 Good morning, good afternoon, or good evening, wherever you are. My name is young will follow me. I'm an editor in a fence microbiology reviews inspector to welcome you today to this webinar on active organic organisms. Fence, as you may know, is the Federation of European Microbiological Societies investing science, using the income from our journals to fund charitable activities and support the community. We provide grants as well to scientists to organize and support conferences and sponsor a wide range of events such as this webinar series today. The fence webinars provide a form for the presentation and discussion of key research, enabling the flow of ideas to continue despite the current cancellation of in person events and conferences. The series has been ongoing since June 2020. If you missed any of the previous sessions, you can watch the recordings on the fence YouTube channel. So it's my pleasure to introduce both speakers of today. We have Walter Mueller, who is a professor in at the department of molecular bio microbiology and bio energetics at the Institute of molecular bioscientists from the young Wolfgang Goethe University in Frankfurt. His specialty is metabolism and biochemistry of anaerobic microorganisms, their metabolic engineering, helophilic organisms and by process engineer. Then there is a sound Islam is a assistant professor lecturing biochemical engineering in the department of chemical engineering and liberal university in Leicestershire in the UK, and his specialty is biochemical bio process engineering, metabolic engineering systems, biology and synthetic biology. So by accident, those two gentlemen will find themselves today on this webinar because they both approach fence microbiology reviews with reviews on ethnic organic organisms. And these two reviews they centered on the problem of replacing both chemicals from petroleum sources to different sources. And one of the organisms or group of organisms that may be interesting for this are acetogenic microbes that could potentially also help to reduce the emission of greenhouse gases. If we manage to actually change the petroleum based economy to something that is a more plant based bio economy. And with the metabolites that are produced by these organisms, we can potentially create a new chemistry. And particularly interesting is also the group of a citrogenic bacteria, because they can use mixtures of carbon dioxide carbon monoxide and hydrogen for the production of acetate and ethanol. So the two webinars that we will hear today will focus on the one hand on the interesting and fascinating biochemistry and by a process engineering perspectives of a citrogenic microorganisms. And on the other hand on the potential biosynthetic biology and genetic manipulation to improve such strains for bioprocess technology. The webinar will proceed as follows. We have both speakers first Volker Muller, then Assam Islam each for about 20 minutes. And after we've heard from the two speakers will open the floor for discussions. You can submit your questions to the online tool where I will see them and then I can pick from those questions and ask the gentleman to reply to them and discuss this. So you can pose your questions anytime during the session they will appear in the question window that I can see and from there I can pose them then afterwards to both speakers. I hope you will enjoy both seminars. It is my pleasure now to give the floor to Volker Muller. Please, Volker. Thank you very much, Jan, for this very nice introduction into the topic and the introduction of the speakers. It's my great pleasure to give you a summary of the recent paper published by Florian Krempp, my PhD student and myself in FEMS microbiological reviews on methanol and methyl group conversion and acetogens, biochemistry, physiology and application. So in the 15 to 20 minutes talk, one can end the highlight some glimpses of the review and you are invited to check out the review for further details. So the acetylgenic bacteria characterized by a specific pathway that would give that pathway of CO2 fixation, which is the two branched linear pathway in which two molecules of CO2 are converted to one molecule of acetyl-CoA. In the method branch of the pathway, CO2 is first reduced to formic acid and formic acid is then bound in an ATP dependent reaction to the cofactor tetahydrofolic acid, a common C1 carrier in U and prokaryotes. The formal group is bound to tetahydrofolic acid and then you can see the intermediates here. It's subsequently water is split off and then it is reduced via the methanol, the methylene to the methyl intermediate. So at the end of this reaction sequence of reduction, a methyl group is produced from the CO2. Then there's one more reaction to prepare the method group for condensation, which is the transfer of the method group to another protein, the coronoid iron-silver protein. In the carboxyl branch, the second molecule of CO2 is reduced to carbon monoxide, which is enzyme bound. And on the enzyme acetyl-CoA synthase COD hydrogenase, the structure of which is shown here on the right-hand side, the two moieties CO and the methyl group N-CoA are condensed to acetyl-CoA. The next step then is the production of acetyl phosphate by phosphatransicidylase, and then the last step is the production of acetate and ATP. So this pathway is used by acetogens, but also by methanogens and sulfate reducers. And the acetogens can also grow by using this pathway and by using reduction of CO2 with hydrogen. So acetogens are autotrophic and they convert hydrogen and CO2 to acetate. And this goes along with a free energy change of only 95 kJ per mole of an acetate. So how much is that? To make an ATP, standard textbook knowledge in biochemistry is 60 kJ, so that is enough for roughly 1.5 ATP per mole of an acetate. So this is not very much if you compare that, for example, to the aerobic respiration that our mitochondria perform. And now if you go into the anaerobic ecosystems where these bugs live in, there's not a lot of hydrogen. There's not one molar of hydrogen around. So these concentrations have been measured to be around micromolar range. And then the delta G is only minus 20 kJ per mole of an acetate, and this is at the so-called thermodynamic edge of life. And this gives only 0.3 mole of ATP per mole of acetate, and our group was interested in the last decades. One can say how this pathway is coupled to the synthesis of ATP. And I'm here depicting the Wurundal pathway again on the right-hand side. And now we have here a cell surrounding the Wurundal pathway. And now you see that 1 ATP is generated in the acetate kinase, but 1 ATP is hydrolyzed here in the formate activation step. So the overall ATP gain is 0. So the big question was for decades, how is this pathway coupled to the synthesis of ATP? And it must be coupled to the synthesis of ATP because these bugs grow on it. Electron comes from hydrogen, and these bugs have a hydrogenase that is electron bifurcating, means that electrons are split. One electron goes up energetically downhill, in this case to NAD, and this energy is used to pump up the other electron uphill to ferredoxin. If you now look at the electron sources, hydrogen in this reaction, the first reaction, NAD here, NAD here, and ferredoxin here, there's a misbalance. There is more ferredoxin than NAD, and therefore the sets need a redox balancing system, and this redox balancing system is membrane bound. Actually, it's a membrane bound ion translocating enzyme. It's called in the CWDRNF, but there is another one in other acetogens, which is an energy converting hydrogenase. So both systems use reduced ferredoxin, and the electron is transferred to NAD in the case of RNF, or to a proton in the case of the ECH complex. And this little energy between these two pairs is used to pump out sodium, thus creating a potential, and this ion potential is then used to drive the synthesis of ATP. I would say that this system is essential, of course, to make ATP during autotropic growth, but it's also equally important and essential for redox balancing. So if you now do the overall calculations, and this is published a couple of years ago in Nature Reviews Microbiology, you end up with 0.3 ATP per mole of an ATP, which fits very well to what I've just shown you before. So let's have a look again at the pathway, the pathway from CO2 and CO2 to acetyl-CoA. This pathway is very well suited for the conversion of other C1 components. Another C1 component is carbon monoxide. Some of them not all, some of the acetogens grow on carbon monoxide, some of them grow on carbon monoxide and CO2 and hydrogen. Together, this mixture is called synthesis gas or syn gas, and that is used in industry at the moment on an industrial level for the production of ethanol. Another compound which is highly interesting for applications is formic acid format, which goes into the pathway at the level of format, as I described before. Form aldehyde can be used by resting cells, but it's highly toxic, so it's not a real good growth substrate, but a real good growth substrate is methanol. Methanol is used by a couple of acetogens, and you see here the example that goes from acetobacterium to crostridium to u-bacterium. And here you see the old Morella thermoethytica discovered by my good old friend Stephen Daniel in 1980, that the growth on methanol you see spoo, musa, and you see thermo-acetogenium pheo. So there's a couple of acetogens that grow very well on methanol. So what is required for an acetogen to grow on methanol? It is required to transfer the methyl group into the central pathway. The final acceptor is THF, and the donor in this case is methanol. For the conversion of the methyl group into the central pathway, three proteins are required. First, methyl transfer is one that abstracts the methyl group from methanol and transfers to the second protein, which is a coronoid protein. And the cobalt atom in the coronoid protein, that is methylated, giving rise to methyl-coronoid three. And now the third protein, which is called MT2, methyl transfer is two, demethylates the methyl-coronoid three to give rise to methyl-tetahydrofolic acid. These three proteins can be three distinct proteins, but they can also be encoded by only two genes, with two of these domains being present on one polypeptide chain. So we have cloned and analyzed and identified the methyl transfer genes for methanol in the CETA bacteria in woody eye. You can see those here, MT1, COP, MT2. And then there's another couple of genes which are related to regulation and which are related to assembly, storage, transfer of the cobalt atom and the coronoid. And this gene cluster is highly conserved among different ethyl genes. So if you now take methyl transferase one as a query and check how many of these methyl transferases are actually present in different CETA genes, you will end up with a surprise. And the surprise is shown here for our pet CETA bacteria in woody eye. In yellow is MT1, and you see there's many MT1 enzymes, genes that have no MT2 or COP counterpart. There's a number of solo MT1 enzymes encoded in the genome, meaning that there's a number of MT1 enzymes that share common MT2 COP protein systems. And this means there's a treasure for hidden growth substrates. Only a few substrates have been identified for these different methyl transferases. So what could be the substrates of these methyl transferases? So far I discussed methanol. A possible substrate is methyl sulfide, and this has been shown by Theo Hansen's group a long time ago that methyl sulfides can be used as methyl group donor. And methyl groups, for example, coming from glycine beta-ine, our pet CETA bacteria woody eye grows on glycine beta-ine. And interestingly enough, it only takes this part of the glycine beta-ine actually missing over here, sorry. But it actually only takes one methyl group and not the second and not the third. So it demethylates glycine beta-ine to demethylglycine. And there's more of these N-methyl groups found in nature. And the biggest group that donates methyl groups from the CETA genes are these aromatic compounds that derive from lignin and from pectin degradation as shown years ago by Bernard Schink and the Cycloid. And two representatives are shown here, three metoxybenzoate, which has metoxygroups, and ferrolate, which has a metoxygroup here. So the very high number of different empty one enzymes in the CETA bacteria woody eye implies that methyl groups are most likely the most preferred substrate for at least this CETA gene in nature. So how are these methyl groups now converted? I discussed what Jungler passed, and I discussed with you the reduction of CO2. Of course, the methyl group is highly reduced and to convert the methyl group, you have to generate electrons first. So the methyl group coming from methyl X, and it's very important to note, methyl X can be methyl hydroxyl. So methanol, methyl sulfide, methyl bound to N or metoxygroups. That doesn't matter at all. So the product in this pathway is at the end methyl tetahydrofolic acid. Methyl tetahydrofolic acid is now oxidized by the same pathway that I described before in the reductive direction. Here it goes in the oxidative direction, generating hydrogen, NADH, and NADH, and these electrons now have to be disposed. And they are disposed by reducing CO2 to CO in the second reaction. So this is a reduction reaction. And here we know in any CETA gene analyzed, the reductant is always the same. It's always reduced ferredoxin. So now we need a system to convert electrons from hydrogen and NAD to ferredoxin. And this is made again by the redox balancing system. And you see here in this example, now the electron transport chain goes backwards. It's no longer an ATP synthesizer system. Now it is an ATP utilizing system. ATP is hydrolyzed, generating a potential, and this potential drives the energetic uphill transport of electrons from NAD to ferredoxin. And now in the last step, when the electrons have been balanced, the carbon monoxide and the methyl group are condensed in module number four, the condensation module to acetyl-CoA. And this has been worked out by our group for acetyl-bacterium woodyi and another few other CETA genes, and you can check out the review. We have all these figures for all the different CETA genes in that review. And at the end, you can of course calculate the amount of ATP produced. And you see here for woodyi, it's 0.83 ATP per mole of an acetate compared to 0.3 when they grow on H2CO2. So methanol is energetically clearly preferred over H2 and CO2. And now I would like to play around with you a little bit. I've discussed the energetics, the mortuids, and now I will show you that methanol metabolism is highly fascinating. That's one of the most fascinating metabolism that the CETA genes have because you can play Lego with the oxidation module, with the reduction module. You can combine and delete and create new passwords. First, if you only take the oxidation module and then produce from all the electrons that are generated, any hydrogen, then the hydrogen is released in the ecosystem. And this is thermodynamically unfavorable and only works if the hydrogen concentration is rather low. And this can be achieved by co-cultivating the CETA genes on methanol with a hydrogen-consuming partner, for example, in methanogen, like methanospiridium hungatei, as shown by Kord-Rubisch and Olivier, but also others again with methanol shown by Theo Hansen and others. So this is known for a long time that these bugs can grow non-acetogenically. They do not produce acetate anymore, but they produce hydrogen and the hydrogen is used by the partner. So another interesting thing is to exchange the reduction module. So you take the oxidation module and now we no longer reduce CO2. So these cells are no longer CO2 reducing, but they can reduce, for example, this stuff here, which is called cafeate, that also comes from lignin, from pectin degradation and lignin degradation. And what you see here is it has a double bond. So if you close both eyes and open one slowly, slowly, slowly, it looks a little bit like fumarate. And what these cells do is they reduce this double bond to then hydrocaffeate, and that's all they can do. They cannot eat the stuff, they can just reduce it and take cafeate as an electron-ceptor. Therefore, we and others, Bach and Fennig were the first to describe this, and we discovered the type of respiration mechanism. So this is called cafeate respiration. And now it goes further. These bugs can grow mixotrophically. And now we can exchange the entire oxidation module. So you see there's no methanol oxidation anymore. And the electrons we can provide, for example, by hydrogen, by fructose, by diodes, by any other compound. And these substances are oxidized and then the electrons are used to reduce CO2 and then acetate is formed. So here no oxidation module anymore, no methanol oxidation anymore. And you can go again one step further and delete the entire oxidation as well as reduction module. And then you have a situation like this, methanol and carbon monoxide. Actually, these bugs perform acetogenesis from methanol and carbon monoxide, and you see there is no longer any redox reaction involved. There's no electron transport chain, but there's a very high ATP yield, this ATP by substrate level phosphorylation here, while acetate is formed. So this is the fascinating aspect on the physiology of acetogens. You can play around with different modules and you can create, let's say, new pathways by creating new modules. And this is what makes acetogens so interesting for biotechnology. And as you've heard in the introduction, like with a ThunderMare, these bugs are already used in industry. Professor Islam will talk about that more in the next talk. And they are used to use hydrogen CO2 and carbon monoxide syngas to produce lactate, acetate, bioplastics at the beginning. I suppose there's one publication or three hydroxypropionic acid. The problem in industry when you grow these cells on these gases is gas fermentation. The low solubility of these gases and very bad mass transfer into the system. This could be improved by using methanol or formate. And actually methanol and formate can be produced easily by chemical means from, for example, synthesis gas. So methanol and formate as well are very promising alternatives for the industrial usage of acetogens while avoiding gas fermentation. I discussed the upper part. The upper part is essential to know if you want to do biotechnology. I told you that the ATP yield has a maximum of 1.7 and if you use methanol only, it has only 0.8 ATP per mole of methanol. So if you want to produce a compound like isoprene, which requires 6 ATP, there's no way to get isoprene as a pure compound at the end of the day. So for metabolic engineering, it is very important to know the biochemistry of these production pathways and to know exactly the electron carriers involved and the ATP demand. And this is what we have outlined in our review for different acetogens. For example, here's urobekthium limosum, but we also have acetobacterium woodyi, we have plusculem dalii and others in our review. And then we have methanol as a substrate and then we outline the biosynthesis of different biotechnological interesting compounds from methanol. And just as an example, here is butyrate butanol formation from methanol by eubacterium. And I'm not going into detail because there's not enough time to do that. Check this out in our review. Florian Krem did wonderful figures on all these metabolic schemes, easy to follow, easy to calculate. And when you calculate this, you end up with, yes, butyrate formation from methanol should be possible on paper and then you actually do the experiment. This was done by my PhD student, Dennis Litti, just published a couple of weeks ago, Microbial Biotechnology, eubacterium limosum, growth on methanol that has been described before. And it produces indeed, produces butyrate from methanol. So the calculations that we give you in our review will actually guide you to products that are possible and will warn you for the production of products that are not possible. So at the end, I would like to finish by saying that especially to the young people in the audience, the most fascinating organisms you can actually work on are acetogens. They have a fascinating physiology. They can use different tricks that other anaerobes cannot do. You can play Lego with different metabolic modules and by doing so, you end up with promising production platforms for sustainable biotechnology. At the end, the hope is to have biotechnology that is no longer based on fossil fuels but can do the same with carbon dioxide. And Professor Islam in the next talk, he will tell you how acetogens can be metabolically engineered and what the molecular toolboxes are for metabolic engineering in acetogens. With that, I would like to thank the coworkers that I mentioned along the talk. And you've seen the paper that I was citing from Alexander Katziff that appeared also this year where you have many of these energetic calculations. We have a fruitful collaboration with the Göttingen Genomic Center of Daniel and Anja Boerlein and the funding of our project is by the European Research Council. With that, I thank you very much for your interest and I'm giving back to you. Great. Thank you very much for a very nice overview of this fascinating group of organisms. Please remember if you have questions, type them in the question window and we can continue with our next speaker. Asham, are you ready? Thank you for the invitation. Well, it's been a privilege to be able to allow the publication of our review in this journal and today I was invited to talk about this review paper. But I was kind of so essentially, this is the review paper that he published in this sort of a thematic issue. And then with Volker and his PhD student, we sort of tried to manage, talk about the genetic and metabolic engineering challenges of a serogenic chastry. I would like to sort of try to go back and provide an overview of my previous experience with the serogens to give you a broader sort of overview of why I think the or how I'm getting interested about the serogens. So this is the outline of my today's presentation, as you can see. So first I'm going to talk about my quintess with the serogens, sort of a personal view and how I got involved with whether it's a problematic thing or not. I'm not quite sure, but let's see how that goes. And then the next thing I'm going to talk about the genetic and metabolic engineering challenges or the tools that are available for serogens and the tools that needs to be developed. And also, finally, I'll show you some of the preliminary results from my PhD students work, who is probably the audience to them. Okay, so during my PhD, I sort of got interested about this particular types of organism who are very important for the viral mediation of toxic chlorinated compounds. So the toxic chlorinated compounds such as trichloroethane, tetrachloroethane and vinyl chloride, these are known carcinogens and these are widely used industrial decreasing agents. And when they're thrown out in the environment, they sort of ended up in the subsurface ground motor contaminants and they remain there for a very long time. So if you sort of leave a glass of chlorinated compounds on your table and come back 100 years later, it remains pretty much like the same. So it's a very stable compound. Fortunately, these microorganisms, these bacteria, the helicocoutis microtea, they can degrade these compounds and produce B9 compounds B9 ethyne. So the way they do is the following. So it's kind of like a chemical process. You can think of that you feed them this toxic chlorinated pollutants and they eat that up and produce this B9 or less toxic products. So how do they do this kind of thing? Well, the enzymes that are important in catalyzing this particular reaction is called reductive dihalogenesis. And those enzymes are essentially degrading this sort of step-by-step reductive reduction reaction and then dechlorinate those higher chlorinated compounds into ethyne. And in other cases, like higher aromatic compounds into lower aromatic compounds. And then there are other organisms which can take up this lower aromatic compounds and then convert them into B9 compounds like B9 ethyne. So now, as you can see that those organisms, the helicocoutis, although they are kind of like the main players, they cannot do this work by themselves. Usually the work in a community and a dechlorinating community involves methanogens and acetogens. And as you could see, the acetogens play a very big role in this dechlorinating community where this helicocoutis and a green geo-vector. So basically these are the dechlorinators that they sort of degrade this chlorinated compounds. So in the sort of the microbial community, we usually feed the methanol and then trichloroethane. And then essentially, you could see that the acetogens and also the methanogens. So the acetogens, they're converting methanol into hydrogen, which nicely mentioned about the conversion of methanol and their production by the acetogens. And sort of they also produce acetate. Now these acetate are also being used by the dehelicocoutis as the carbon source and then hydrogen as the energy source. So acetogens are very central to these organisms. And the other important thing is that this dehelicocoutis bacteria, when they're degrading or they're reductive dehelicinous enzymes by which they degrade these toxic compounds, those enzymes require coordinate co-factors or the cobalamin co-factors, which are obviously they cannot produce themselves and then it help from the serogens. So serogens are a very big player in this dechlorinated community. And that's how I got involved with this serogenic metabolism and their understanding. So next, when I move into my post-prol research, I sort of tasked with developing new commodity chemicals production from gases. So sort of the vision that we'll try to replace these with this kind of organisms and convert these gaseous substresses into these commodity chemicals, or probably as an added sort of side-by-side with this chemical industries, we can use these types of smaller scale fermenters probably and then use the waste gases from them and then use these to produce this kind of products. So in order to do that first, this is the workflow that I tried to follow. So essentially, we need to figure out the different types of biochemical reaction pathways first that can be designed and then we need to figure out that how we can design those pathways because once we design this, then we can implement synthetic biology or genetic engineering and ultimately metabolic engineering to produce these compounds. So this is sort of the workflow that I tried to follow. Then the strategy that I wanted to work on is obviously Morella thermoesthetica. Now, why is that? Because Morella thermoesthetica, as Valka mentioned in his presentation as well, is a fascinating organism and it's very hard to work organism as well. And it has, as you can see that the metabolic diversity is really huge and it can grow both autotrophically and hydroprophically and it can use a number of different types of substrates. So, you know, there's the possibility that you can use different types of substrates as feedstock to convert them into different types of products. And at the same time, these are thermophilic organisms, which is also industrially very important and relevant because the thermophilic nature can actually reduce the capital investment cost, like energetic cost, and also the medium contamination problem, so and so forth. So these are really useful organisms. So, in order to understand more about the Morella thermoesthetica, I developed a genus scale sort of the metabolic model in details where this is shown here. And so, when I was building this model, at that point, Valka's paper about this different understanding of the biochemistry and different, you know, the detail explanation of those biochemical pathways in the audio pathways and their energy days. Those helped me a lot and I tried to implement some of those techniques and some of those understandings when I was developing this model and analyzed all those with the help of the novel pathways that I developed. So, if you want to look at more about this paper, you can follow this and look at more details in there. Now, the pathways that I tried to develop kind of leveraging the Oudlundl pathway of serogens. And so I was trying to use a serial query as my central or starting compounds and how we can convert that into my target compound ethylene glycol. So I was trying to follow this scheme where obviously, you know, this pathway is already in the organism. And I was trying to figure out the two main hubs, glyceride and ethanol, and then design the engineered pathway to convert them into black allyldehyde and from which there's another one step pathway that can convert to ethylene glycol. So I followed this scheme and tried to figure out these different pathways, which obviously starting from glyceride and then producing black allyldehyde. And I mentioned that as a pathway one, then I developed also pathway two. And pathway three is also starting from glyceride and going back here through hydroxypyruvate producing glycol allyldehyde. And then finally, this pathway is pathway ethylene glycol pathway four that is converting glyceride to L serine via hydroxypyruvate to black allyldehyde. And then the next hub was our ethanol. So for ethanol, we have identified three pathways. Then of these three pathways, I mentioned them as five, six, seven. And after identifying these pathways, then the next step was I was inserting those pathways into the Morella metabolic model and also this Clostridium lundale metabolic model. This metabolic model I was trying to identify to sort of get a sense of which chassis can give better yield, molar yield in terms of the pathways. So I was analyzing all those design pathways, the novel design pathways as well as the pathways that has been published and implemented in E. coli. So I was trying to sort of take those pathways and insert them into Morella as well as in Clostridium lundale. And identified the more yield. And as you could see from this figure that for the molar yield on pathways on the CO carbon monoxide, the Morella thermo city is showing a better results. Then similarly, I was trying to analyze their molar yield on CO2 and hydrogen. And that also shows that somewhat similar Morella is mostly the better choice compared to the Clostridium lundale. So that sort of interested me when I started my independent position here at Loughborough University to sort of invest time and energy to engineer this pathways into Morella. And then there comes my PhD student Barbara Borgett who is a really very industrious and hardworking PhD student. And I was looking for someone who can actually sort of hammering down these ideas and inserting these pathways into Morella. However, when we start working with Morella, we sort of hit the huge wall, sort of a brick wall and we are trying to figure out how can what technique or what should be the main bottlenecks and how can we approach those different types of bottlenecks. So we needed to sort of figure out a systematic way and we was kind of thinking, well, probably we should address these challenges first and then probably addressing these challenges step by step. We'll be able to develop a genetic system that could be ultimately be used for engineering those pathways. So we started from the very preliminary stage and that sort of, you know, encouraged or inspired us to come up with this review papers idea. And there we sort of discussed what we sort of faced during this journey of engineering Morella thermostatica basically. So we sort of discussed how to overcome these different physical and biochemical barriers in order to genetically engineer Morella. So first is DNA transfer into an acetylgenic host. Obviously, acetylgenic are all gram positive organisms. So their cell layer is very thick. So that's a big challenge. And then for the, in terms of the Morella, they are also, you know, synthetic organisms. So thermal stability of exogenous enzymes is another important issue. And then also there are, you know, when you started working with Morella, still, there is no genetic system that is available through which you can use this replicating plasmid. So the gram positive replicating plasmid is also a very big bottleneck. And it's also important because the replicating plasmid can be used for CRISPR-Cas applications as well. Right. So then the other things we also discussed about the optimization of different transformation protocol. Then we discussed about the methods for genetic manipulations. We sort of divided them into manipulating gene expression as well as the methods that are involved or used for modifying post genome. So in those two categories. And you can have a detailed look at those discussion in the review paper. So these are the sort of the figures that we presented in the reviews and said that, well, you know, what are the main barriers? As I just mentioned, alluded to you that the gram positive barrier and the thermophilic requirements as well as the, you know, the requirement of a replicating plasmid. And another very important thing that we discussed is this plasmid methylation, which is to evade or sort of, you know, do the restriction modification systems of these organisms, which is really a big problem, you know, in order to engineer the serogens. And then obviously we need to, we discussed the different transformation, the different parameters that I need to be, you know, that are required to be optimized in order to get a good transformation efficiency. So then, again, here we discussed different genetic manipulation tools that could be used for, you know, changing or manipulating gene expression levels. So we discussed the promoters and RNA based regulations and also discussed about the ribosome binding sites. How can you modify those and what are the tools that have been applied and that are available in the review paper. So then finally, we also discussed about the, how can, you know, what are the tools that could be used to achieve the genetic modification and some of the newer tools that, for example, obviously the CRISPR-Cas is really widely available for different organisms. However, for serogens in terms of, in particular, morella thermoesthetica, it is not yet developed. And then we sort of discussed what are the challenges associated in developing such tools for thermophilic organisms and what are the useful or sort of successful applications in terms of application of CRISPR-Cas9 in morella. So after discussing those, we also discussed about these different metabolic engineering efforts in serogens and then how the different types of metabolic engineering techniques has been implemented so far and what are the achievements in that area. So you can have a good look at in the paper that we also provided this kind of tables showing that how and what types of grand positive replicas has been used and engineered in serogens, in different serogens. And we also discussed, you know, the genetic tools that are available for the serogens. We provided details of the different types of tools that have been implemented in serogens as well as, you know, that could be potentially implemented. And finally, we also showed that the metabolic engineering studies that have been implemented in different serogens. For example, as you could see here that the ethanol production has been implemented in morella thermoesthetica and also electric production in morella thermoesthetica. So now, finally, I'd like to share a few slides for the morella thermoesthetica shuttle vector that we tried to develop in my lab and then on the Barbara invested quite a huge amount of time. So we sort of tried to engineer gram positive replicas from thermotourga petrofida and then also inserted thermostable kinomics and resistance genes. And this is sort of the figure that shows the PMTL KTP shuttle vector that we developed. So that includes the thermotourga petrofida, this gram positive replica over here. And then this is the morella thermoesthetica promoter. This is the thermostable kinomics and resistance gene. And there's a gram negative replica that is also provided here so that, you know, you can insert both the methyl transferase genes as well as your gene of interest in the same plasmid so that you can first methylate using an E. coli methylation stream and then insert this methylated plasmid into the morella chassis. Okay, so these are the sort of, we use that particular PMTL vector to express both the morella thermoesthetica native ALDH gene and also clostridium autoethanogenum adh gene. And these genes, we express those because morella thermoesthetica cannot produce natively ethanol. We're just trying to see if the promoters that we have developed is actually functional and we have seen ethanol production. And interestingly, we have seen that when the culture is static, it produced less ethanol compared to the agitated culture, which probably kind of shows that the gas diffusion is really more fission in the agitated culture or could be another thing. So finally, just to summarize that as Volker mentioned nicely is really the sedogens are very important chassis for the biotechnological applications and more efficient genetic tools are required in order to engineer them. And obviously, you know, for the metabolic engineering applications or for more industrial applications of feed, definitely there is no alternatives to get better genetic tools. So we sort of started our journey towards that direction by creating a replicating plasmid enamel genetic system for the morella, but more optimization of the developed system as required. And then finally, I just like to acknowledge the funding that I've received so far. And also, you know, my collaborator, Dr. Nigel Minton, the University of Nottingham, who provided a really good support for this project here. Thank you. Great. Thank you very much. And thank you very much Volker. This was a great experience, actually. So for the people who are listening, both articles are open access, as far as I can see. So you should be able to enjoy yourself by reading this in detail and finding your way through it. Be carefully edited so that you should be comprehensible to all non specialists like myself and enjoy this thing. So there's quite a number of questions. Some of them are very detailed. I maybe as a chairman, I would like to take up with a very general question. Like, I mean, either a son or a balker can can reply to this depending on what they think. So is there a realistic industry interest in this biotechnology? If not, what does it take that this principle takes off at a wider scale? I mean, we all hope to go through a bio based economy and biotechnology that replaces petroleum, right? So is this already being implemented or what will it take? Yes, there is industrial interest and not only interest. For example, there's the, I would say the world leading company Lanzatec has a couple of demonstration plants and is now building production plants in China and one in Belgium and in Ghent. Of course, at the moment, we cannot compete with ethanol produced from by yeast from sugars, let's say, but this is a matter of time and price and political willingness. So we are facing one of the greatest challenges in human history, the global warming. And if you want to combat global warming, we have to reduce CO2 emissions and we have to find a way to reduce the CO2 concentration. And this is one of the ways. So by, let's say, political willingness to to enforce that, if you, for example, increase the prices for carbon dioxide, then this process comes compatible and doable. And apart from Lanzatec, there are companies actually working on it. And as you've heard in the in the second talk, the Mullika toolbox is steadily increasing. So metabolic engineering is possible. And Arzan has a couple of examples in his review where, where it is shown that value added compounds can be produced. So yes, there are companies interested for my feeling there could be more companies interested. But that is, I would say, a matter of time. So how much is this limited by, you know, the fact that you have that you're using natural strains and genetically modified strains would make a real killer advantage? Or is it just slight optimizations? Or is it a reluctancy to use genetically modified strains in industry? What is the barrier there for your progress? So I would say, I don't think there's a sort of, you know, huge sort of, you know, problem in terms of using genetically modified organisms, because these are mainly used for biochemical productions or biologics productions. I think the main barrier here is to get an optimized cell factory at an optimized cell factory that can compete with, you know, in terms of the productivity or in terms of the tighter, because when you try to place your case and take your case to an industrialist, all they try to look at pretty much like what is the, you know, the tighter, whether it's the viable industrially, and if I'm making a huge plant, would I be able to, you know, get a break even or something, those kind of something like that. Now, for that point of view, I think the upstream engineering challenges is really huge, because if we can have a really good optimized cell factory through developing good genetic tools, obviously that can increase the productivity and tighter, which can ultimately sort of, you know, translate into other sort of engineering challenges, sort of downstream challenges, but those downstream challenges will be less complicated if we can sort of enhance our efforts in the upstream development. But going back to that question, the Volcker nicely answered that, you know, Lanzatec is really showing the way of going forward towards this direction. However, I think if you just simply think about, for example, I was working at, during my postdoc, I was working with the Saudi Aramco basic industries corporation, right, the Sabi. So they're kind of like sitting on oil. And the question is, why do they're interested about this green chemicals and this kind of, you know, technologies. So the reason is, everyone is trying to look at from different angles, whether these technologies are viable, whether there could be an opportunity out there. Now, all these things are really sort of interesting, because if you're thinking about, can we compete with these bulk industries? Well, if you think about that, we cannot compete directly. However, if you think about from the selectivity point of view, selectivity of catalyst, selectivity of biochemical systems, then definitely there's a huge opportunity for this system and definitely where the future is towards this direction. Right. So one of the questions in the forum is, for example, it's a question from Avantika Ghosh, who asks about the low growth rates of acetogens. So how can you overcome the low growth rates of acetogens for industrial purposes? Is this a real issue? This is a real issue. Of course, this is a real issue. However, what we could try to do, we are, you know, obviously trying to achieve different metabolic engineering techniques by providing different types of, you know, substrates and a different changing the media composition and this and that. However, now there are some inherent limitations, some thermodynamic limitations, which you cannot overcome, definitely. But if we are trying to implement sort of the different strategy, for example, one strategy is the mixotrophy. If we can implement both mixotrophy, how can we sort of try to use both glucose substrates as well as the gaseous substrates, combine them together, activating both the autotrophic and heterotrophic metabolism, combine them and enhance their growth rate? Well, these techniques have been sort of explored. And also, as I said, it's working with anaerobic patients. And that's one of the reasons why at the beginning of my talk, I sort of tried to elude towards this point, whether it is a good choice or bad choice, whether working with Morella, you know, especially if you're a new faculty, thinking about your tenure and other stuff. But I'm really fascinated about this organism because this is sort of the types of organisms because of their embedded ureodontal pathway, the pseudo-fixing pathway, which is the thermodynamically the most efficient non-photosynthetic pathway, carbon-fixing pathway. So, going back to the question, can it be achieved to, you know, the enhanced growth rate? Well, it can be achieved that is sort of bounded by the thermodynamic limit. If we try to compete with E. coli or is, well, definitely we are not going there. However, it has other advantages. Sure. So, to get to this industrial side of things in terms of the product outputs, there's also several questions about that. First, maybe a question of Steven Daniel, who asks to Volcker, like, okay, if you're going to produce something, like, what's the maximum concentration of methanol that can be tolerated by acidity rectivoonie and other acidity gens? That is a question, can't be answered that easily. So, there is a nice study on the methanol adaptation of growth of acetogens to methanol so we can adapt it over time and then they would tolerate more. But the tolerance is not, one has to say, not very high. In the unadapted state, for example, in acetobacteria woodyi, we are talking about a couple of hundred millimolar. So, not molar concentrations. And the e-adaptation system, you would not get very high. So, for example, if you compare this to ethanol, at the beginning, people thought that bacterial ethanol producer maybe may have a higher tolerance compared to yeast, but this turned out not to be true. There's an intrinsic property and this intrinsic property is determined by the cytoplasmic membrane. So, at a certain point, and all these cytoplasmic membranes are in principle organized the same way, the cytoplasmic membrane collapses and then it's the end of the story. Okay. And there's a question also related to production from Anke Neumann who asked, like, is ethanol really the preferred product and what about acetate as an intermediate? And if you would use a second stage fermentation, can you produce other by high value products? Or is there any thermodynamic feasibility or so or limitation to do this? So, the first part is ethanol is the preferred product. No, it is not. If acetates want to produce ethanol, they have to have certain enzymes called AOR and what they do usually is they do produce acetate first. Thereby, they produce the ATP in the what Jungler passed me that I described. And then they use reduced pherodoxin to reduce acetate back to ethanol. Ethanol producing the citrogens do have the AOR system. The citrobacterium woolly eye does not and it cannot produce ethanol, only acetate. So one way of producing of making ethanol producers would be to put in the AOR plus in aldehyde diadrogenase. So ethanol is not the preferred product because cells lose normally if you would go from acetyl-CoA to acetate aldehyde and then to ethanol cells would lose one ATP. Only if they can use the AOR, then they have the same amount of ATP let's say left, but then they need additional reductant. The second part of the question was if actually you would use acetate as an intermediate and then use a second stage fermentation to produce high value products. Yes, that is a very interesting idea. And companies are interested in the group of Dr. Leumann I guess has research on it and papers on it. So what you could imagine is a two-step process where for example the acetogen produces acetate first and then you feed this to a yeast. Yeast is pH tolerant, grows at low pHs and eats the acetate and produces some goodies out of it. If you want to do the same with let's say just one group of with the acetogen that would not work. There are a couple of acetogens that could oxidize acetate but very poorly. What you could do is experiments for example that Lars Anzhen is doing very elegantly also chain elongation. They produce acetate, you feed acetate from the outside and then they would produce C4, C5, C6. So that that could be done by feeding acetate to just previous. So that exact same method that Volcker you described we sort of implemented when I was in Gregus de Farnanpolis's group. We sort of produced, used morella to produce acetate and then feed that acetate into urea lipolytica to produce the fats in large amount. And we tried to also we showed how you can sort of manage or we call this a doping technique, sort of manage the substrate, coordinate the substrate and manage their metabolism and enhance their growth rate. And in that case we pretty much used wild type acetogens, wild type morella thermo-cytica, wild type urovia. So just to manage the substrate concentration by feeding, you know, how to change the feed concentration by managing those, we were able to show a really nicely that you can have a good high titer and good yield. So that paper we published in Nature Metabolism, it was in last year. So it's a nice example showing how you can use acetogens acetate into as an intermediate and then produce high value products in the second fermentation. There's a comment also here by Michelle Bradley who says that who was a CTO for an industry in the UK. And she's mentioned that one of the biggest problems in implementation here is the scale of costs for the fermentations and the gas fermentation. Because it requires so much basic financial investments. So yeah, she says great benefits but high risk of failure because of investment challenges. Yeah, that's a very useful comment I think. Maybe you could think about switching to methanol. So it's sort of easy to produce methanol from syngas and then you don't have all these technical problems anymore. So you can use the same box to convert methanol that comes from syngas. But this is one of the biggest factors in implementing such a system. Actually as a microbiologist, I have a question for you as well. So Falker, if you say that there's so many different possibilities, these legos that these acetogens can do. Is there any evidence that they also do job sharing in a population so that some bacteria, some cells do this and another cell does that? Or is that not possible thermodynamically? I mean, it's known for all the bacteria that there is job sharing. Not everybody is doing the same. Some single cells can do more of this and others do more of that. Is that possible for acetogens? I don't know. There's no studies on that topic. People my age, we were trained to work with pure cultures. And now the mixed cultures, this is a very hot topic. But there's very little studies at least that I know on acetogens. It's still a pure culture, but it's just individual cells doing different things. I mean, if you grow a population of bacillus, they will do different things. I know, but I guess here, I don't know anybody that has actually looked at that. And I guess for that, I'm not a geneticist, but I would guess for that you need a much better genetic toolbox. You need some sort of fluorescent markers to see differences in cells in one population. But thermodynamically, I would not see a problem that, for example, this sharing of hydrogen that could work also with cells of the same species in one population. Right. So would you be ready for some more technical questions on like metabolic pathways, thermodynamics, and so on? Yes. So there is a question from Chen Zhang, who asks, if I interpret correctly, are there cases that formates can be directly converted to energy age rather than through hydrogen? Yes. So the due to the lack of time, I couldn't describe the fascinating variety of biochemistry of acetogens. Cetogens are not a phylogenetic group. You could see this from the different names already. There's 23 described genera at the moment. And the overall principle is the same, two CO2 to one acetate, but the enzymes involved and the electron carriers involved are very different. So the enzyme that I was showing that produces hydrogen, we discovered that and we named this hydrogen-dependent CO2 reductase, the classical enzyme would be a formative hydrogenase. And cells that oxidize formate, they can have formative hydrogenases coupled to NAD, coupled to NADP, coupled to ferredoxin. They can have different cofactors. Formative hydrogenases that work in the direction of CO2 reduction cannot work with NAD because this is thermodynamically restricted, but they could work with ferredoxin or NADP. And the group of Reuf-Tauer, they also described electron bifurcating formative hydrogenases that work together in context with hydrogenases in which NAD and ferredoxin are used as electron donor. So there's a huge variety of different formative hydrogenases. The enzyme that was on my slide, the HVCR, that has been found biochemically in two species, in Cetobacterium and in thermo, anaerobacter kivui, and from the genome in let's say around 10, 15, 20 other species, but many, many acetogens have classical formative hydrogenases. All right, great. Thank you. And then there's a question to Assam from Luca Pere, who asks if the genome manipulation or engineering that you are intending or that you have done so far, does it have any stability on the strains in fermentation processes? Now, we haven't been explored that far, to be honest. We still kind of like, as I said, we are still preliminary stages of this development. And one of the problems with this, you know, the optimization of the development of the genetic tools is to make sure that, you know, the mutant that we are developing or the change that we are trying to impart into the HSE is stable. So those areas we haven't explored yet, but we are still, as you could say, that's our next step. So I cannot answer right now. That's fair enough. I'm reading the question to you. And there's a question for a Volcker from Ari Satanovski. He says, thanks for the fantastic talk. Fascinating metabolism. I have wondered for some time why apparently in nature the methiline tetrahydrophyroid pathway is exclusively converted to acetyl-CoA by a methyl tetrahydrofolate and carbon monoxide dehydrogenase, and why is there not a glycine synthase glycine reductase pathway? Is there two more dynamic reasons for this? This is a very detailed question of a knowledgeable person. Yes, so the glycine synthase glycine reductase pathway is sort of a bypass to the woodluminal pathway. And indeed, there is a recent publication from our Korean friends that demonstrated that this pathway does exist in, for example, Prostridium, if I remember correctly, Drache. Yes, it does exist. And this pathway has also been elaborated by the group of Alphons stumps in diesel-for-vibrio species and was postulated by Arembar even for some acetogens. Yes, this pathway does exist solo, but also in combination with the woodluminal pathway at least in some. The enzymes are not present for the glycine synthase or glycine reduction in, for example, acetobacterium woodyi, but some do have that. So there's a few questions that ask about acetogens in environmental conditions. So Abiet Singh is asking to either of you, how would you study acetogens in the environments like in biogas systems where they will be mixed with other bacteria? Well, that is a very general question. It's a question is on how to detect by molecular probe acetogens in a system that is sort of difficult. People have thought about it for a long time and I guess I'm not a microbiologist, I guess, but the molecular probe that people use is on the formulatetahydrofolate synthase gene enzyme. With that people, I guess, think that they can detect acetogens in environmental samples. If you want to study acetogenesis in environmental samples by classical techniques, you take a microcosm, for example, put in radioactive CO2 and see whether you find radioactive acetate. And by labeling experiment, you do find out whether this acetate is produced by the Wojongrad pathway. So you can actually detect this biochemically. And with molecular probes, I'm not sure, but I guess there are ways to do that. Right. And do they play any other roles in the environment? I mean, their numbers must be reasonably dependent on information. One of the things that I mentioned in my presentation, as you have seen probably, that acetogens, when I was working with this dechlorinating microbial community, they sort of supply the corinoids or cobalamines, because acetogens are kind of like a, they are kind of like a corinoid factory. And they can produce more than 30, 40 different types of corinoid factors. And these are really, these cobalamines are really, very important sort of, you know, metal ions. And acetogens can play a big role in this kind of, you know, dechlorinating microbial communities from my experience that, but I've noticed. So definitely these communities, we sort of studied on, we collected these communities from the contaminated sites in microbial that are contaminated with this TCC, this dechlorinating compound, or this chlorinated solvent contaminated sites. And naturally, we have seen that there are acetogens out there. So definitely, the acetogens are playing a big role. And obviously, you know, one thing that, if you think about that, sort of their origin, this kind of like a soil bacteria. So definitely, you know, if you think about their origin, then they can play a different types of roles in the microbial ecology as well. Right. Okay. Maybe last question by Frank Mengelsdorf, who asks to Volker. Thanks for the great talk introducing the Lagomodule Contact. Today, you haven't talked about the role of electron bifurcating transhydrogenesis, NUN. In some acetogens, the respective enzymes are known, and in some, the respective genes have not been found. Could you please speculate if all acetogens would need such an enzyme, or if they could make a living without enzymes such as NEN or NUN? Yes. Electron bifurcating transhydrogenesis are known in acetogens, well known in acetogens, and we have currently two classes. One class is the NFN. So what is for the general audience, Dr. Mengelsdorf is an expert in the field. For the general audience, what is a transhydrogenase? The transhydrogenase is an enzyme that interconverts NADP and NAD with reduced ferrodoxin as a mediator, let's say. The transhydrogenase in E. coli is also present, and in E. coli, the transhydrogenase uses the memory potential to drive the reduction of NADP with NADH as a reductant. So that is the reaction that is important, and it is the more important if the Rundberg pathway has an enzyme that is NADP dependent, so then you have to have a way to make NADP from NAD. And therefore, all the bugs that have NADP dependent enzymes in the Rundberg pathway have to have a transhydrogenase. And of course, these cells have to make biosynthesis. This is what I always tell my students. If you look at the Rundberg pathway, this is just a pathway to produce ATP when they grow autotrophically. But of course, they have to make biosynthesis of lipids, of DNA, of carbohydrates, of all this kind of stuff, and so therefore you need NADP. And currently, we know the NFN type transhydrogenase. And then we discovered and published also the first author on that paper, Florian Krem, published a paper on a novel type of transhydrogenase, which we called Spurumusa type transhydrogenase. So the answer to that question is yes, transhydrogenases are required for the acetogens. Every acetogen is looked at has a transhydrogenase and the transhydrogenase can be either of the NFN or STM type. I think we will stop here. This was a great experience. I thank you very much, Athan and Volker. This was extremely interesting to listen to. It's also a very lively field. I think the audience for the very lively discussion as well. It looks like a field that is really growing in importance even more. And I hope that several of your ideas will make it to industry applications so that we can work on climate and better chemistry and production and so on. That is all very good for our future in that sense.