 Good afternoon everybody. I would like to start by thanking the organizing committee for this nice opportunity to be talking here in this very nice conference. And today I would like to share with you some of our latest results on biodegradation using pseudomonas putida as a microbial chassis. So now we will move from scientists, which was the main subject of Ericus talk, to biodegradation. So I would like to start just by devoting a few minutes to what is the path of a very typical synthetic biology procedure. So what we normally do is to select biological parts and using genetic tools create new genetic devices that we plug in in a biological chassis. So in the first part of my talk I would like to pay some attention to the selection of an adequate chassis for synthetic biology. So we have heard so far that there are many types of microbial or biological chassis anyway, and we have heard nice examples of yeast, E. coli, and bacilli. Anyway, today I would like to stress some of the interesting features of environmental bacteria as microbial chassis for biodegradation. Okay, so if we take into account the metaphor of cells, living cells, like computers making more computers, one can think of a chassis like the structural scaffold in which one wants to implement genetic devices in the same way that the hardware executes the software. In that sense, what we would like to have in a nice microbial chassis is a number of interesting features, such as in the first place it has to be safe from a biological point of view, that is to say it shouldn't be a pathogen. It has to display a very robust metabolism and some of the results I'm going to talk about are closely related to this point in particular. The chassis has to be very resistant to stress either endogenous or exogenous in the bioprocess, and of course it will be very desirable that we can actually manipulate the chassis the way we want, in particular in terms of genetic engineering. And last but not least, it has to be stable. So that means that if we engineer something in this chassis, it should be maintained like that for the longest period of time possible. In this sense, in our lab we advocate the use of microbial chassis derived from bacteria that come from natural environments, and in particular we are very interested in using bacteria that comes from contaminated soils, which for us is like a treasure trove for microbial chassis. In particular we use a bacterium that was isolated from contaminated soils like 30 years ago, which is Pseudomonas putida. And the reason for which we like this bacterium so much is that it is naturally endowed with some of the properties I have been just discussing. In the first place, and very important for biotechnological applications, Pseudomonas putida is a non-pathogenic bacterium. And in particular the type strain of Pseudomonas putida, which is called KT2440, is not only non-pathogenic, but it also has been given the GRAS certificate, which means that this chassis is generally regarded as safe for biotechnology procedures. On the other hand from 2002 we have the complete genome sequence available, which means that we know all the genes encoded in this bacterium. And over the last 25 years or so it has been used for a number of applications, such as in situ bioremediation and industrial production of polyhydroxial canoids, which are biopolymers. There are several companies that are presently using Pseudomonas putida for biopolymer production. But what is very interesting as well is that since you can find this bacterium in heavily contaminated environments, it is naturally endowed with a number of pathways for degradation of very difficult to degrade compounds, such as aromatic compounds. Pseudomonas putida is able to degrade toluene, xylene, and other aromatic compounds as well. All these features speaks in favor of a very remarkable adaptability to thrive in very different and harsh environments. In other words, this bacterium has to be endowed with a very high metabolic versatility. And in particular, our main interest in the lab is to tame Pseudomonas putida to be used for a number of applications. As a first step in that endeavor, what we want is to be able to refactor the central metabolic pathways of Pseudomonas. And just at a glance, it might seem easy, but as you will see in a moment, we discover some features in the central metabolism of Pseudomonas putida that are very unique and different when compared to other bacteria that are normally used as you see in biotechnology. So of course, the ultimate goal here is to use it as a predictable biological chassis for biodegradation and biocatalysis. So if one has a look at the central metabolism of Pseudomonas putida, as I said, there are a few remarkable things to mention. So in general, if one thinks about bacterial metabolism, one will think about the use of a carbon source to convert it into biomass and to produce energy during this process and to generate reducing power during catabolism. A remarkable property of Pseudomonas putida is that it can produce a lot of NADPH. The rate of NADPH formation in Pseudomonas putida is orders of magnitude higher than in other bacteria. And this is one of the reasons for which these bacteriums is so resistant to the stress produced by solvents and to oxidative stress. And of course, this is a feature that we want to exploit for biocatalysis purposes, because NADPH is of course a very important cofactor in many biotechnological processes. If one takes a closer look at the central metabolic pathways in Pseudomonas putida, you will see more or less this mess over here. So let me wide you through the main steps in the upper catabolism of Pseudomonas putida. In the first place, when cells grow on exoses, glucose for instance, there are two possibilities for the processing of the substrate. On the first place, there is a phosphorylation pathway that starts with glucose, and of course the first step is phosphorylation to glucose-6-phosphate, but also, and that's something very typical of Pseudomonas in general, there is an oxidation pathway that oxidates glucose into gluconate or keto-gluconate before phosphorylation. So there is a branching point of exos processing as a first step in the catabolism. But the very remarkable thing about the central metabolism in this bacterium is that it operates the endardular of catabolic pathway, which is composed of just two enzymes, EDD and EDA, and it transforms 6-phosphogluconate into two trioses, glyceride through phosphate and pyruvate. On the other hand, another remarkable thing that makes Pseudomonas different from other typical bacteria, such as E. coli, for instance, on other interbacteria, is that there is an incomplete MDMA of Parmas pathway, the linear glycolysis, typical of E. coli. The reason for that is that there is an enzyme which is missing here that converts fructose-6-phosphate into fructose-1-6-phosphate. So what happens here is that there is only one enzyme which is gluconeogenic, but the enzyme that goes down here is missing. So with these features, one of the first things that we asked was, what was the central metabolism regulated in this bacterium? I will not enter in the technical details in here, but I will be happy to discuss with you this if you are interested. But what we found is that there is an important reason for which the linear glycolysis is missing, and that is that this bacterium operates a cycle for exos-processing, and that makes it very different to most of the biological chassis used in biotechnology. So in Pseudomonas' beauty, the glucose is first phosphorylated. Remember that there were two possibilities, phosphorylation or oxidation, and after it gets phosphorylated, it uses the endodular pathway to produce two trioses, but a significant part of the trioses are recycled back to exoses. So there is part of the glucose that ends up in lower catabolism after trioses formation, but part of the trioses are recycled back. So the interesting thing about this metabolic architecture is that the three main catabolic blocks described for bacteria work together because we have components of the pentosphosphate pathway, the endodular pathway, and the endomeric pathway, and that's a good reason to explain why Pseudomonas doesn't have this step here downwards. But there is a further consequence of this cycle here. As you can see, the reaction catalyzed by glucose-6-phosphate dehydrogenase produces NADPH. So in each turn of this cycle, this bacterium is able to produce one NADPH molecule. And in any case, under conditions in which the cells are growing on glucose, there is a catabolic overproduction of NADPH. But even more important, and I will not talk about that today, but it's an important feature of Pseudomonas as well, when the cells are exposed to oxidative stress, the amount of carbon that is recycled back using this metabolic architecture goes up to 6 to 10 fold, which favors NADPH formation under oxidative stress conditions. The important conclusion about this very atypical metabolic architecture here is that it enables Pseudomonas to live in environmental niches that are characterized by very stressful conditions. Because using this metabolism, it can overproduce the NADPH, it also needs to counterfeit the stressful conditions. So this is one of the reasons for which we are so in love with Pseudomonas' putida. It has very interesting characteristic to be used as a chassis. But today I would like to focus on one of the features of these species that we want to manipulate as well. Pseudomonas' putida is not able to form strong biofilms. The biofilms it forms are kind of weak, which is an stark contrast as compared to other Pseudomonas' species. For instance, Pseudomonas' aeruginosa is known to form very strong biofilms. That's not the case for Pseudomonas' putida. And there is a further thing. The ability of Pseudomonas' putida to form biofilms varies from isolate to isolate. So it's a trait that is closely related to the particular isolate you are working with. And of course, for the reasons I will discuss in a moment, that that was something that caught our attention. We have heard something about biofilms so far, but there are three important features I would like to stress out today. In the first place, the initiation of biofilm formation is an stochastic process, not totally understood, by the way. Of course, we know that it's a multifactorial phenomenon. It doesn't respond to just one signal. It's a multisignal regulation that ends up in biofilm formation. And the intracellular regulatory network that finally results in biofilm formation is very complex as well, and not totally understood. And in the case of Pseudomonas' putida, it wasn't studied in detail anyway. But of course, biofilms, as we have heard so far, are negatively regarded in, for instance, medical setups. But there are some conditions in which biofilm formation by bacteria can be advantages. And biocatalysis is one of the examples, one of these setups. Why is that so? Because one can use biofilms as catalytic platforms. The reason for which we wanted to have bacteria catalytically active forming biofilms is that in the first place, cells in biofilms show enhanced resistance to external stress, so we can further improve the natural resistance of Pseudomonas' putida if the cells form biofilms. There is a large surface to exposure to substrate, which is, of course, important, but there is another trait which makes catalytic biofilms desirable, and that is that there is a very limited diffusion of metabolic intermediates. Since cells are closely attached to each other, the diffusion of substrates and in metabolic intermediates is favored from one cell to the other, because they are physically together as compared to planktonic cultures. And on the other hand, cells in biofilms are known to display very low cell-to-cell phenotypic variability, so they are more synchronized from a metabolic point of view, which, of course, in a biotechnological setup is very important. So what we wanted here was to be able to reshape biofilm architecture using some genetic tricks that I will discuss in a moment, and what we wanted, basically, was to implement a synthetic morphology approach for biofilms. That is to say that we would be able to externally control the way in which the cells stick to each other and the way they attach to surfaces. If one thing about this stochastic process that I was talking about for biofilm initiation, one thinks on something like this, like a very messy arrangement of cells just sticking together and sitting on a surface. But what we want is to be able to change this situation to a more controlled situation in which, through engineering of the cell surface and rewiring the cyclic DGMP-dependent biofilm formation regulatory network, we can get into something like this, a situation in which planktonic cells can be told when and how to stick together and to sit on a surface for the sake of biocatalysis. Today I will basically focus on this part of our approach that was to rewire the cyclic DGMP regulatory network that ends up with biofilm formation in Pseudomonas Putida. So just a few words we have heard in some previous talks about cyclic DGMP regulation, but the biochemistry behind biofilm formation has to do with this secondary messenger, cyclic DGMP. So as we have heard before, there are a number of signals that determine cyclic DGMP formation and there is a cascade of regulatory processes that end up in very different phenotypes in bacteria when the intracellular concentration of cyclic DGMP increases. For the sake of my talk today, there are three things that we took into account. In the first place, upon an increase in cyclic DGMP concentration in the cells, there is a decrease in flagellular motility, so there is the switch between planktonic to cesile state, and there is an increase in the formation of fimbria and pili and in the formation of exopolysaccharides that increase the thickness of the cells to surfaces and other cells as well. As we were discussing this for a while, there are mainly two mechanisms for cyclic DGMP turnover in bacteria. Proteins that have the GGDEF motif are D1ylate cyclises that produce cyclic DGMP out of GTP and there are other type of proteins that display the EAL motif that are phosphodiesterases that degrade cyclic DGMP into GMP. So, what we did was to identify two genes encoding a D1ylate cyclase and a phosphodiesterase from E. coli to be overexpressed in Ptida to control cyclic DGMP formation and degradation and therefore biofilm formation. But before doing so, and coming back to my first slide, what we needed was to have once we had the biological parts identified these two genes from E. coli, what we wanted was a genetic tool to get a fine control of the transcription of these two genes that result in the production and degradation of cyclic DGMP. In order to do so, what we did as a first step was to standardize a new expression system that depends on cyclohexanone as the inducer. So, in order to get this expression system running, what we did was to take some inspiration in other environmental bacterium, which is Acinetobacter Johnsoni, which is a non-pathogenic soil bacterium that has an interesting feature that makes it very similar to pseomonas as well and that is that it can grow and degrade a number of different substrates, among them cyclohexanone. Acinetobacter has a whole pathway for cyclohexanone degradation and what was interesting for us is that there is a transcriptional regulator and a promoter driving the expression of one of the enzymes involved in this biodegradation pathway that had been identified in such a way that this regulator activates this promoter in the presence of cyclohexanone. So, we had these two elements identified to construct an expression system. So, we did, we took the regulator and the promoter, we standardize the promoter and the regulator in such a way that it fits in what we call the standard European vector architecture, which are a series of plasmids constructed in Victor's lab in which all the the enzymes are known, the restriction enzymes are known and all the modules of the plasmids can be interchanged as needed. So, what we did was to transform this that comes from a natural bacterium into an expression system that fits in into the standard European vector architecture. This is what we did, this is the resulting plasmid and what we did first was a very short and very quick characterization of the dynamics of the system using a fluorescent protein. As you can see in here, in the absence of cyclohexanone as an inducer, the regulator and the promoter are completely shut down. So, the response of the system is very dependent on cyclohexanone. When you induce the system and as you culture the salts in the presence of cyclohexanone, there is a more or less linear response of the promoter up to getting a 30-fold increase in fluorescence after four hours. So, that was the type of dynamics that we wanted for the expression of these enzymes that control cyclic DGMP formation and biodegradation. So, the next step was to use this genetic device to overexpress those enzymes from E. coli in P. butia. This is a very simple experiment in which we qualitatively measured biofilm formation using the crystal biolate assayed in a P. butia strain that caries the empty vector, the vector with the oneylate cyclase and the vector with the oneylate cyclase induced by cyclohexanone. As you can see, there is an increase in the amount of cells that can stick to the glass surface. But of course, we quantify that and as you can see in here, this is the quantification of biofilm formation in a strain that caries the empty vector and in a strain that caries the vector with the oneylate cyclase as plotted as the concentration of inducer used to activate the system. As you can see in here, as we observe with the fluorescent protein, there is a more or linear increase in biofilm formation upon addition of cyclohexanone. This is on glucose as a carbon source, but more or less the same situation happens on succinate. So we can induce biofilm formation either under glycolytic or gluconeogenic growth conditions and it can be externally controlled. So in the situation in which we induce the system with five millimolar cyclohexanone, we got a threefold increase in biofilm formation, which for pseudomonas putida is a huge increase. So these are the results with the cyclase, but we also have a diesterase that degrades cyclic DGMP. So what you have in here is more or less the same situation in which we tested biofilm formation by cells carying an empty vector, the cells carying the same regulatory system we engineered, but in this case, driving the expression of the diesterase. So there is, of course, a decrease in biofilm formation, irrespective of the carbon cells. And just to illustrate that this expression system is tightly regulated, we repeated this experiment with another expression system that we already had in the lab, the LacIQ PTRC system, and as you can see as compared to the newly engineered system, this other one is more LacIQ. So now we have the chassis defined, now we have the parts, now we have the genetic device to overexpress these parts at the user's wheel. So what we did was just to combine everything in the chassis. In this case we use a derivative of a strain KT2440 that has been deleted of all the prophages. So that results in a more genetic stability in the chassis and the ability to propagate plasmids more easily than in the wall type of strain. So what we did in this case was to tag the cells with a fluorescent protein that is constitutively expressed so we can follow individual cells and we transform these cells with the expression system driving the expression of YEDQ, which is the D1ylate cyclase. What we did in here was to grow the cells in liquid culture and to submerge in the culture a glass cover slip to see how cells stick to the surface. In the first place of course we run the control with an empty plasmid, then we run the experiment with the genetic device for biofilm formation and induced and in here you can see that when the system is induced you can easily detect micro colonies on the glass surface. So it is really working, it is really affecting biofilm formation not only from the microscopic point of view in the sense that we can detect it by a crystal violet but it also forms micro colonies on a glass surface. What we wanted to see next was what were the reasons for this microscopic and macroscopic behavior. So what we did was to stain the cells with Congo red which gives an indication of exopolysaccharide formation and cellulose formation by the cells. In here you have the cells transformed with the empty plasmid, this is the morphology you get in Congo red plates and then cells that have the D1ylate cyclase induced as you can see the colony gets a completely different morphology with these blobs over here in the colony that can be also seen at higher magnification and in the same cells overexpressing the phosphodiesterase you see a more blurry morphology in the colony border. When we quantified exopolysaccharide formation we saw an increase of exopolysaccharide formation in the cells that have the D1ylate cyclase and of course we saw a significant decrease in EPA's formation in the cells that overexpress the phosphodiesterase. But of course all this was just evidence that the actual reason for the phenotypes was cyclic DGMP formation or degradation so what we wanted was to quantify it in a way and as we heard yesterday in Minxtok it is quite difficult to quantify cyclic DGMP. In the case of pseudomonas one of the reasons is that the concentration of cyclic DGMP peaks at a point during exponential growth and then it goes down very rapidly so it's very difficult to detect it by HPLC-MS and we tried a number of different protocols but we couldn't so we resorted to the biosensor. So what we did basically was to take some inspiration of a regulatory circuit that takes place in pseudomonas aeruginosa. In pseudomonas aeruginosa this operon here the PEL-A-B-C-D-E-F-G operon encodes all the enzymes needed for a particular type of exopolysaccharide formation which is the PEL-exopolysaccharide. This system, these genes over here are repressed by the FLEQ transcriptional regulator which is a FIS-like transcriptional regulator and it represses the transcription of these genes so that it blocks exopolysaccharide formation in pseudomonas aeruginosa. But in the presence of cyclic DGMP this repression is relieved, this operon gets expressed and cells produce the exopolysaccharide. So what we did was to take the promoter that drives the expression of PEL-A as a promoter to be using a biosensor to detect cyclic DGMP formation. So this is the genetic device we constructed is a TN7 transposon that contains the PEL-A promoter, this one from pseudomonas aeruginosa and of course it drives the expression of a fluorescent protein. There is a translational coupler here to enhance the translation of the construct and what we did next of course was to test it in cells that overproduce either the diwanylate cyclase or the phosphodiesterase. So this is the level of fluorescence one gets when this TN7 transposon caries the biosensor is integrated in the chromosome and when the cells caries the empty plasmids. So this will be the normal condition and these fluorescence correspond to the normal level of cyclic DGMP. When cells were transformed with the phosphodiesterase the fluorescence went down, indicative of a lower concentration of cyclic DGMP and there was a significant increase in the signal when cells overexpress the diwanylate cyclase. So that was an indication that we were actually modifying the turnover of nucleotides in pseudomonas and so that it produced more biofilm for that reason. Now we had this strain engineer to produce biofilms and what we did is to use it for biodegradation purposes and the model we decided to use was haloalkenes. This is a very complex family of compounds but all of them are very toxic and one of the interesting features of some of them is that they are xenobiotics so they were introduced in nature after the human race started to produce them. They weren't there in nature before industrial activity at all and as a consequence of that there are very few degradation pathways that can be found in nature for these compounds. The compound we use as an example and as a model is 1-chlorobutene which of course is a xenobiotic and is used a lot in dry cleaning. It is produced more than 50,000 tons per year worldwide and the thing is that of course it's a very nasty compound because it's potentially carcinogenic and it's very resistant to biodegradation. There are no means to remove 1-chlorobutene from soils for instance which is the place in which it ends up. Fortunately we were able to identify a biodegradation pathway from a pseudomonas species not surprisingly it is a pseudomonas as well so this species that was isolated from contaminated soils has a pathway for 1-chlorobutene and other haloalkanes biodegradation and complete mineralization. But the thing is that this natural strain that is able to degrade this grows very slowly in most of the biotechnological setups and industrial setups so it's not a very good chassis to work with in biodegradation procedures. But it is a good source of dialogenesis of all these compounds and in the case of 1-chlorobutene there are two dialogenesis that have been identified that can be used to degrade the compound. So what we did was to construct a synthetic operon using these two dialogenesis and what we did next was to combine all what we had so far so we got the chassis we transformed it with the genetic device for biofilm formation we also transformed it with this haloalkane degradation operon with enzymes from pseudomonas pabonacea and what we did was to grow it in multi-world plates and after the cells established a biofilm we exposed them to 1-chlorobutene and after 48 hours we removed the planktonic cells and biofilm cells and we measured the dialogenase activity and the removal of 1-chlorobutene and these are the results of course this is the control in here you have the planktonic cells growing either on succinate or glucose this is the activity ratio normalized to the total protein content and what you see in here is that most of the dialogenase activity was recovered in cells that come from biofilm so the cells in the biofilm were not only catalytically active but they were more active than planktonic cells in such a way that more than 65 percent of the total dialogenase activity was recovered in the biofilm cells moreover after 48 hours all the chlorobutene 0.5 millimolar was completely removed in this setup this looks like a very low concentration 0.5 millimolar but this is the highest concentration you can use you can expose yourselves to without damaging the cells so this is the maximum concentration they can tolerate but after 48 hours they can degrade everything so I hope I have convinced you that in this example we follow different steps in a way that in the first place we design it a microbial chassis that can be used for biodegradation we also contributed new synthetic biology tools that can be used for manipulation of the chassis but a part of the metabolic engineering of the enzymes the enzyme activities that are involved in biodegradation there is a new twist that we think it has to be taken into account which is the synthetic morphology approach to modify the physical arrangement of cells in a biodegradation process in order to get a superbug okay so this is an approach that we think it will become an integral part of a biocatalyst design in the sense that normally we are used to use planktonic cells in bioprocesses for industrial processes right so we think that synthetic morphology the way that cells really are arranged in a three-dimensional pattern is important as well so there are a few things that I would like to remark just to end up there are a number of approaches that we want to follow now that we have these and these preliminary results let's say we would like to use stronger cyclase enzymes we know that the d1 elate cyclase from caulobacter crescentus is a more active enzyme than the one we use from E. coli we would like to be able to switch the cells back to the planktonic state after they establish a biofilm that can be useful in a number of setups and finally since cells in biofilm are normally subjected to oxygen limitation what we are actively working at is to change the lifestyle of pseudomonas putida putida kt2440 is an obligate aerobe so we are trying to identify the elements needed to change that lifestyle completely dependent on oxygen to an anaerob that will be very useful in biofilm setups in which oxygen can get a limiting factor with that I would like to thank the financial support by the maric reactions and the european molecular biology organizations and of course I would like to thank the people who actually conducted experiments ilaria and angeles hueso in the lab with this I will end up here and thank you very much for your attention