 Hello everybody. You're very welcome to this webinar, which is hosted by FEMS East Research in collaboration with the East Lippard Conference. My name is John Morrissey. I'm editor-in-chief of FEMS East Research and I'm co-chairing this session today, along with Adrogh Kropov and Vrenna Severs from Chalmers University. So, just before we get going into webinar itself, I thought I'd just say a couple of words about FEMS and FEMS East Research before I hand you over to my co-chair. So, I expect most people here, but maybe not all, are familiar with FEMS. It's the Federation of European Microbiology Societies, and you can see it's a network of over 30,000 microbiologists, right across all disciplines of microbiology. And a big part of what FEMS does is it connects microbiologists through organizing conferences, sponsoring conferences, sponsoring conference prizes, and publishing journals. And here's a list of seven journals that FEMS publishes right across the spectrum. We like to encourage people to publish in these journals, just society journals, and that means that any income that's generated from these journals goes back to the community. So, we think that's a worthy cause to be supporting with your publications. As I mentioned, I'm editor-in-chief of FEMS East Research, and we publish, sorry, I thought I had another slide in there. So, we publish articles right across the spectrum of yeast physiology and genetics covering fundamental biology and applied biology covering pathogens, saccharomyces, non-pathogens. So, any papers that advance the knowledge of yeast physiology and genetics and application, you're eligible to be considered by FEMS East Research. We also publish various thematic issues and special editions, and I just wanted to mention this one that I think you will have seen in any case, which are registration. We've just published a virtual special issue where we pulled together a collection of papers on both fundamental and applied aspects of yeast liquid research that were published in FEMS over the last 12 to 18 months. And this is really just, I mean, partly because these are interesting papers, but also hopefully to give attendees here who are of course specialists in yeast liquid biology, an idea of the types of papers that we can publish, and hopefully some of you here will consider FEMS East Research for your own papers. So, I will stop sharing my screen now, and I'm going to hand over to one of my two chairs, Dr. Edward Cuckoven from Chandler's University in Gothenburg, who is also one of the co-chairs of the yeast-slipit conference that you would all be familiar with, and Edward is going to take over now. All right. Thank you, John, for the welcome to this webinar. So, I will say a few words about the yeast-slipit conference, and this is a conference series that has been going on since 1995 already, and it's been happening since 2001. It's been happening every two years, and the last time was then actually in 2019 in Ljubljana, Slovenia. And this is a conference series where the topic is basically any aspect of yeast or fungal lipids, and this can be metabolism of phospholipids in sterols, protein membrane interactions, yeast as a platform for lipid production, yeast as a model organism for human pathogenesis related to lipids, or, well, any other aspects related to this. And we particularly want to encourage young scientists like these students and postdocs to present their research during these conferences. So, last time it was the 14th yeast-slipit conference, and in 2021 we were planning to organize the 15th yeast-slipit conference, but you can imagine that this was not happening this year. We decided to delay it one year, and it will actually take place in 2022, the first to the third of June. It will happen at Chalmers University of Technology in Gothenburg, Sweden, and here we will have three sessions, one related to phospholipids, sterols, and metabolism, protein membrane interactions, and inter-organal membrane contract sites, and yeast as a platform for lipid production. Well, just a few words about Gothenburg. It's the second largest city in Sweden, and it's located on the west coast, as you can see here. It's about 600,000 inhabitants, and actually this year it's existing 400 years. I think it's foundation. Just a few pictures of beautiful Gothenburg, which is located on the coast, and it has canals that were built by the Dutch many years ago, so you see a lot of water in the city. We also have a theme park that is located in the city center, and there's a lot of nice Swedish and particularly west coast delicacies to be had. We have an exciting list of speakers for this conference that have already all confirmed their participation, and we have received funding from FEMS, actually, to authorify travel grants for early career scientists. So, I very much want to invite you to, if you are excited after today's webinar, to also join us in Gothenburg in 2022 for the in-person yeast lipid conference, and here's a link to our website, and also you can sign up to the mailing list to make sure that you stay up to date. But of course, so we delayed the conference by one year, but we did not want to completely have no yeast lipids in people's lives in 2021, so we decided to team up with FEMS for a webinar that's happening today, and for this we have four speakers divided into sessions. The first session is more about fundamental yeast lipids, and the second session is more about yeast as for production of lipids. So, I guess we can just get started with the first session, and there I can say that we advertise that the first speaker of today would be Professor Maya Schildinger from the Weizmann Institute of Science in Israel, with a talk titled Regulatory Mechanisms Underpinning Direct Mitochondria Nucleus Contacts in the yeast cell, but unfortunately Maya Schildinger had to at last minute excuse herself for presenting a work because of personal reasons, but we actually will hear from a talented postdoc from her lab, Dr. Michael Eisenberg, who's the first author of the work that Professor Schildinger was applying to focus on, so I think we have a very high quality of replacements for her stats, and I would say without further ado, I hand over to Dr. Eisenberg, and looking forward to hear your talk. Can you hear me? Yeah. Okay, cool. So first thank you very much, and yeah, I'm not Maya, I'm Michel Eisenberg-Bord, and I'm doing a postdoc in her lab, and first Maya sends our apologies for not being able to join us today, and I hope I will be able to step into her shoes, and I want to thank the organizers for giving me the opportunity to share with you my science today. So, I'm trying to move the slides ahead. Okay, so this is my son, Lavi, and he turned three the other day, and I decided that this is a good time to start teaching him some cell biology, so I downloaded these coloring pages for him, but I was quite surprised to see that they don't really accurately represent the cell, because in these coloring pages, it looks like each organelle does its own thing, and that organelles work in isolation, but today we know that this is completely not the case, so we know that organelles can't function alone in isolated environments, and they need to work together in cooperation in order to have a functioning cell unit. Therefore, organelles must communicate between them. Now, one way of communication between organelles is through membrane contact sites, so these are areas of close proximity between the two organelles, allowing transfer of lipids, ions, and other molecules between them, and don't think that these are areas where the organelles are randomly bumping into each other, because in these contacts, the organelles are actively tethered together by tethering machineries. So, in recent years, it became clear that these contact sites are fundamental for cellular health and for maintaining cellular homostasis, so it wasn't a surprise to find that they play key roles in many different diseases and this list is only for mitochondrial contact sites and the full list is even longer. So, in our lab, we're interested in understanding contact sites' biology, and as it became clear that many different organelles can form these contacts, many new questions emerge, such as what are all the contact sites in the cell, and also which proteins can act as tethers and regulators in these contact sites and carry different functions in the contact. So, since contact sites are evolutionary-conserved, and they can be found basically in every cell type, we are studying in them in the yeast, the model organism, the baker's yeast, the Charomyces cerevisia, and first, as I mentioned, we wanted to understand what are all the contact sites in the cell, we wanted to have this comprehensive map of all the different contacts. But the problem is that contact sites are quite small, so only 10 to 100 nanometers are separating the two organelles. So, they're below the diffraction limit of fluorescence microscopy, and they're also quite rare to find, so it's hard to discover them by electron microscopy. So, to overcome this, Nadav Shai, a former PhD student from the lab, together with Enats Alskva, who is a research associate in the lab, they developed an approach that allowed them to visualize contact sites in an unbiased manner without any prior knowledge on them, and they use a split fluorescent approach to visualize these proximities between the organelles. So, the idea is quite simple. We take a fluorescent protein, in this case it's a venous protein, and we split it to two halves. We tag organelle one with one part of the venous, the VN, and we tag organelle two with the second part of the venous, the VC. And you can imagine that these VN and VC are coating the entire organelle. Now, only in areas of close proximity between the two membranes, as is happening in the contact, the full venous protein would form, and we will get the fluorescent signal. So, that is a way to mark specifically these proximities between the organelles. And we call the E-strain that has both the VC and the VN in it, the reporter strain, for its ability to report on the contact site. And this is how it looks. So, we have here our cell, and we have the green dots here that are marking the contact sites. They are marking where the contact sites are found. Now, amazingly, when Nadav used these pairwise combinations of reporter, he was able to see that basically every two organelles have a contact site between them. So, here we have VN and we have different organelles of the cell here, and we have the VC with different organelles here. And you see the cells in the green fluorescent signal marks the different contact. So, you can see that basically every two organelles in the cell can form these contact sites. And this was later also shown in Eastern in mammalian cells. Okay, so, now we know that basically every two organelles can form these contacts, but we wanted to understand what are the proteins that can act as headers and regulators and carry out the specific functions of the contact. So, this is a work that is done by Nesh Kaspo, she's a postdoc in the lab. And the idea, again, is quite simple. We have our yeast strain that had the reporters. So, we have the reporter marking the contact. And we can take another yeast strain that has a cherry-tact protein. And then we can look for collocalization between the cherry-tact protein and our reporters. And if we see that this protein is collocalizing with our reporter, we can say that it is a contact site resident and it is found in the contact site. So, this is basically the approach that we decided to use. But the thing is that, as you can imagine, yeast have about 6,000 genes. And tagging every one of them with a cherry tag one by one can be a very time-consuming mission. But luckily for Neshi, she could use a library that was recently developed in the lab by Ido and Uri, two former PhD students. And they created an yeast library where every single yeast protein is tagged at its end terminus. So, here we have, for example, the gene. You have a GFP tag for this gene and a constitutive promoter. And this was done for every one of the single genes in the yeast genome. The cool thing about this library is that it is made with a technology that is called swap and tag, or SWAT. And this was done in collaboration with Mifal Knoop. And this strategy allows us to make basically every library that we want in a rapid, efficient, and easy way. So, here, for example, we can create a library where we have every single protein tagged with cherry. And it is expressed under the teft promoter. So, this is a constitutive strong promoter. And basically, now we have this collection of yeast where every single gene is both tagged with cherry and overexpressed. So, now what Neshi could do is she can take this reporter strain that I mentioned that marks the different contact sites in green. And she can integrate it into the library where every single colony marks one gene that is both overexpressed and tagged with cherry. And then, using our automated microscopy system, she can look for these cases where she see collocalization between the cherry protein and the reporter, as I mentioned earlier. Now, as you can imagine, she has to visualize thousands of different strains. But in our lab, doing a high-content screen is actually a child's play. And I'll show you now how Rotem, Nomi, and Matana are doing a screen in our lab. You have the banners explaining a bit what they're doing. Okay. So, using this automated microscopy system, we were able to uncover hundreds of new contact site-resident proteins and regulators. And we have this list of hundreds of proteins. But I think going over the list one by one might be a bit boring. So, I decided that in the rest of the time that I have, I would focus on one specific contact site that is my personal favorite contact site. And that is a project that I did together with Nama Tsung, who is a PhD student in the lab. And Nama and I are interested in the contact site between the mitochondrion and the nuclear ER, or mitochondrion nucleus contact sites. And for many years, people treated these contacts as a type of mitochondria, ER contact sites. So, it seems like sometimes mitochondria can form contacts with tubular ER. So, here we have the mitochondrion and the tubular ER, and this is the contact with the tubular ER. And also, sometimes they form contacts with the nuclear ER, but they're all practically the same. But in recent years, it became clear that these mitochondrion nucleus contacts actually have special roles in special tethering machineries. For example, a role for the transfer of cholesterol was suggested in mammalian cells, and also a role for the transfer of heme was suggested for yeast. So, Nama and I decided that we want to find what is the tethering machinery for these mitochondria nucleus contact sites in our model organism. And we also wanted to understand what is the function for these contacts. So, to do this, we decided to use the same approach that I already mentioned. We have here our yeast strain that expresses the mitochondrion nucleus reporter, and we integrate it into the library where every gene is both overexpressed and tagged with chem. And then we image these strains in the same way that I just showed you in the movie. And following this screen, we have our list of hits, in this case, 55 hits. But we decided to focus on our favorite protein, which was a protein that was never studied before. It didn't even have a name. So, we decided that we are calling it CNM1, and the name stands for contact nucleus mitochondria 1. And I hope that by the end of the talk, I would convince you that this name is appropriate for this protein. So, this is our mitochondrion nucleus reporter. This is how it looks. We have our cells, and we have the fluorescent signal. And we have here cherry tagged CNM1. And I hope you can appreciate that it is nicely co-localizing with the signal of the reporter, so showing that it is a resident of the contact. But the cool thing was that when we overexpressed CNM1, so this is overexpressed CNM1, and we compare it to the control, we see that the reporter signal is much stronger and elongated. And the reason for that became clear. When we just tagged our nuclear, we are in with green, and we have a mitochondrial marker in red. And these are the control cells. But when we overexpressed CNM1, now we see that mitochondria are clustering around the nucleus, forming these very large proximities. And this is just by overexpressing CNM1, so we don't have the reporter here, we have nothing, we just overexpressed CNM1. And we see that mitochondria are clustering around the nucleus. So, then we decided to go for, to do some electron microscopy, because electron microscopy is basically the gold standard in studying contact sites. So, here we have electron micrographs, and this is the control strain. We see the nucleus and we see a mitochondria here, and we have a quite distinct contact site, but honestly they were quite rare to find. But when we overexpressed CNM1, now we see that the mitochondria are forming these huge contacts with the nucleus, they are surrounding the nucleus, forming these very elongated contact sites. Again, only by overexpressing CNM1. And this, the amazing thing is that this happened in every single micrograph that we checked. So, we checked tens of different cells, all of them show these elongated contact sites that were very abundant in these cells. Okay, so due to the lack of time, I won't go through everything that we did, but we next wanted to check if CNM1 can act as a bona fide tether for the mitochondria nucleus contact sites. And a few years ago, we published what we think are the standards for protein to be called the tether, and we wanted to see if CNM1 can fulfill these requirements. So, first we found that it is a nuclear membrane protein, and this was done by Leila from the lab of Toronwapapot. We next saw that it is specifically located in the areas of contact between the nucleus and mitochondria. And finally, as I showed you, it can exert a tethering force, so it has the ability to cluster mitochondria around the nucleus. Okay, but how does it function? How is it doing what it is doing? So, to answer this question, we decided to do another screen. In this case, we have our strain that overexpresses CNM1, and we have our nuclear marker in green and a mitochondrial marker in red. And because we're overexpressing CNM1, we see that mitochondria are clustered around the nucleus. Now we're integrating it into our deletion library. So, this is a collection of these colonies, every single colony has one deletion of a gene or a mutation. So, this is basically covering the entire east genome. And then we imaged them using the same approach that I showed you. And now we're looking for cases where we no longer see this clustering around the nucleus anymore. So, we see that mitochondria are not clustered anymore. And we can assume that if this is a result of a deletion of a gene that we deleted the gene and we no longer see this clustering, we can assume that this gene is somehow involved in the contact site formation by CNM1. So, one of the hits that we got, a very interesting hit, is a protein called Tom70. And here we have the control cells and we see mitochondria surrounding the nucleus. And when we delete Tom70, we see that we no longer see this clustering of mitochondria around the nucleus. And Tom70 is actually part of the translocase of the outer membrane, so part of the Tom complex. And this is a complex that is responsible for import of proteins into the outer mitochondrial membrane. So, you can ask, okay, this can be either a direct effect that Tom70 can bind CNM1 and they form the contacts together. But this can also be due to an indirect effect, because it might be that Tom70 is responsible for importing a different protein into mitochondria. And that is why we got it as a hit. So, as you can see here, Tom70 has a transmembrane domain and we did several experiments to check if this is a direct or an indirect effect. And to make a long story short, we found that it is a direct effect and we think that Tom70 can directly bind CNM1. So, one example for such an experiment that we did, we deleted the transmembrane domain of Tom70 and we tagged it with GFP. So now we see that it became cytosolic and we also see again that mitochondria are not clustered around the nucleus anymore. And when we overexpressed CNM1, we now see that Tom70 is basically dragged to the nuclear ER. So, we see the fluorescent green signal around the nucleus. Basically, it is CNM1 dragging Tom70 to its localization around the nucleus. And we did additional experiments. We did also some CoIP to confirm that indeed we have a direct interaction between Tom70 and CNM1. But because this is a lipid seminar, I want to end my talk with three hits that we got that are actually related to lipid biosynthesis. And in this case, we're talking about the phosphatidyl colline, PC, biosynthesis pathway, E02, CHOT2, and OP3. So, you can see that in these delitions, although we still overexpressed CNM1, we don't see the same clustering of mitochondria around the nucleus as we see in the control cell. So, these proteins are actually, as I mentioned, involved in the biosynthetic pathway of PC. So, we have here PS forming PE and then PC. And E02 is a transcriptional activator for OP3 and CHOT2. And one thing that was really interesting about these hits is that it was already known that the PC biosynthetic pathway relies extensively on ER mitochondria context sites. And it was shown that the Hermes complex, which is a known mediator for contacts between the ER and mitochondria, has a role in the transfer of PS and PE between the ER and mitochondria. So, next, when we have PC forming in the ER, it is clear that it has to go back to mitochondria somehow because mitochondrial membranes have about 44% of PC. So, we hypothesized that maybe CNM1 has a role in these contacts to transfer back PC from the ER to mitochondria. Okay, so we know that deletion of E02, CHOT2, and OP3 result in less of these contacts. And we wanted to understand why, why do we see less of them? And the reason for that is that when we abolish CHOT2, OP3, and E02, we see reduced levels of CNM1. So, here we have CNM1 tagged with GFP in the control. And when we delete CHOT2, OP3, or E02, we see that we have less CNM1. And the cool thing about the piece is that we can bypass this pathway so we can add external calling to the media. And then the yeast would utilize it through the Kennedy pathway to form the novel PC. And indeed, here shown in this western plot, we can see that when we delete CHOT2, OP3, and E02, we have less CNM1. But when we add external calling to the media, we can restore the levels of CNM1. They are becoming more abundant in the same like in the control. Okay, so now that we have delta CHOT2 and delta OP3 overexpressing CNM1, we see less of the contacts. And when we add calling to the media, we now also see that we get the contacts that are restored. We get back the contacts, mitochondria clustering around the nucleus again. So restoring PC levels enables CNM1 mediated contact sites to form again. And this also suggests that it is the PC itself and not the proteins. It's not CHOT2 or OP3. The proteins are the PC levels that are regulating the abundance of CNM1 and the contacts. So to summarize what I showed you today, I hope I convinced you that CNM1 is a novel tethering machinery for the mitochondria nucleus contact sites. And it works together with Tom 70 to form these contacts. And that CNM1 levels are regulated by PC. And I can just say that we know that this is happening post-translationally. Okay. And with that, I would like to end. I would like to thank Maya, the most amazing mentor. All of the people here involved are people that the work was mentioned. And I want to thank you, especially for the amazing drawings. And thank you very much for your attention. And I was up to get your questions. All right. Thank you very much. Michael for a very exciting talk. I forgot to mention to everybody listening in that you're of course welcome to post any questions that you have for the speakers. But I see that we already got the question in. So I'll just get started with that one from Varinia Lopez Ramirez. She's asking in what development stages do the contacts between organelles, do you have the contacts between organelles? Are they more frequent or are they random at certain stages? So if I understood correctly the question, we see them basically all the time. Most of our work we're doing in the east that are grown in the logarithmic phase. So basically they're always dividing. So we see them, but also in stationary phase when basically you see less of the division of the cell. This we still see contacts. And actually we have several contacts that are more abundant in logarithmic phase and some of them are more abundant in stationary phase. And you have different contacts forming according to the metabolic state of the cell. But they're basically always there. So you can find them all the time. So are these then also very in a way rigid contact sites? Or is it more even though you see them throughout the whole cell cycle? Is it that contact sites come and go all the time? I'm not sure I got it, but basically we see them all the time. I mean these mitochondria nucleus contacts, we see them more in logarithmic phase. And then when cells enter into stationary phase we see that actually we have less contacts. So I'm not sure I answered the question, but we do see them all the time, just sometimes more abundant and sometimes less. As a question here, that in the screen there were 55 hits. And are the others and not just CNM1, are they also involved in this contact site? So yeah, that's a great question. Actually we had, we narrowed down the list to seven proteins that might have roles in this contact. And some of them we're still working on to find out more about them. I can just say that some of them seem to regulate the contact's extent, but we were focusing more on tether. So we wanted to find the actual tethering machinery for this contact. So that is why we focused on CNM1. And another thing that we noticed is that some of these hits actually affected also mitochondria ER contact. So for example, if we got a hit we checked how is it affecting the Hermes complex, which is a known mediator for the ER mitochondria contact. And if we found that these hits are affecting the Hermes complex we decided not to focus on them because we wanted to find something that is separated from the known tethering machineries and we wanted to find new tethering machineries for these nuclear ER or nucleus contact sites. That's clear. I have another question from Christian Ungerman. Is CNM1 in contact with the lipid synthesis machinery at the ER? And does that mean that CNM1 is a PC transfer protein? So that is something that I would love to answer, but sadly we still don't know. We haven't done any in vitro experiments already. We haven't done that yet. I mean we're now still testing the idea that that can directly bind maybe PC or affect the levels of PC in the different organelles. But this is something that we're still testing. So I can't say if that is for sure. We know for sure that PC levels are affecting CNM1 and by that it is affecting the contact. But we don't know yet to say for sure that this contact is responsible for the transfer of PC directly. And this is still a hypothesis that we have. So we still haven't tested it yet to say. Thank you. I have a question from Fando Yuk and the question here is would like to ask about the meaning of the contact between the mitochondria and the nucleus. Do they exchange something through the contact or what do they exactly do? So our hypothesis for now is that they might exchange PC, so phosphatidylcholine, between them. So once PC is formed in the ER, it has to go back to mitochondria somehow. So we think that this might be a way to transfer this phospholipid. And we know that transfer of phospholipids occurs in other contact sites in the mitochondria ER contact, for example. And we also found interestingly that some of the enzymes that are responsible for PC biosynthesis, both through the regular pathway and the Kennedy pathway, are actually enriched in the nuclear ER. So that is also something that we find that is interesting that it might be that PC from the nuclear ER specifically can move to mitochondria through the contact. But I also can mention that these contacts were studied also in other contexts. We have, we know that in mammalian cells they can be used for transfer of cholesterol and they have a role in the retrograde response that communicates mitochondrial stress to the nucleus. We also know that it was suggested to have a role in cellular inheritance in different organisms. So they were kind of suggested to have different roles throughout the years in inheritance and retrograde and heme transfer also I mentioned. But for this specific machinery that we found for the CNM1, because we see that it is regulated by PC levels, we think that it might have a role in the transfer of PC between the organelles. Right. These are more questions coming in and so related to whether there's transfer or binding with PC. So it seems that this is very much on people's thoughts. But you're saying that you are, you don't have any proof for this now, but you're looking into it. I can't give you this proof. I would love to give it to you, but sadly for now we still didn't see it. I want to finish off with one other question is what if you knock out CNM1? What is the phenotype of that? So we were able to show that when you just add coline to yeast cells, they form more of these contacts between mitochondria and the nucleus without even overexpressing CNM1. I mean, I believe that they upregulate CNM1 and they form these contacts and then when we delete CNM1, we don't see this upregulation of contact between mitochondria and the nucleus. But other than that, if you're asking about growth, let's say growth curves and growth on plates, we don't see any specific phenotype. And I also should say that, I mean, as I mentioned, these mitochondria-nucleus contacts, we think that they're not very abundant. I mean, they can be found, I guess, in every cell, but it's not like they are super abundant. So it might be that when we delete CNM1, this can be, for example, maybe rescued by other contacts. Maybe the mitochondria-ER contacts can rescue for that. And we know already that different contact sites can sometimes do compensations. Loss of one contact can be compensated by another contact. So this might be also the reason why we don't see any specific phenotype for the loss of CNM1. All right. Thank you very much for your presentation today and the exciting discussion afterwards. There are more questions, but unfortunately, in the interest of time, we're going to have to move on to the next speaker. So I just want to thank you again, Michaela, for presenting today. Thank you very much. So then I would like to say that, as I said before, at the Youth Lipid Conference, we very much want to encourage young researchers to also present their research. So that's why we've also invited Paulina Kanoviciova from the Communion University in Bratislava, Slovakia, where she is a PhD student, to present her work as well. And the title of her talk is the Role of Pospatidine Glisseral in the Manifestation of Mars Syndrome. So with that, I just want to hand over to Paulina. And again, if you have any questions, just join the presentation. Talk to them already in the chat. All right. Can you see my slides? I hope so. Okay. So good afternoon, everyone. Thank you very much for this opportunity. Metacondrial phospholipids have been the object of interest for many scientists because of their medical significance. And the best characterized is cardiolipin, since its impact metabolism has been associated with aging and diseases such as cancer, Alzheimer's, Parkinson's, and also type 2 diabetes. And the best example of cardiolipin importance is a life-threatening disease called Barx syndrome, which is directly caused by cardiolipine deficiency. But what about other mitochondrial phospholipids? Do they also play roles in disease manifestation? We focus on phosphatidine glycerol. It's an anionic phospholipid. And it's well known as a cardiolipin precursor. However, in various organisms, it also performs functions outside of mitochondria. It's one of the major bacterial membrane phospholipids. It's the only phospholipid in teloquid membrane. And it is also crucial anti-inflammatory component of lung surfactant. In the east, especially saccharomyces cerevisiae, its levels are tightly regulated and kept very low. And even at this low level, it is still important for function of some mitochondrial proteins such as cytochrome, c-oxidase, or for activation of inositol-swingolipid phospholipids, IC1. Redulation of phosphatidine glycerol metabolism in yeast has been studied in our laboratory for several years now. And this study started by discovery of phosphatidine glycerol-specific phospholipids C called PGC1. PGC1 is an interesting protein that hydrolyzes PG to a glycerol-free phosphate and diaceglycerol. And in case of its absence, PG is accumulated and spreads out of mitochondria evenly to other membranes. Based on our results, we also proposed a model of regulation of activation of PGC1. It's been previously localized to lipid droplets, however, it's not active there. It's only stored there and protected from degradation. And activation of PGC1 and degradation of PG were only observed in mitochondria and or ER, which are phospholipid bilayers. But what is the reason for this regulation? We know that both lack and excess of phosphatidine glycerol are harmful for cells. In case of PG abundance in PGC1 mutant, we observed fragmented mitochondria and impaired oxidative phosphorylation. And these results suggest that phosphatidine glycerol levels may be also important for proper mitochondrial morphology and function, similarly to cardiolipid. Cardiolipid is also an anionic phospholipid, but it has two negative charges, 450 SO chains, and it is conical shape. And due to these specific properties, it is involved in most mitochondrial processes. The major cardiolipin is its fatty acid composition is tissue specific, but it contains mainly unsaturated fatty acids. And double bonds in unsaturated fatty acids are very prone to oxidation. And therefore, cardiolipid undergoes process of remodelling in which saturated fatty acids are cleaved, monolysocardiolipin is formed, and then protein taphazine, it's unsaturated fatty acid. In case of BART syndrome, mutation because of mutation in gene TAS, taphazine is not functional. And therefore, monolysocardiolipin is accumulated and the total amount of cardiolipin is decreased. And this leads to symptoms of BART syndrome, which are cardiomyopathy, scaldomyopathy, neotropenia, growth delay, and so on. Yes, the saccharin mindset service here has served as a powerful tool of to study cardiolipin a biosynthetic pathway, mainly due to the existence of viable mutants. And yeast TAS mutant has been used in several studies that significantly contributed to our knowledge of BART syndrome. However, often yeast TAS1 model showed only mild defects that were observed due to TAS mutation in other organisms. And what are the differences between yeast TAS mutant and some mammalian BART syndrome cell culture models is that these mammalian cells accumulate not only monolysocardiolipin, but also phosphatidylglycerol, which is kept low in yeast TAS mutant. And therefore, we decided to look at what is the effect of phosphatidylglycerol accumulation in yeast BART syndrome model. And we prepared double mutant PGC1 TAS1. In this picture, so you can see comparison between human cell line TAS mutant and double mutant PGC1 TAS1 prepared in yeast. And the mitochondrial phospholipid profile in, I don't know if you can see this, in yeast double mutant is much more similar to TAS mammalian cell model than is TAS1. And subsequently, we looked at how increased PG levels affect some mitochondrial functions in TAS1. Mitochondriomorphology often reflects their function. And previously, abnormal mitochondrial ultrastructure was observed in a fibroblast from BART syndrome patients. And they observed this abnormal circular arrays in Cercristi. And we saw similar on-line structures in our yeast model of BART syndrome. And using fluorescent confocal microscopy in here, we also observed this circular shaped mitochondria. And importantly, their frequency was doubled in a double mutant accumulating PGC1. Then we looked at what other mitochondrial functions are worse on the reflected in the double mutant compared to TAS1 strain. And we observed a decrease oxidative phosphorylation of double mutant compared to TAS1 or val type. And this drop in oxygen consumption might be caused by decreased activity of CO2 activity of complex form. And this change in activity was not caused by a change in protein level. And next, we figured out why yeast TAS model of BART syndrome does not accumulate PGC1 as was observed in semi-million models. We measured decreased, in TAS1, we measured decreased mitochondrial activity of PGC1, which is the same necessary for synthesis of PGC1. And we also measured increased degradation of PGC1 in this strain. And these results indicate the tendency of TAS1 to keep low PGC1 levels and to avoid damaging effects of PGC1. So phosphatidyl glycerol accumulation caused further deterioration of mitochondrial functions that we tested. And we think that this could be explained by different shape of phosphatidyl glycerol, because it is the lact of chronically-shaped cardiolipin that plays an important role in onset of BART syndrome. And the accumulation of cylindrically-shaped PG could further impair thermodynamic stability of damaged membrane. And the other explanation might be connected to the importance of PG in function of somatocondrial protein, which we know it could be cytochrome c-oxidase. So we hypothesized that phosphatidyl glycerol metabolism could be one of the physiological modifiers that affect the severity of symptoms of BART syndrome patients even with the same mutations. And next, we plan to study regulation of phosphatidyl glycerol metabolism in mammalian cells. And in the end, I would like to thank you for your attention. I would like to to my co-workers and this work was supported by following grants. Thank you. All right. Thank you very much for your presentation. Again, if you have any questions, leave them in the chat. Then we have a discussion about that. I'm very excited to hear or to see a present about how yeast can be used to study human disease. I'm just wondering, do you know whether, so you showed this impaired oxfoss activity in yeast, how does that relate to the symptoms in BART syndrome in humans? Do you know that? Well, because in these cells, with test mutation, there is NADF-NAV cardiolipin, which is important for the function of probably all proteins that are involved in oxfoss. Even oxfoss in BART syndrome patients is decreased. So this leads to formation of NADF-NAV ATP, which is responsible for symptoms that occur in BART syndrome. Or disorganization of ATP metabolism? Yes. Yes. Then I have a question coming in here that you showed an effect on complex four of oxidative phosphorylation. Is it very specific to this complex? Or did you also look at other complexes, maybe complex three as well? We looked also at complex three and we didn't observe any changes, but we observed slight decrease in activity of ATP synthase complex five. There are any other questions from the audience? I think it was a very clear presentation. I think it's also very exciting to see how a lot of us focus very much on just yeast, maybe for production of lipids. It's very nice to see that you use it looking at human disease as well. You see that it's more than just yeast, but they're interested in. So I'd just like to thank you again for your presentation and exciting discussion afterwards. Thank you very much. Then I will hand over to my co-host, Serena, who will lead the rest of this seminar. I would like to welcome you to the second part of this webinar, where we move into the more applied field. Our first speaker, our keynote speaker in the session, is Rodrigo Ledescu. He's a group leader at Imperial College in London, and he will talk about the sustainable production of lipids and lipid-derived molecules in Yarovia Lipolitica. Thanks, Serena. Can you see my screen? Can you see my cursor as well? Yes, we can also see it. Okay. So thanks for the introduction. Serena, thanks as well for the invitation to talk here today and talk a little bit about this applied part of the webinar, where we're going to see in this particular about Yarovia Lipolitica. We'll introduce to Yarovia a little bit later, and we're going to see how we can use this yeast to produce lipids. So first, I would like to introduce to the group. So we are at Imperial College in London, in this campus, South Kensington, which is central London, really close to Hyde Park, and all the museums. Serena is really nice around here, especially days like today that are sunny, which is quite unusual. We are partners of the Imperial College Center for synthetic biology, which is a multisplinary research environment with people from different departments, spanning from mathematics to biology or bioengineering, such as my case. And the research that we are doing is at the interface between synthetic biology and metabolic engineering. And because the webinar is a bit broad, and there might be people who are less familiar with these terminologies, especially those coming from more fundamental or this is related lipid yeast research, I'm going to introduce you to these two concepts. Synthetic biology is a discipline that is trying to bring engineering principles to biology, and in particular, principles coming from electrical engineering and mechanical engineering, which aims to make biology more controllable, predictable, standardized, and modular. While metabolic engineering is the tuning of metabolic flaxes in order to make a specific product, and it also brings in engineering concepts to biology, but in this case, these concepts come from a different engineering field, which is chemical engineering. So in a chemical plant, you have different tanks where different chemical reactions happen, and in the end, you make a product. In metabolism, this happens very similarly, but instead of tanks, we have reactions catalyzed by enzymes that change and metabolize into others, and in the end, they make the product. So when I talked about this interface between these two fields, synthetic biology and metabolic engineering, I want to make an analogy to understand better, which is what happened in our current production plans, and if you imagine any chemical production plans for making paper or materials, you have a series of vessels where this reaction happened, but everything is controlled by different tubes and valves, and everything is integrated and regulated and making it reliable by electrical circuits that control the processes. So the idea is to try to make this in the cells, and well, in cell factories, so we can use this fine tune and control from this synthetic biology into the metabolic flaxes to regulate them to make the product of interest. So the aim of bringing these two fields together is to move to the bioeconomy, with basically trying to replace fossil fuel-based chemistry to make all the materials that we need, like food, materials for construction, or chemicals, pharmaceuticals, and so on. So one way to get around fossil fuels is using lipids, so luckily lipids can be converted into a variety of end product, like a pharmaceutical food, bioplastic, chemical fuels, and this can be either biologically catalyzed or chemically catalyzed starting from fatty acids. So where we can find these fatty acids is in vegetable oils, animal fats, but also in microbial oils, and this is what we are going to focus on today. So luckily all these oils or lipids are more sustainable than fossil fuels, and that's why there's the interest in moving towards lipids-based chemistry, and in particular microbial oils, they have some advantages. Some microbial oil is basically an oil produced by a microbe by fermenting a specific carbon source or substrate and making this oil, and this will happen more efficiently in organisms called oleogenias, and it can also happen by converting a cheap oil, like waste-cooking oil, into a high value oil. The advantage of microbial oils is that they don't compete with food, which doesn't happen with animals and plants. They have short-term process cycles, they are independent of the season, climate, or geopolitics, they can be easily scaled up, they can use cheap carbon sources, and we can do metabolic engineering, which means we can specifically make oils that have a specific function or value. However, there are also some disadvantages with microbial oils, like they need a certain condition, so these micro micro are prone to contamination, they require sometimes expensive substrates, no money sugars, then they all need to be extracted from the cell, it can be an expensive process, and basically it's easy to produce oil, but it's not so easy to produce high-value oil. Luckily, in the past couple of decades, we've been developing the phyllo synthetic biology that allows us to engineer this organism to fight these disadvantages and make it more feasible when we move them to industry. Something that is key when we want to make microbial oils is to select the right organism, so here I present a bunch of microorganisms that we work in our lab, and in particular, I want to draw the attention to the oligenous ones that are here, that mainly are cerebellia politica, rhodoporinthroloidus, these two organisms accumulate more than 20% of the cell's right weight as lipids, and in particular we're going to talk today about cerebellia politica, that is an organism that, as we will see, has a capacity to produce lipids in high genes. So here I'm going to introduce you to this yeast, so it's a yeast that is well studied nowadays, specifically in relation to lipid metabolism, and specifically as well for the idea of bioproduction, we'll see later. So cerebellia politica can produce a decent amount of lipids when it's grown in sugars, for example, and here you can see the lipids stained in green, so these are the lipid bodies stained with podipi that are mainly made by triacic visors, but cerebellia is also good at consuming lipids, and that's why it's called politica, and it's because it can degrade this lipid very efficiently, and here you can see an electric microscopy image where you see the yeast cell consuming these lipids that were present in the media. So more recently cerebellia has been regarded a very, very good host for biotechnology, and specifically for the production of these lipids that can be then converted and released in fields or chemical. So a little bit of history of cerebellia, so it is known for many, many decades, but its relationship with industry started in 1950s when it's used to produce protein, single cell protein, a source of protein for feeding. Even before that, we have identified in cure meat and tissues, so it has been helping us humans to make the product since early, early times. In the 1970s, more applications of this organism were explored, especially the production of organic acid, citric acid, which is something else that cerebellia is naturally good at doing, because it's able to degrade oils. It has been also proposed for bioremediation, for remote oil spills, for example, and from the 2000s, groups like Claude Guéderdan of German Nicode in France, they started to develop genetic engineering tools to modify this organism, and they grow and developed, and there was a huge interest, and you can see how the publications in the Arabia started to rise around that time, when there is an interest in making an alternative to petroleum for fossil fuels, and this goes up in the 2017, more or less, when the price of the petroleum drops, and the scientific communities tend to look at what other things we can do with the Arabia, and there's a shift towards high value limits. More recently, the past decade, there has been many groups developing synthetic bioregual tools to more easily engineer this host, and we developed things like Golden Gate libraries, there has been groups developing efficient CRISPR tools to engineer metabolism by knocking out gene, but also by expressing or repressing genes by CRISPR-A and CRISPR-I, groups that are doing this, for example, I am well done in the University of California, Riverside, there are genomes here, metabolic models of the Arabia, and Edouard Coven, one of the organizers here, developed a very, very comprehensive model for it, so we have a variety of tools, and we know how they live in the devil's work, kind of, so we can use these to make products, to make units, and now I'm going to focus the rest of the talking, going through some examples of the Arabia to make units. So there has been a lot of works, and you can see here the numbers, this is not completely up to date, but there are around 200 papers in the Arabia published yearly now, and this curve is growing exponentially, and there are also many companies working more and more with this organization, as you can imagine, there are a lot of works that have been working in in this lipid metabolism, and you need to make different products, so there has been many works about trying to increase the yields of lipid being produced, and there are reports that say that up to 90% of the cell weight can be lipids in the microbial rigidity, so basically all of the cell weight can be just from the lipids, which is really good news. There are groups as well trying to fight some of the disadvantages I've mentioned before about microbial oils, such as the requirement for accepted conditions, and the group of Stéphanoculus in MIT worked in engineering in the Arabia to be able to use phosphate instead of phosphate in order to grow less clean conditions, let's say. There has been a lot of works, like hundreds of works almost per year producing different types of lipids, high values among them unusual lipids, and they have been also quite a good amount of research in engineering in the Arabia to use low cost substrate. There are a few works as well in facilitating the secretion of this lipid to facilitate or also to fight one of these disadvantages we've mentioned in the beginning, that is the need to extract this lipid from the cell. And now we're going to delve a little bit more into some particular examples coming up from lab and using the Arabia to make lipids in the most sustainable way, and when we talk about sustainability we should not only think about the environment, but also about the economics of the process. And here I present a typical process from the upstream part where you have the substrate, when it's loaded with the fermentation, and then when your lipid is produced they need to go to a downstream part where they need to be recovered, insulated, purified and polished. And we need to consider that all these parts contribute to the cost of the process, so if we want the overall process to be economically feasible we need to think about decreasing the cost of each of them. And of course by increasing tighter yields and productivities we can make fermentation more efficient, but we also need to consider which is the substrate we are using, if it's cheap enough, and how we can recover the product in an easy way. So I'm going to bring some examples about each of these sections. And we're going to start by the first one, by the upstream process, and how to expand the range of substrate that the Arabia can use. So I should mention the Arabia is naturally good at producing, at using different current sources like, like visceral glucose or hydrophobic substrate like alkanes, or even fatty acids. But we wanted to expand this to use cheap substrate that are usually wasted in industry, like starch, which is the example that I bring. So starch are these granules, it's the second most abundant polymer sugars in earth, and it's made of sugar units, but in a very complex arrangement, so they are very inaccessible to bags, so they, microorganisms, if they lack these acetic and enzymatic activities, alfamilase and glucomilase, they are unable to use them. And the Arabia lacks these two enzymes. So in this work we express these two enzymes, we secrete them out, and we found that we were able to to see how the Arabia was expressing these two enzymes, actively in supernatant, degrading these granules that were unable to be degraded by the white area. And we further engineered lipid metabolism by enhancing the Kennedy pathway and blocking bed oxidation, to in the end produce these lipids from raw starch or from effluent, from bioethanol, a company that was a waste, contained starch. So another example, a series of examples, I'm going to talk now about the use of silos in the Arabia, and the Arabia can use glucose, but cannot use silos, and these two sugars as the main components of limousine losing material, that as you may know, it's one of the most wanted substrate for biotechnology. So the idea was to transform them into lipids, and for that we first need the Arabia to use silos. And so in the first work, we identified the reason why the Arabia was not growing in silos is because these two first enzymes in the pathway were not really highly expressed. So what we did is to take these two enzymes, the silos for the taste, and see if the hydrogenates from an organism that is well to utilize silos. And we put them in the Arabia together with the over expression of the native cellular kinase, and we found that the Arabia was able to grow and was able to grow as good as in glucose. So this was good news, and we explore the production of lipids in different media containing silos to make like 40%, almost 40% of the cellular weight as lipids from a silos only. In a follow up work, we wanted to use the native genes, so we over express the native genes instead of using heterogonal ones. And we saw that these genes were able to sustain growth in silos as good if not better as the heterogonal genes. And then we use that strain not to use only silos, but a real lymphocelulosic hydrolysis. In this case, the one coming from the tequila manufacturing. So for every liter of tequila, 10 kilograms of cellulosic biomass is wasted. And this waste can be converted into glucose and silos by hydrolysis. And by doing a little bit more metabolic engineering in this pathway, we were able to convert the whole glucose and silos extraction from this into lipids. And in this work, we reached the maximum theoretical view. So one thing that we observed when we're trying to grow the Arabia in this mix of glucose and silos coming from immunological hydrolysis is that the Arabia prefers the use of glucose. So as you can see here, in green, glucose is consumed first. And when most of the glucose has been consumed, silos started to be taken up. And this is considered to be negative for by a process. So in this collaborative work, we did an adaptive laboratory evolution of the Arabia that was engineered to be able to use silos to be able to also utilize silos in the presence of analogous glucose. And after the evolution, we saw how both glucose and silos were co-consumed. And these strains were not only able to produce lipids, but a little bit of further modifications. They could also make other products out of silos. And in this collaboration with Vinod Kumar from Cranford University, we used the same strain that we developed with a little bit of further modifications to make saccinic acid and cellulite. The next example is about how to make something that is a little bit higher value than just the neutral lipids, just 3-acelucidols. And I'm going to talk about foetacalotine, which is an adipate soluble molecule that is antioxidant, vitamin precursors, the precursor of vitamin A, and it's also a cholera. So it has many applications in pharma, cosmetics, food and food industry. And here we express different enzymes that were required to complete the pathway towards foetacalotine from the mevalonic pathway that was native in Yarobea. So these two enzymes were required heterologous lip, and these two were expressed in this cassette together with the last step from the mevalonic pathway here, DSS1. And by putting this cassette into Yarobea, we were able to see this orange color that is typical from foetacalotine. So we did several modifications, and in the end, we wanted to optimize the flux towards this pathway. And what we did is coming back to this cassette here with there are three key genes controlled by three different promoters. We wanted to optimize this cassette to maximize the fluxes, and we did that by a combinatorial library assembly, where we basically make a library where every promoter in the library was able to control every of the genes. And in the end, this library could be transformed into Yarobea. And because luckily, foetacalotine can be easily screened by eye, we should be able to select the most orange genes to identify by sequencing which are the key promoters in the cassette that drive the highest expression of the foetacalotine. We did that. We obtained something similar to what we were expecting. We sequenced the best cassettes. We reconstructed the strains that after incorporating several copies, we took to the bioreactor, and we reached a production of 6g of this relatively high value compound. We follow up that work, and we're still working on this area. This is a collaboration with Jose Luis Martinez in NETU, where we use high-to-put bioreactors, micro-scale fermentations to optimize the culture conditions, and we find out how drastically production can vary by controlling environmental factors rather than genetic factors. So there's a lot of optimization that can be done after your strain has been maximized in flask or standard bioreactors. So the last example today is about the downstream part of the process. As I mentioned before, one of the limitations of microbial oils is the destruction of the snippets from the cell. We need to break down the cells using either mechanical, physical, or chemical agents that can disrupt the cells and allow the structure, or basically the ozone solvent, that are kind of not really good for environment, to extract this product. So we're trying to get around that by engineering anaerobic to secret these fatty acids. So the neutral lipids, the main lipids, storage lipids in anaerobic, the acid glyceros cannot cross membranes, and they are well stored in these lipid bodies that we've visualized before with these greening droplets. But what we did is to block the activation of free fatty acid and the metaphylation of these free fatty acids of red consumption or degradation in order to convert the neutral lipid moiety into amphipatic lipid moiety, and we know amphipatic lipids can cross membranes. So we did several approaches, one strategy where we enhanced the Kennedy pathway to make more of the neutral lipid first and then overexpressed different intercellular lipases to maximize the production of free fatty acids. And the second strategy where we basically blocked the production of free acid visuals at all, and we expressed tire stresses that basically convert the acyl-CoA moieties into free fatty acid moieties that can cross. We found similar results of secret fatty acids, around three gram per liters in each case, and we optimized the libid conditions in fermentation. And what we saw is that when there are enough lipid secreted outside, this lipid aggregates and they are very easily recovered, and you don't really need a lot of energy to do this. Also, what means it can be achieved in the cell bioprocess, if this is industrialized. And here we were just fishing this lipid with a loop. And here you can see how the wild type accumulated lipids inside the cell, but how the engine used strains was secreting all these big droplets outside. And this allows us, as well, to uncouple biomass formation and lipid production, and we reach an equivalent of 120% of the cell dry weight as lipid, so something that is unable to be achieved only within the cell dry lipid production. So I think here I'm finishing my presentation today. A common message is, I hope you've learned a little bit about Jarovia, the potential that it has. And I must say that this is not a close topic. The field is growing exponentially, more and more groups, more and more industries are developing technologies for these organisms. So I'm sure you will hear more about this in the following years. And the other thing is like whatever your process is, think not only about yield-stighted productivity, still about the economy of the process and how viable it is going to be. And for this, the best thing is to directly talk to companies which have these expertise. So just so quickly, I'm going to mention that in the lab, we are working in other things that are related or unrelated. We are working in Jarovia, the political engineering for the production of all the things, like terpenoids or flavonoids. But we are working also with Cerebici and all the many other organisms that you show in the previous slide. We are also working in developing new synthetic biology tools for metabolic engineering, like technologies for multiplexing CRISPR. And we are also exploring microbial communities as a new tool to make bioprocesses. And with this, I would like to acknowledge all the members of the group, all the funding bodies, the organizer of the webinar and all of you for your attention. We will be happy to take any question. Thanks a lot, Voduhigo, for this really nice overview talk. The first questions are coming in. So we have one question from Alefandra Sanchez on the use of other yeast. So do you think that Jarovia is the best yeast to produce microbial lipids compared to other ones? And if yes, why or why not? So it's a good question. It's a good one. And for each particular lipid, probably you will have a better one. So there are some organisms that without any modification, like rhodotratauloides, can produce much more amount of lipid than Jarovia. So I think one of the advantages of Jarovia is that we have converted it, like a whole synthetic community working on this. We have converted it into conventional. And now we have a lot of tools that we can use to engineer it. And that makes it a very good organism to produce these things. There are limitations still in Jarovia. Sometimes it filaments, sometimes it produces too much waste products, like citric acid. So it is still a good one. Despite the limitations, there are some others that are emerging ones without the potential. It depends on your specific product. I would recommend to select one or another. Great. Then we have a question from John Morrissey. Are there any or many lipid products made commercially at this point from Jarovia? I would say no. There are a lot of companies starting now to work with Jarovia. There are several startups, but there is also some big company that has been exploring Jarovia for commercial purposes. One example was DuPont several years ago, which got approval to make Omega-3 fatty acids. They go to the point where they scale the production and they start to produce, but at some point they decided to stop the process. So that's not happening anymore. I know of others that are trying to get to scale, but I'm not aware of many that are successful. Thanks. Then we have a question from Jan Meiju. How do you detect lipid content in the yeast cells? Will temperature affect lipid production in Jarovia? And when will the lipid content reach the highest level in the cell? I assumed during growth, which growth stage? Regarding the how we measure lipid, we measure lipid with GCMS or GCFID. So we extract the lipid, we trust, ventilate them, and we quantify that with a standard standard. There are other ways to estimate by fluorescence or something like that, but they are not as accurate. So in the end we always go to GC. Regarding the temperature, it's a good question. There are variations with the temperature in production level. There are even more variations in the profile of fatty acids. So there are more unsaturated acids being produced at lower temperatures to keep the fluidity of the membrane. So again, depending on your product of interest, if it's more unsaturated, you might have better ratios when you grow at lower temperatures. And the third question was about the face of the culture. So normally we work in nitrogen limitation conditions. So at a certain moment in the culture, nitrogen is limiting and there's a still carbon source present. And then it's when there is a highest increase in intracellular lipid production, which is a natural response of the cells to store this extra carbon wasted or leave it for other competitors in the environment. And some of the modification that we have done in Nairobi allowed Nairobi to start accumulating more from the very beginning. So depending on the strain, some of them just started accumulating early on, some of them were nitrogen repletion happen. We also had a question on toxicity. So do you have any problems with fatty acid toxicity at these high production levels? So in general, we haven't observed a lot of toxicity with fatty acids. And this is because as I mentioned, Nairobi, the political kind of evolved to utilize fatty acid as a main kind of source. We have observed toxicity with specific lengths of fatty acids, like C10 for example, toxic Nairobi. But in general, like oligases or things like that, it can tolerate as much as you want to give it. What's the approximate yield of lipids from glucose or one other carbon sources? Yeah, I'm not sure I have the right numbers in my head. I would say, correct me if I'm wrong. I would say it's around 2.3 is a maximum of 3 taken yield from glucose around that. That's difficult to get. I mentioned in the slide that we achieved that, but I wonder if I was cheating a little bit there because it was a complex hemocelulosic hydrolysis. So this yield was calculated from glucose and silos only, but there are other things there like acetic acid that Nairobi can also use to contribute to that. Concerning yield, I have a question on my own also, because for example, for the beta carotene production, you're basically competing with neutral lipids for precursors because they both use acetyl-CoA as precursors. But on the other hand, I guess the neutral lipids are also beneficial for storing the beta carotene. So what would be the best way to balance this, having still some storage compartment, but on the other hand also increasing the yields for beta carotene production? Yeah, that's a good question and we still don't know. We are working on that. What we observed in that work is that the more lipid we had, the more beta carotene was produced. What was not expected in the beginning, and the answer is what you just mentioned, storage, and we know the beta carotene, if there's not enough lipid bodies, it will go to the membrane and would disrupt the membrane and the toxicity. If you have enough lipid bodies, it can tolerate much better this beta carotene and it doesn't increase or anything that can disrupt other parts of the cell. There might be a sweet spot somewhere between one or the other. We tried with extracellular lipids to control the intracellular level. It doesn't work because I think somehow they produce at the same time to prepare the store. But yeah, there should be a balance that we would define it sometimes. Also, a quick question maybe at the end was like, have you tried to produce the short chain fatty acids? A question by Tomik. So we tried briefly with these diasterases that are specific for short chain fatty acids, like coconuts. We didn't succeed in the experiment. We secreted the fatty acids. We thought, okay, diasterases are not specific of length. We might be able to preferentially create a specific length from them. It didn't work like that. There are other groups working in Nairobi, as we're working on short chains and they're good results that they have achieved with different lengths from 12 to 8 to 6. Great, a lot of more interesting questions and they are like, they continue popping up but unfortunately I think we have to move on in the program. So thanks a lot again for a really nice talk, interesting overview talk and also to the audience for posing the questions. And we will, thanks again Rodrigo and we will continue in the same line. So that was a nice introduction also to the following talk, which will be by Carolus Petkevisius, who is a PhD student at the Novonautus Foundation Center for Biosustainability at the Technical University of Denmark. And he will talk about the biotechnological production of the European corn borer sex pheromone in the yeast Jaroveale politica. So please go ahead. So hello everyone and thanks for introduction. Yes, so my name is Carolus and I will be talking about insect sex pheromone production in yeast Jaroveale politica and specifically I will focus on production of European corn borer sex pheromone and the reason why this insect is interesting, it is because it is the main pest of the Maze worldwide, it is distributed in North America, North Africa and in the Europe. And if measures are not taken up to 20% of the crops might be lost due to damage caused by this insect. And currently to solve this problem, chemical insecticides or genetically modified crops are used. However, there are other alternatives. For example, one could use insect sex pheromones to solve this problem. And specifically Ostrinian bilalis uses insect sex pheromone blend, which is composed of E11 and Z11 tetradesynyl acetates. And here is the biosynthetic pathway, how this insect produces this fatty acetic mixture. So everything starts from Palmettoilka way, which undergoes one cycle of beta oxidation and meristic acid is generated, which is then desaturated at position number 11. And this desaturation stuff provides the mixtures of E and Z11 tetradecanoic acids, which are then converted into corresponding alcohols and finally acetylated. And the ratio between these isomers is dependent on the strain, which is known that Ostrinian bilalis has two strains, E-strain and Z-strain. And E-strain primarily uses E-isomer of these acetates, while the Z-strain primarily uses Zet isoform of these acetates. And we, as a group, we were interested in production of Z11-14 acetate because it is the most prevalent Ostrinian strain in the world. And for that, we have chosen the East Jarovia lipolytica, because as you can see from this review, if someone wants to produce relatively expensive fatty acid derivatives on high titers, Jarovia seems to be, seems to be very good option for that. So, can you see my entire screen? Sorry. We see the entire slide, but not everything is displayed yet, so. Yes, okay, so here on the right side you can see which steps have to be taken in order to produce Z11-14 acetate. And as you can see, the first step is production of meristic acid. And in general, in yeast, Danova production of saturated fatty acids relies on fatty acid synthase, which is a huge protein consisting of alpha chain and beta chain, alternatively called FAST1 and FAST2. And a couple years ago, quite nice discovery was made that if you mutate keto-synthase domain, you can increase meristic acid production. And we were inspired by that. And we replaced isoleucine with phenylalanine. And we increased production of meristic acid by ninefold. So that was great. That was the first step towards insect pheromone. And as you can see, the next step is introduction of double bond at position 11 in meristic acid. And as I mentioned previously, we were interested in the production of Z-izomer. So theoretically, the saturate should produce mostly Z-izomer and only small amounts of E-izomer. So here on the left side, you can see the screening of fatty acid desaturated. So as a control, I took the strain which was engineered for meristic acid production and introduced seven different de-saturated which have been described in literature. And from this screening, you can see that de-saturated LBOPTQ produced the highest amount of Z11 tetradecanoic acid, while some of the de-saturated, they were not active or they were less active compared to LBOPTQ. And the next step was, and in the next step, we were interested to see if this Z11 tetradecanoic acid can actually be converted into corresponding alcohol. So for that, I took LBOPTQ strain and expressed four different reductases on top of LBOPTQ strain. And here you can see that two candidates appear to be quite good. So it's reductase LITPG-FAR2 and reductase HARFAR. And even though LITPG-FAR produced the highest amounts of total fatty alcohols, we have selected HARFAR as the best candidate because as you can see tighter wise, there was not a big difference. But however, HARFAR produced slightly higher purity of our target compound which was Z1114 alcohol. So yes, in summary, LBOPTQ and HARFAR were selected as candidates for production of Z1114 alcohol. So after this enzyme screening, we moved further and then we did the small round of metabolic engineering where we took high meristic acid producing strain. We introduced two copies of reductase, two copies of de-saturase, and finally we have regulated fatty acid synthase subunit one from Erovia Le Politica. And here you can see that the best strain is strain 9253 which produced in small scale around 100 milligrams per liter of our product. And once the small scale screening was done, this strain then went into one liter fermenters where compared to the small scale, we actually managed to increase the tighter by two fold. And we obtained the tighter of 200 milligrams per liter of Z1114 alcohol. And after that, as the final step in order to convert this alcohol into active pheromone ingredient into acetate, we extracted fatty alcohol from fermentation broth and then chemical acetylation was done. And the chemical acetylation of fatty alcohols yield in this fatty acid mixture our target product Z1114 acetate comprised around 7% of total fatty acid content. And the ratio between E and Z isomer was roughly 9 to 1. And so as the last step, we tested biological activity of yeast derived insect pheromone. And this was done by our collaborators in Democritos in Greece. So it was done by Dr. Maria Eleni and Petri within the framework of your funded olefine project and experiment they did what is called the wind tunnel experiment where you have insect on one end of the tunnel and where you have the pheromone blend on the other side. And then once you have insect and pheromone in one in wind tunnel, then you apply the wind towards the insect and you observe how insect behaves. So in this case two parameters where monitored its approach and landing. So approach means and the question is if insect at all flies towards this pheromone blend and the landing was quantified based on if the insect approaches then how for how long the insect sits on this yeast derived pheromone blend. So here you can see the control which is only organic solvent without no pheromone. Then as a positive control it is the native pheromone blend of Ostrina nubilalis. And here by a fair is our yeast derived pheromone sample. And you can see that compared to the negative control yeast derived pheromone blend definitely elicited and elicited insect behavior. However it was not as strong compared to the behavior which was which was which was observed when the positive control was was used. So even though there is a still place for improvement for example the fatty acidate mixture contained only seven percent of our target compound and the ratio between E and Z isomers it was it was not the most optimal but yeah I think these results could could be considered as success and it can show that yeast derived pheromones could actually modulate insect behavior and they could for in the future they could be used for insect insect management. And so yes at the very end I would like to say a big thank for the people who contributed to this work and also for the Bioferro Innovations Fund and Olifine who financially contributed to this work. Thanks Carlos for a very clear presentation very nice result. Are there any questions in the chat? John Morris says very nice talk. Are you close to field trials? Honestly I I don't think so because the amounts for the field trials they they have to be increased and the yeah the sample which we have was yeah simply simply too small for that. Yes thanks then I have a question more concretely about the the pathway you chose or the the enzymes when we selected or tested different reductases because I say reductases can be found in all kinds of organisms but you specifically selected those from from insects why do they have a yeah more specific substrate spec from or? So the very very first thing which is is evident when you so these enzymes they're characterized and very first thing which has to be considered that all of these reductases they are not taking c18 fatty acids which are the most abundant. So by excluding c18 fatty acid conversion into alcohols we can already get a much higher purity of c14 fatty alcohol for example. Yes that's that's a very good reason I think so but then you do the last step the the esterification or the acetylation you do chemically but would there theoretically be also enzymes that could do that in vivo in the yeast? So once again I would be fan of using let's say acetyltransferases from the particular insects because I expect they might be also more specific but so far no acetyltransferases from the insects have been characterized and there are several attempts which which failed to find enzymes which can convert fatty acid fatty alcohols into fatty acidates and there's actually ATF1 acetyltransferase from saccharomyces which could do the job and previously my supervisor have tried but once you express we cannot we cannot obtain full conversion so some of the fatty alcohols they are still there while chemical acetylation we obtain full conversion from alcohols to acetylates and as you can see it was done in one hour with the relatively cheap chemicals so it's like simply easier to to extract fatty alcohols and the chemical conversion rather than to look for the enzymes. But Jarovia doesn't have any acetyltransferases at all that so that you see a small portion of esters coming up you only see the alcohols? Yes, yes. Then you showed that in your fermentation your you get titers up to up to 200 milligrams per liter what would be what would be the aim you're you're looking for? So in in our group like we have the paper on different pheromone which is on the other hand very similar which is Z1116 basically two carbons longer and here we produce around two grams per liter so I think yeah like somewhere in in the range of grams per liter yeah would be yeah would be definitely good I would be satisfied with that as we like achieve the titer as it was for different pheromone which is already published. How much time do you have left in your phd maybe you can still? So I started in 2019 June and I have three years so I will start I will finish the 2022 in the mid-summer. So sometime left to reach the grumper? Fingers crossed, fingers crossed. Great, any more questions not at the moment but if not then I would thank you and all the other speakers again for great talks and I hope that those talks were a real nice appetizer for the Yeas Lippert conference next year so I hope we'll see you all in Sweden and in Gothenburg in 2022. I don't know if John wants to say anything else? Yes? I think Sabrina I think you know I always want to speak so I would maybe we can stop sharing screens there now so I'd like to add my congratulations to all of the speakers and indeed to our session chairs for selecting the speakers and for keeping us on track with the questions. I think that was really interesting I'm no doubt that the talks will have wetted appetites for the Yeas Lippert conference next year. I think it's really interesting to see I mean that mixture of fundamental research research related to human diseases and then you know very interesting applications. I think for Carlos I mean I think we will demand that by next year he has at least you know doubled his yield I mean he can't do that in a year but only joking you know I'm sure the project looks like it's going really well and so I enjoyed those presentations. Obviously you've seen the website for the Yeas Lippert conference for next year so I would encourage you to log on and get on that mailing list. I just want to say as well sometime in the coming months Thames Yeast Research will launch a call for papers for people who are interested in submitting papers on Yeas Lipperts and you'll be able to submit your papers to the journal in the in the normal way and we'll collect those papers and publish them as a special issue around the time of the Yeas Lippert conference but there'll be typical normal research papers like the papers you can see in the virtual special issue that that's that's for this conference. So I think everything we heard today it's you know the kind of papers that were very suitable for Thames Yeast Research and we'd love to have some of those high-quality 200 papers a year that have been submitted that Rodrigo told us about on on Eurovia and I'm sure as well by the time we come to Yeas Lippert conference the road of Spuridium Yeas Lippert researchers will be fighting back and will be presenting some of their exciting work as well on that particular platform which I think is also very exciting platform into the future for bi-technological production of Yeas Lipperts. So I think that oh actually one further point I forgot to make but I've been reminded in the chat by the technical team there were some questions that we didn't get to earlier because we had a lot of questions for some of the speakers and we just didn't have time to address all of them so we will pass those questions onto the speakers and the speakers might be able to allow the email addresses of the questioners and so the speakers will be able to respond directly to the people who ask questions so apologies that we weren't able to get to everyone's question as we went through and it just wasn't enough time and of course when you come to the Yeas Lippert conference next year you'll be able to chat to those same people at a coffee break and at a free beer session that Reina is going to organize for everybody and that's sort of a plenty of time to have additional discussions. It'll have to be free because I know anyway it will be free I was going to say something negative for so with that I'll thank the speakers I'll thank the attendees we had well over 200 people attending I'll thank Sarah and Joe behind the scenes from FEMS and from Oxford University Press who gave all the technical support that made this run run pretty smoothly very smoothly and I'll let you know as well that this is recorded so it will be appearing on the FEMS YouTube channel in due course along with the other webinars that we've had so you know if you want to listen back to the talks or if you want to recommend the talks to any of your colleagues who weren't able to attend you'll be able to do that. So Reina and Ed are you happy or do you is there anything you need to add or will we wrap up? No just also for me thank you and also you and the team at FEMS for organizing this. Okay so we're done and dusted thank you everybody and over and out.