bioreactiles inside cells. But it turned out this really never got anywhere. And I thought these proteins assemble inside the cells but could we actually use them outside the cell and use them as materials? As you can see here, inside the cell, they already assemble in very robust scaffolds. And if you purify these hexameries scaffold proteins, these UDM scaffolding proteins, they also assemble into materials outside and through very large scaffolds. They assemble into hexameric tiles and arrays. Can be easily manufactured in E. coli. And as you will see later on, they are robust. You can heat them up to 50, 60 degrees. They remain stable. They're stable under various conditions. And we thought this would be an ideal platform for the design of a protein-based material. So how did we go about this? It's a protein, you can modify it. In this case, we fused tags to the carbonyzymes and to the hexameric protein units, the UDM hexamer. We use the well-known spy catcher spy tag system. If these two domains come together, they form very rapidly an isopeptide bond for covalent immobilization. Of course, there's many other opportunity to do this in a very similar way with other tags. We show it here. We show that it works. We here is a very rapid immobilization with to the spy tags spy catcher system. And these are the scaffolds that are formed by... Oh, there's no pointer. I can't get the pointer to work. There are these scaffolds that form, in this case, the spy tags spy catcher system basically immobilizes the GFP onto these scaffolds. So on the right top side, you see some of these fibers that are formed. And on the right side, there is a spy tag GFP loaded onto these fibers. Doesn't work. It's too far away. So to demonstrate utility of this scaffolding system, we immobilized one of really nice dual enzyme cascade, a self-sufficient chiral amine synthesis cascade that was published by the Turner group. And it's a hydrogen borrowing cascade where the alcohol dehydrogenase and the amine dehydrogenase work together and shuttle the cofactor around to produce a chiral amine. We use this scaffolding system with the spy tag spy catcher system to immobilize these two enzymes on it. You see this STS page that's actually the immobilization occurs very rapidly. We vary the protein building box to enzyme ratios and optimize these ratios. And you can see on the right side on the TEM images some of the fibers at the lower bottom, you can see that they're coated in these enzymes that are attached to them. And if you do the reactions, immobilized reaction and the non-immobilized reaction compares them to each other, you can see that the scaffolds shorten the reaction time to reach 90% conversion in 24 hours while the free enzyme reactions took 48 hours. So we reduce the reaction time here, the reaction efficiency by 24 hours. And this was mostly due to stabilizing the enzyme on providing a much better reaction microenvironment for the enzymes. From there, we created actually a toolbox of scaffolded building blocks for other homologs because in many of the enzyme immobilization, if you use enzyme immobilization matrix, the microenvironment of the matrix is actually very important for the enzyme, not only the stability, but also the activity of the enzymes. And the idea is here if you can build different types of scaffolds with different similar building blocks, but they have different electrostatic properties, we could use this to tailor or create a toolbox of these scaffold building blocks that can then be used to create different types of materials for immobilization. So the idea is you have these different types of UDM, basic scaffold building or homologs that have different, create scaffolds with different properties and you can pick and choose and use this for your enzyme immobilization. Here's an example to just show that it's not just acute protein-based material, but it can be produced in really scale. You see here a 500 milliliter of an E. coli culture makes significant material of these protein-based materials. Here are some examples of these types of scaffold building blocks that we cloned from basically by looking at homologs what's out there and cloned those, expressed them, purified them and you can see that they built different types of scaffolds here, TM images. They also scable up to 60 degrees and we tested the function of these scaffolds with one of the enzymes that be used in our cascade reaction and this is an alkyl dehydrogenase and tetrameric alkyl dehydrogenase and we notice that this enzyme, if it is not immobilized, is rather unstable. It is not very stable and our goal was to see if we can, as we have seen already in our cascade reaction, can we actually see a difference with our different types of scaffold building coms? Can we stabilize the enzyme similar to what we have seen in our dual cascade reaction? So, here is our enzyme, we modified it again with a spy tag and then tested out its immobilization of these different types of scaffolds, the different types of homologs that we have cloned and expressed and purified and you see here that in all cases that are shown here the enzyme again codes very nicely the fibers. Here's the example to show that the spy tag alkyl dehydrogenase by itself is rapidly basically inactivated which in 12 hours or 24 hours the enzyme has almost no activity left while when you immobilize them on these protein scaffolds they remain stable, they remain stable longer than 48 hours and in most cases, or some of the scaffolds here actually increase the activity of the enzyme and in the case of one of the scaffolds the SA scaffold there the enzyme is very, very stable and we only show here 48 hours but it becomes stable for days. With that, we thought so now is it possible to go from just the 2D scaffolds that we are building with the enzymes? Can we go from the nano to the micro to the macro scale and build scaffolds that self-organize them into 3D structures? The motivation is to build these hierarchical materials not only for enzyme catalysis I only show you here some of the work that we do with enzymes but you can also think about because it's proteins you can attach any type of domain or tag to these scaffold building blocks to also incorporate potentially what we do electron photon transfer you can make the biosensing materials materials for biomedical applications and make different types of materials one example that we are using here is building 3D architectures we integrate the cargo proteins and here's an example where we build these building blocks basically a synthetic biology approach where we modify the cargo protein with different types of tags in this case we are using a spy tag and a snoop tag so get some orientation in the building of the materials in a cognate tag on our scaffold building blocks and basically to fine tune the spatial organization of enzymes you have also incorporated building blocks that are not modified by co-expressing and basically assembling these entire building blocks assemblies you can control how much of the enzyme you incorporate, how much to space them apart and how these scaffolds will grow here's an example to show that it works you can actually easily follow this but just by STS page analysis that these scaffolds build up larger and larger structures as they click together and then if you interface it with unmodified building blocks it's shown here, the ones with the red are modified, they have the spike catcher to for cargo attachment while the ones in gray and light gray are unmodified by doing this, by co-expressing them with additional ratio because basically you can control how you load the cargo or what the cargo density is on your scaffolds shown here again there's Eppendorf tube that shows there that this is actually materials that forms and I have no time to really go through in all of these details but what we have done is basically systematically explore different types of expression levels of these building blocks and co-express them with different types of ribosome binding sites to really characterize in depth the protein expression how much protein, how much scaffold you produce control the molar ratios of these building blocks and also look at what the size of the scaffold are going to be we also investigate pH and temperature effects on these scaffold assembly and here are some examples shown how these scaffolds look and these scaffolds actually form in this system with this by-text by-catcher system they form tubes and they form massive tubes that are here cross-linking or cross-link with each other with in this case GFB as our model cargo protein and here's another example just to show you the scale of the scaffold they are massive we are actually deluding them out because they build huge scaffold huge materials we are just showing here the scaffold in this case with GFP interface integrated into these scaffolds here's an example you can actually see if you zoom in these are microscopy images if you zoom in you can actually see that there's tubes growing out of them that are modified for the scaffolding protein here's an example where we have another enzyme integrated where we have a phosphide dehydrogenase for cofax recycling integrated into the scaffolding system just to show that with the spy-text system on the readily immobilized which in a minute and then on the right bottom you can see that the enzyme does not lose if it is spy-text we are now basically building these scaffolds for biosynthetic pathways for different types of enzyme cascades and also integrating very in a very modular fashion different types of cofactor recycling systems so with that I like to shift a little bit and show you some work that we do in building biocomposites and integrating a combination mechanism into these building these types of materials and I really became interested in looking at biocomposite structures in nature and you see here some of the examples silica, aragonite, apatite and sponges, the specules of sponges, a nacre of mollusks or the bone structure, NML corals, the skeletons or the bones and all these mineralized structures key to all of them are proteins proteins control the mineralization processes and proteins are key components of these materials that are important for their unique mechanical properties so we thought is it now possible to integrate our protein base materials and integrate mineralization mechanism materials to build biocomposites and here is an example where we actually are interested not only using enzymes of proteins and building these biocomposites but interfacing actually with cells, these microorganisms to build engineered we call engineered living materials or ALMS and one of the motivation is to either make materials that you can use in a reactor system that have mechanical properties plastic encodings so we had this crazy, this was back then a crazy DARPA project to engineer this and the goal was to actually make a living materials, not just a material that you mineralize but the component of the material has to survive the entire process so we engineered a living material using a bacillus bacillus we engineered to secret our scaffold building blocks and the scaffold building blocks would then crosslink with our cells, cells would display a spy tag on their flagella and then attach to the spy catcher on our scaffold building blocks and then we would modify our scaffold building blocks with mineralization tags that would force them to build a silica material and the final test for this that it actually is living you had to basically show that you make a material and you can regenerate from this material the entire system again so first challenge was to secret the scaffold which is not easy we used bacillus subtlis, secret the scaffold is not easy to do with the cytoplasmic protein that once assembled inside so we showed secretion of the scaffold building blocks shown here so we tested different types of systems and different types of media conditions and successfully secreted our scaffold building blocks we then had to do some strain engineering to make sure that our cells don't kind of just form spores but remain intact and then we also would like to have the flagella just display, flagella be not on the polar ends of the bacillus we then integrated the spy tag display system, the flagella spy tag system which was quite challenging to find a position on the flagella in bacillus where we could actually display a spy tag many locations were not permissible or not functional here is finally shown our flagella, the flagella are labeled with a cysteine stain, you can see that they are now polar and also the kind of boxed images there show that we have now spy tag which actually bind a spy catcher modified tomato fluorescent protein we then integrated biomanalization, silica biomanalization on our scaffolds by incorporating a biomanalization tag we tested many different types of tags and here's one that's caught B1 is a spore protein peptide from another bacillus that basically achieves silica mineralization on our scaffolds with these mesoporos nanomaterials now formed in the presence of silica and then we put the entire system together to demonstrate that we could make a biocomposite material we have all the building blocks expressed, secreted, mineralization tags, the spy tags display and prevention that the bacillus lizes after spore formation and you see on the bottom a block that forms with the bacillus incorporated the final test of course that we had to show for DARPA can you actually revive your entire system can you cure your silica blocks that you have formed now in this mold and can you take a dry silica part of this and re-inoculate and do the same thing all over again and that was the final test that you can see on one of the ends that in the dish it forms this cross-linked materials with silica also incorporated for the fun of it another function by doing a consortia building a consortia where the second strain that would also attach to the scaffolding system would actually express a purple purple protein just to show that you can easily incorporate other functions in there and then make a purple material where you have these functions basically built into a consortia so with this I like to conclude my talk and hopefully I've shown you some capabilities and how you could merge synthetic biology together with material science and help with design of new types of biomanufacturing platforms especially for in vitro and cell free applications but also because everything is genetically programmable you can not only do this and program this in a cell but you can also do this in vitro through transcription and translation systems our goal is to build these self-organizing systems for the design of some advanced functional materials and with this I like to acknowledge all the lab members that were involved in this especially Sun Young Anaya then Ji Zi Wang Zhang Zang, Riji that have been instrumental in building these types of materials so I would like to thank my funding and thank you for your attention Hello, excellent talk so you kind of alluded to this but how do you prevent the self-assembly inside the cell as they're expressing and would that cause issues with cell viability? In general the self-assembled structure as you could see in the E. coli perhaps they make very robust structures to prevent some cell division but generally they're not toxic for the cells but in the case of the bacillos we really had to fine tune the expression level so that we could basically couple secretion and expression transcription and then translation and the secretion so I didn't show but there was a lot of work in optimizing secretion of the scaffold building blocks Hi, Claudia, good talk I was wondering have you tested the structural integrity of these scaffolds? Are they very resilient? Are they strong? Do they fall apart easily? Do you have any data on this? So in terms what you see normally in our images that I show you we have to dilute them somewhat so that you actually get some insights in the TEM otherwise the grids break but normally if you have they are pretty robust making really materials so it's a very robust materials and actually they self-assemble into structures very quickly so it is somewhat difficult to break them back open or actually mix them together that's one of the reasons why we actually do the co-expression you actually have to co-assemble them into these scaffolds Flat structures and then there were some tube structures can you predict what you're going to get? So the compartments they actually do have another shell protein part of it this is a U2S shell protein which actually provides the curvature to it the U2M that we are using is actually the main it's actually the main shell protein but it is formed in the flat the flat sides of these scaffolds and we realize when you over express that you get these flat scaffolds that is something that we cannot directly predict but it has something to do with a pH with an electrostatic environment that these scaffolds form and I really would like to use some of the machine learning or some of the AI to have some predictions of some modeling capabilities to see how you can predict their assembly. Thank you very interesting talk I'm thinking about this from an industrial immobilization perspective it doesn't immobilizing the enzyme onto U2M just sort of kick the can down the road as it were because then you still have to immobilize the scaffold. The scaffold you wouldn't need to immobilize they are materials you can basically centrifuge them down or separate them on a membrane it's like immobilization like a bead you can do the same thing they are not soluble they are insoluble. Thank you very much you investigated the ratio of scaffold and enzyme and could you explain more about how the ratio affects the stability of the enzyme and performance of the immobilized enzyme. So we found that you have to increase the scaffold you actually have to fine tune the ratio between the scaffold building block and the enzymes so you find I don't know what this but 1 to 5 1 to 9 scaffold to enzyme ratio so you have to increase the scaffold regions to actually spatially separate the enzymes out and the scaffold otherwise you get some kind of constraining of the enzymes too close to each other. But you want to increase the enzyme ratio for the the enzyme ratios we also optimized. So what we have done we first in vitro optimize as you do for cascade reactions you optimize the enzyme ratios to each other and then you find the best scaffold ratio to your enzyme catalyst. So you optimize the enzyme ratio first and then introduce the scaffolding and see that you get the optimal ratio with the scaffold and the enzyme system. But you don't want to separate two enzymes far away so that the cascade reaction doesn't proceed. So you mix the enzyme together and then immobilize. How far is the best if there are two enzymes you don't want to make it very far so that there are no effect of immobilizing together so I wonder how you determine the distance between two enzymes. So in the case of the amine and the cascade reaction the dual enzyme and the cascade reaction we first determine what would be the best enzyme ratio to two enzyme ratios and then we use that system once we figure that out that is the optimal ratio enzyme for the cascade reaction we then optimize it to the immobilization to our scaffolds. And it's not that we used one enzyme here and one enzyme here actually it's often been shown that doesn't really matter it's more the clustering of the enzyme that matters in a cascade reaction. So they can operate together but then you have to see that you get the spacing right in the entire system. Thank you very much Claudia.