 and kind of a tight schedule today, only about 30 minutes for each one, so I'm gonna ask the speakers to kind of stay on time with that, so we've got four speakers and a career panel that's been organized by the students and then we'll be having a lunch afterwards. Our first speaker is Professor Jun Ogawa from Kyoto University in Japan and he's the head of the laboratory of fermentation, physiology, and applied microbiology. You know, in the US, most of the prestigious universities are private universities, but it's actually reverse in many of the Asian countries, for example, so the public universities like Kyoto and Tokyo University are the most prestigious ones there and he is a well-renowned, highly-renowned professor and he has won a number of awards just recently and he's won the Ching Hu Biotechnology Award. He's a fellow of the American Oil Chemistry Society and the Chevrolet Medal by the French Association for the Study of Lipids. And I have one more comment before I introduce him and that is, as you listen to Professor Ogawa's talk, to look for the Eastern approach to science. Something I had never really considered but it's something that I've discussed with him several times, what is the Eastern approach to science? I mean, the contrast is with the Western approach to science. I think the Eastern approach is more holistic and interconnected in contrast to the more analytical and separation of phenomena that we typically do in the, in Western science. So if you look at even the name of his laboratory, fermentation physiology. I think of a fermenter as a bunch of isolated bacteria but he's thinking about it as an organism and organism has physiology that changes a completely different view of what a fermenter, what's going on in a fermenter. So the title of this talk is from function to genes, enzymes and communities, creating novel biotechnology tools. Welcome Professor Ogawa. So, thank you Chairman for your kind introduction and thank you for Professor Romas Kazuruskas and other organizing member from Minnesota University and also Dr. Shotaro Yamaguchi and other organizing members from Amano Enzyme Company to invite me here and giving me the great opportunity to talk our current activity related to something, enzyme and function of microorganisms. Today I'd like to talk about this title from function to genes, enzymes and communities, creating novel biotechnical tools. So some are strange title but I make a thought, finally you all understand this concept. Of course, biotechnology is a key tool for keeping healthy us. Biotechnology for example, bioprocess using biocatalyst is important for future society. For example, replacing chemical process by bioprocess using biocatalysts can reduce the energy consumption and also reduce the generation of waste and carbon dioxide. Future sustainable and eco-friendly society requires much more diverse biotechnology tools, of course. So how to expand biotechnology tools, this is our aim. I think microorganisms are important sources of novel biotechnology tools such as biocatalyst. So as Romans mentioned, I'm a researcher in fermentation physiology and applied microbiology, so mainly focused on these. This is our strategy for the screening of biotechnology tools in microorganisms. So we usually start from collection stage that is analysis of natural environment, next microbioconsortiae, and isolated microorganisms and make lively and moved to the analysis and development stage of metabolism, genomes, next enzyme, gene, and finally use these results to develop a process for useful compounds production, et cetera. Modern omics technology supports these strategies and together with synthetic biology, we now make possible a little to synthesize artificial microorganisms with hopeful function. What is next? Maybe design and development of microbial functions expressed by microbioconsortiae. In these, our strategies, we usually start from analysis of function, especially metabolism. Then resulted in obtaining novel things, for example, genes, enzymes with novel function, and also functional communities with design members. This is the title from the function to genes, enzymes, and communities creating novel biotechnology tools. So I would like to introduce our current activity, regarding to screening in microbial metabolism, and the finding of genes and enzymes with novel functions and constructing functional microbial consortia with design members. Also, I'd like to mention about utility of omics technology. The first example is amino acid metabolism, and finding of amino acid hydroxylase, together with mention on the genomics for diversification of biocatalysts. What is the target function in this case? The function is for hydroxylase in production, and target, analysis target is L-isolucine metabolism in microorganisms. What is for hydroxylase? For hydroxylase in HRL, is a potential drug candidate for the treatment of diabetic and obesity. It is contained in fenugreek seeds. This is hard for curry, but the amount is too low. So, enzymatic process are promising for HRL synthesis that needs high stereo and functional group selectivity. We analyzed L-isolucine metabolism in microorganisms. This used actually long, long days, for example, in this case, seven years. But finally, we found a very unique L-isolucine metabolism like this. This is the best way for producing AMKP. This is an antibiotic, but the first reaction is very hopeful to hydroxylate L-isolucine, radio and stereo-specifically. So, we analyzed the enzyme catalyzing the first reaction, and revealed that this is an FV2 alpha-ketoglutarate dioxinase, novel type one. This enzyme IDO catalyzes stereo and radio-selectivity hydroxylation of L-isolucine and produced 2S3R4S HRL-specifically. Very unique enzyme and useful enzyme. We established a practical process for HRL production by modifying the metabolism of E. coli because IDO, this kind of dioxinase needs alpha-ketoglutarate as a co-substrate, and we provide this co-substrate by a modified CCS cycle which likes alpha-ketoglutarate, the hydrogenase, and coupled with this IDO reaction which generates succinate from alpha-ketoglutarate coupling with our hopeful reaction. So, this system can provide this co-substrate from glucose, and we established a very, very practical process with huge amount with good yield and good selectivity. So, the enzyme is very, very useful. So, this is a finding of such kind, new kinds of dioxinase, and we now know the zinc sequence. So, based on this zinc sequence, we searched the homologous enzymes in genomic information. So now, we have a library of homologous enzymes acting on aliphatic amino acids like this, and applied these enzymes to make a chemical library of related hydroxylated amino acids, chiral hydroxylated amino acids which start from various amino acids as a substrate. So, not only aliphatic amino acids, we also found unique enzyme act on cyclic amino acids like this. This enzyme has a bit different sequence, and we also try to establish library of this kind of enzymes. So, using these newly found dioxinase, we now can make many kinds of useful hydroxyl amino acids. And this is very, very unique cyclic hydroxylated amino acids. So, this chemical library now commercialized to find out some new target. For example, chiral blocks for pharmaceutical industry, something like that. So, this is an example of finding new enzymes in microbial metabolism with the support of genomics for diversification of biocatast. So, next example is alpha-aphradisobstitutaminosid hydroxylase. I talk with the example using proteomics for the enzyme system identification. In this case, our target function is D-mesyl alpha-methyl disaline production, which is the chiral intermediate for various kinds of peptide and amino acid-like drugs. The analysis target, of course, the alpha-aminoisobuturate metabolism. This also needs very long way to find this metabolism, but luckily we finally found this unique AIB metabolism in lotococcus species. This is a novel metabolic pathway of AIB, but the first enzyme, our target reaction, the purification of the enzyme is very much difficult. It is not easy and unsuccessful. The activity was lost after cell disruption, so how to identify the enzyme? Then we asked the proteomic analysis for enzyme identification. Luckily, this is inducible enzyme, so the addition of AIB to culture medium enhanced the production of enzymes, so we can make comparative proteomic analysis. This is a volcano plot, and we picked up these very unique expression enzymes and find out these gene cluster. This AIB gene cluster has something hydroxylating or oxygenating groups. This is pherodoxyne, and this is pherodoxyne reductase, but this monoxyneous part is annotated as amide hydrolase. Is it monoxyneous? Anyway, we expressed these enzymes in lotococcus aceropolis. Yes, actually the gene product expressed in this strain surely produced, certainly produced, alpha D-metallicene from amide butylate, and with good stereoselectivity. So the gene AIB H1 and H2, the amide hydrolase homologous gene product surely act as monoxyneous with this electron transport system. Now we're analyzing this totally new monoxyneous by crystallographic method, and now we know the monoxyneous is a novel type of non-hem di-iron monoxyneous. We now analyzing the homologous gene using the genomic information and evaluating the function of these homologous genes. Anyway, omics technology make possible to identify multi-component enzyme systems which is very difficult to purify. So I would like to introduce the examples of such approach in gut microbiome metabolism. The first example is glucosionate metabolism. Glucosionate cut into isochocyanate. Isochocyanate is very bi-active compound showing anti-inflammatory and anti-oxidation activity, et cetera. The human cannot catalyze this reaction and only gut microbes can do it. However, what kind of enzyme and what kind of gene involved in this process is not reviewed because the purification is very much difficult. Yeah, again, after this eruption, we cannot identify the activity. So then, again, apply the proteomic analysis like this. The initial stage of cultivation, the activity is not expressed, but after the glucose consumption, the strain start to degrade these compounds and the enzyme activity comes out. So comparative proteomic analysis of this stage and this stage pick up the candidate's gene like this. So again, we express this candidate's gene in E. coli. The E. coli surely showed the activity to decompose glucosionate, in this case, senigline, and produced corresponding isochocyanate. This is the result we obtained. The senigline transformed to its phosphate compounds via PTSD sugar transporter with this phosphate chain, transforming chain. And the phosphorylated substrate then creeped into these compounds inside the cell. Anyway, so this is very compressed enzyme system. So, of course, that cell disruption totally lost this activity and very difficult to find out, but the new technology, for example, comparative proteomics can possible to identify such complex system. So, analysis of homogenous gene distribution in gut microbes in relation with human health now ongoing, and we also use this result to produce supplement for human health now. Next example is very similar and also is analysis of gut microbial metabolism. Elastic acid metallurgy to urethane. Urethane is like this derived from elastic tannins via elastic acid. We identify, this is very unique dehydration reaction under anaerobic condition. We identify the electron transfer system with hydrogen molecule as an electron donor and also identify the initial enzyme catalyzing this elastic acid to urethane M5 as lactose, hydrolytic enzyme. But the successive reactions the hydroxylation is a very complex system and enzyme activity again lost by cell disruption. Then we again apply the comparative proteomic analysis like this and luckily find out the target gene cluster and expressed in E. coli and identified this very complex enzyme systems which catalyze a unique, very unique dehydration reaction like this. So analysis of homogasic gene again, gene distribution again carried out in gut microbial organisms in relation with human health and also the result applied for the development of such kind of supplement. So now with the two example, I introduced that gut microorganisms are good source for novel biotechnology tools, especially for food purposes. We are now analyzing gut microbial metabolism of food derived compounds to find new metabolism and enzymes. So I'd like to move to next story about gut microbial fatty acid metabolism that is pure for saturation metabolism. And after this finding together with a metabolomic analysis for we established a chemical libraries of metabolites to develop many new health supporting compounds. This is our result for unsaturated fatty acid saturation metabolism find in gut lactic acid bacteria. It is very complicated reaction. Apparently the reaction is saturation of this delta 12 double bond. For example, in the case of linoleic acid, the metabolism generated olic acid, so saturation reaction. But the reaction is very complex. Metabolism is very complex involving one, two, three, four enzymes catalyzing one, two, three, four, five, six reactions. The first reaction is hydration at delta nine double bond and generate this hydroxy fatty acid. Then the hydrogenating into oxo and the migration of double bond that's in the enone type with metabolites. And this enone type is saturated like this. And the reverse reactions of the hydrogenation and hydrologation finally accomplish this saturation reaction. These metabolism in both very unique newly found metabolites. So such finding or much component enzyme system is useful to design cascade reaction. So using designed cascade reactions, we now established the chemical libraries of these unique metabolites. Not only from linoleic acid, but also from alpha-linoleic, gamma-linoleic, and so on. Using these chemical library as a standard for the analysis, we established a novel dipytonic platform for gut microbiology fatty acid metabolites like this. And analyzed the intense now bacterial lipid metabolites to evaluate their health supporting activity. So together with scientists in nutrition and pharmaceutical and so on, we reported various kinds of bio-activities of gut microbiology fatty acid metabolites. For example, intense now and this bow epsilon barrier protection, enhancing gut hormone secretion and anti-diabetic activity, anti-inflammatory and immune control effects, high epidemic effects, anti-oxidative effects, anti-bacterial activity and so on. I'd like to show one example about anti-diabetic activity of this linoleic acid metabolites. Hydration product, this is 10-hydrolysis-delta-triable octatacenaic acid, we call these compounds as HYA. So gut microbiology flora prevents host obesity by metabolizing linoleic acid, for example, in cooking oil, and produced this metabolite in human gut. And this product enhance the hormone secretion and affect the homeostasis of human being. Then act as anti-obesity compounds. Not only this effect, these compounds act as an effective dual controller for both for micro flora. The change is something interesting. The addition of this HYA enhance the growth of similar microorganisms which can have the same activity. So amplify the amount of these metabolites. Anyway, this is very health-promoting activity. So now we established the industrial production process for this HYA by using lipase because this hydration occur only with free fatty acid. So vegetable oil is cut into free fatty acid by lipase and we use this excellent enzyme, lipase AY-amano-satis-SD. And together with hydration activity of lactic acid bacteria, we finally produced food-grade HYA like this and a commercialized as a food supplement. So through metabolism analysis and amplification and intermediate identification, that means the metabolite identification and evaluation of the metabolites by activities, we reviewed metabolites responsible for the health-promoting effects for gut microbiome consortia. So I'd like to move to the story of the consortia. Application of functional microbiome consortia is important for industries. Just now I introduced the use of gut microbiome flora for medical or health care. The next example is how to make functional microbiome organisms to design the consortia for environmental control, et cetera, using the effect of use of spare microbiome consortia, especially nitrification by these bacteria. This is nitrification. Important for plant growth. Organic nitrogen compounds transformed into inorganic nitrogen compounds like ammonia, nitrite, and nitrate. This is carried out by nitrifying bacteria in soil, but soil system is very difficult to analyze. So we transform these microbiome consortia in water system. It makes easy to analyze chemical and microbiota transition, but these AOV and NOV is very difficult to isolate and cultivate. So we established not isolation, but enrichment method by controlling the organic nutrients amount and the aeration conditions successfully. So after this enrichment, we can clearly observe the generation of ammonia, nitrite, and nitrate together with the transition of microbiome consortia. For example, ammonia generation catalyzed by this specific strain, and the nitrite, this strain, and the nitrate, this strain. So it's representative of heterotrophic bacteria and orthotrophic nitrifying bacteria acting in each ammonia, ammonification and nitrification stage are identified, then reconstructed of single microbiome consortia for nitrification like this. We selected the basilar strain as a heterotrophic and the nitrofonosomonas and the nitrobacter for nitrification. This is enriched consortia, but artificial model designed consortia act much efficiently. So this is four times faster than enriched consortia. This is also applied for denitrification. So nitrification coupled with denitrification means electron donation, and we selected biodegradable polymer as an electron donating compounds and screened several biopolymers. So this is a good example, and this is not so good, and comparative metagenomics carried out, and we selected the microbes act as good ones, and selected good biopolymer degraders and the combination with good nitrifying bacteria like this. So selected isolated strain, the combination, account for this good denitrification process. This is again the artificial consortia together with nitrification consortia. So as I mentioned, microbial ecosystem, microbial consortia is useful for human health, crop production and water treatment, and this is also available for synthesis of useful compounds. Now we are trying to coupling the baker's yeast, ATP-generating activity together with some enzymes, needs ATP for synthesizing something. So now we are going to artificial microbial consortia, application for production of useful compounds. So I would like to move to summary. This is a strategy of the development of microbial function. In future, together with modern omic technology and IT technology could expand the biotechnology tools together with the strategy I described. So the researchers presented here, researchers presented here stemmed from the screening of novel microbial function, especially metabolisms. The research presented here were expanded by the observation supported by modern omics technologies. So this research will contribute to future sustainable society through application of unique microbial function as biotechnology tools. My professor say like this, this is something like Eastern philosophy for research. Tenkouken kun shi motte tsutomete yamasu. The working of nature are healthy. We should follow the nature and work hard to learn more from nature. This is the research. So I would like to say thank you to my collaborators and funding supported research, and also like to say thank you for your kind attention. Thank you for an incredible talk. So you went quickly through the proteomics optimization of the expression. Can you elaborate a little bit more on the details there? Please. So for example, in this case, the target microbes cultivated in the medium with inducer in this case, the inducer is this substrate itself. And without inducer, the cell doesn't show activity. That means under the condition cultivated with substrate, it showed activity, but without substrate, no activity. This clearly shows the difference of protein expression. So we rise the cell and extract the protein and cut into peptide by for example, trypsin, et cetera. Of course, we should know the genome sequence of this strain. After then, we can understand what kinds of peptide or protein, peptide derived from protein expressed or not. So it needs genome information, of course, and such conditions on and off, and can compare the expression level of some genes. But to identify the much interesting enzyme, we know the another information. For example, some of it related to oxidation system. This is the clue for understanding reduction and this deductive system is nearby, but annotated to different enzyme, amide hydrolase. But we try to identify it. So this is trial and error. Luckily, we can successful to identify the enzymes. But the important is how to know the expression difference pattern and also genome information. Thank you for your presentation. I wonder if you can comment on how you keep the microbial consortia stable, the ones that you construct, if you can comment a little bit on that. Yes, it's very important. Not so stable, of course, but this is not so stable in the sense of members of microorganisms, but stable as activity. So this is very important to keep the activity. We find out the conditions, environmental conditions. In this case, important is amount of nutrients and oxycytes amount. So if we keep this, yeah, of course, slight change of consortia members, but keep the activity. So environmental factor is important.