Synthetic Morphology Approaches in Pseudomonas putida for Bioremeditation of Haloalkanes
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Transcript: English(auto-generated)
00:19
Good afternoon, everybody. I would like to start by thanking the organizing committee
00:24
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 viodegradation using Pseudomonas putida as a microbial chassis. So now we will move from
00:41
synthesis, which was the main subject of Eriko's talk, to viodegradation. 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
01:06
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
01:23
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
01:47
of environmental bacteria as microbial chassis for viodegradation. Okay, so if we take into account the metaphor of cells, living cells like computers making
02:02
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
02:27
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
02:43
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
03:01
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
03:21
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
03:45
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
04:00
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,
04:22
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
04:47
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.
05:02
And over the last 25 years or so, it has been used for a number of applications, such as in situ bioremediation and the industrial production of polyhydroxyl canoids, which are biopolymers. There are several companies that are presently using Pseudomonas
05:21
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
05:40
compounds, such as aromatic compounds. Pseudomonas putida is able to degrade toluene, saline, and other aromatic compounds as well. All these features speak in favor of a very remarkable adaptability to thrive in very different and harsh environments.
06:01
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
06:22
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 discovered some features in the central metabolism of Pseudomonas putida that are very unique
06:41
and different when compared to other bacteria that are normally used as chassis 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
07:03
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
07:22
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
07:41
is orders of magnitude higher than in other bacteria. And this is one of the reasons for which this bacterium is so resistant to the stress produced by solvents and to exceed active stress. And, of course, this is a feature that we want to exploit for biocatalysis
08:04
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 wither you through the main
08:26
steps in the upper catabolism of Pseudomonas putida. In the first place, when cells grow on exosus, glucose for instance, there are two possibilities for the processing of the substrate.
08:43
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
09:01
that oxidates glucose into gluconate or ketogluconate before phosphorylation. So, there is a branching point of exos-processing as a first step in the catabolism. But a very remarkable thing about the central metabolism in this bacterium is that it operates
09:24
the endodural catabolic pathway, which is composed of just two enzymes, EDD and EVA, and it transforms 6-phosphogluconate into two trioses, glyceraldehyde 3-phosphate and pyruvate. On the other hand, another remarkable thing that makes Pseudomonas different from
09:45
other typical bacteria, such as E. coli, for instance, and other enterobacteria, is that there is an incomplete endomere of Parna's 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
10:06
6-phosphate into fructose 1-6-phosphate. So, what happens here is that there is only one enzyme, which is gluconiogenic, 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
10:28
regulated in this bacterium? I will not enter into 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
10:41
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 putida, glucose is first phosphorylated.
11:07
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.
11:28
So, there is part of the glucose that ends up in lower catabolism after a triosis formation, but part of the trioses are recycled back. So, the interesting thing about this metabolic
11:40
architecture is that the three main catabolic blocks described for bacteria work together because we have components of the pentose phosphate pathway, the endodular pathway, and the MDM pathway. And that's a good reason to explain why Pseudomonas doesn't have
12:01
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.
12:28
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
12:42
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 six to tenfold, which favors NADPH formation under oxidative stress
13:07
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
13:24
very stressful conditions. Because using this metabolism, it can overproduce the NADPH. It also needs to counterfeit stressful conditions. So, this is one of the reasons for which we are so in love with Pseudomonas putida. It has very interesting characteristics
13:45
to be used as a chassis. But today, I would like to focus in one of the traits, one of the features of this species that we want to manipulate as well. Pseudomonas putida is not able to form strong biofilms. The biofilms it forms
14:04
are kind of weak, which is in stark contrast as compared to other Pseudomonas species. For instance, Pseudomonas aeruginosa is known to form very strong biofilms. That's not the
14:22
thing. The ability of P 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 was something that
14:41
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 a stochastic process, not totally understood, by the way. Of course, we know that it's a multifactorial phenomenon.
15:06
It doesn't respond to just one signal. It's a multisynial regulation that ends up in biofilm formation. And the intracellular regulatory network that finally results
15:20
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 negative regarded in, for instance, vertical setups.
15:40
But there are some conditions in which biofilm formation by bacteria can be advantageous. 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
16:03
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 P putida if the cells form biofilms. There is a large surface to exposure to substrate,
16:25
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
16:44
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
17:03
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
17:22
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
17:44
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
18:02
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.
18:24
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.
18:41
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
19:03
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.
19:26
In the first place, upon an increase in cyclic dGMP concentration in the cells, there is a
19:42
and there is an increase in the formation of fimbri and pili, and in the formation of exopolysaccharides that increase the thickness of the cells to surfaces and other cells as well. As we also were discussing this for a while, there are mainly two mechanisms for
20:07
cyclic dGMP turnover in bacteria. Proteins that have the G-G-D-E-F motif are deionylate cyclases that produce cyclic dGMP out of GTP, and there are other type of proteins that display the
20:27
E-A-L motif that are phosphodiesterases that degrade cyclic dGMP into GMP. So what we did was to identify two genes encoding a deionylate cyclase and a phosphodiesterase
20:46
from E. coli to be overexpressed in Pseudomonas putida to control cyclic dGMP formation and degradation, and therefore biofilm formation. But before doing so, and coming back to my
21:02
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 control, to get a fine control of the transcription of these two genes that result in the production and degradation of
21:24
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
21:41
expression system running, what we did was to take some inspiration in other environmental bacterium, which is Acinetobacter johnsony, which is a non-pathogenic soil bacterium that has an interesting feature that makes it very similar to Pseudomonas as well, and that is that it can grow and degrade a number of different substrates, among them cyclohexanol.
22:04
Acinetobacter has a whole pathway for cyclohexanol 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
22:22
had been identified in such a way that this regulator activates this promoter in the presence of cyclohexanol. So we had these two elements identified to construct an expression system. So we did, we took the regulator and the promoter, we standardized the promoter and the
22:46
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 enzymes are known, the restriction enzymes are known, and all the modules of the plasmids can be
23:05
interchanged as needed. So what we did was to transform this that comes from a natural bacterium into an expression system that fits into the standard European vector architecture. This is what we did, this is the resulting plasmid, and what we did first was a
23:22
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 cyclohexanol as an inducer, the regulator and the promoter are completely shut down, so the response of the system
23:43
is very dependent on cyclohexanol. When you induce the system and as the culture dissolves in the presence of cyclohexanol, there is a more or less linear response of the promoter up to getting a 30-fold increase in fluorescence after four hours.
24:04
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 putia. This is a very simple experiment in which
24:24
we qualitatively measured biofilm formation using the crystal violet acid in a P putia strain that carries the empty vector, the vector with the guanylate cyclase, and the vector with the
24:41
guanylate cyclase induced by cyclohexanol. As you can see, there is an increase in the amount of cells that can stick to the glass surface. But of course, we quantified that, and as you can see in here, this is the quantification of biofilm formation in a strain that carries the empty vector and in a strain that carries the vector with the
25:06
guanylate cyclase, as plotted as the concentration of inducer used to activate the system. As you can see in here, as we observed with the fluorescent protein, there is a more
25:22
linear increase in biofilm formation upon addition of cyclohexanol. 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,
25:42
and it can be externally controlled. So, in the situation in which we induced the system with 5 millimolar cyclohexanol, we got a threefold increase in biofilm formation, which, for Pseudomonas putida, is a huge increase. So, these are the results with the cyclase,
26:05
but we also have a digesterase 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 carrying an empty vector. The cells carrying the same regulatory system we engineered, but in this case,
26:25
driving the expression of the digesterase. So, there is, of course, a decrease in biofilm formation irrespective of the carbon source. And just to illustrate that this expression system
26:40
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 leaky. So, now we have the chassis defined,
27:02
now we have the parts, now we have the genetic device to overexpress these parts at the user's will. 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.
27:21
So, that results in a more genetic stability in the chassis and the ability to propagate plasmids more easily than in the wild-type 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
27:40
individual cells and we transform these cells with the expression system driving the expression of YEDQ, which is the dewannylate 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
28:02
stick to the surface. In the first place, of course, we run the contour 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
28:22
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 crystal violet, but it also forms micro colonies on a glass surface. What we wanted to see next was what were the
28:41
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 an empty plasmid,
29:01
this is the morphology you get in Congo red plates, and then cells that have the dewannylate 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
29:24
same cells over expressing the phosphodiesterase, you see a more blurry morphology in the colony border. When we quantified exopolysaccharide formation, we saw an increase of exopolysaccharide
29:42
formation in the cells that have the dewannylate cyclase, and of course we saw a significant decrease in EPase formation in the cells that overexpress the phosphodiesterase. But of course, all this was just evidence that the actual reason for the phenotypes
30:06
was cyclic dGMP formation or degradation, so what we wanted was to quantify it in a way, and as we heard yesterday in Minh's talk, it is quite difficult to quantify cyclic dGMP.
30:20
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,
30:40
so we resorted to a vile sensor. 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.
31:06
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
31:24
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 used in a vile sensor to detect cyclic dGMP formation.
31:46
So this is the genetic device we constructed. It's 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
32:05
construct. And what we did next, of course, was to test it in cells that overproduce either the diwanulate cyclase or the phosphodiesterase. So this is the level of fluorescence one gets when this TN7 transposon carrying the biosensor is integrated in the
32:27
chromosome, and when the cells carry the empty plasmid. So this will be the normal condition, and this fluorescence corresponds to the normal level of cyclic dGMP. When cells were transformed with the phosphodiesterase, the fluorescence went down,
32:46
indicative of a lower concentration of cyclic dGMP, and there was a significant increase in the when cells overexpressed the diwanulate cyclase. So that was an indication that we were actually
33:01
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 haloalkanes. This is a very complex family of compounds, but all of them are very toxic,
33:25
and one of the interesting features of some of them is that they are cenobiotic. 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
33:47
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-chlorobutane, which of course is a cenobiotic and is used a lot in dry cleaning. It is produced more than 50,000 tons per year
34:06
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-chlorobutane from soils, for instance, which is the place in which it ends up.
34:24
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-chlorobutane and other haloalkanes
34:43
biodegradation and complete mineralization. But the thing is that this natural strain that is able to degrade this growth very slowly in most of the let's say biotechnological setups and industrial setups. So it's not a very good chassis to work with in biodegradation procedures. But it is a good
35:05
source of deallogenesis of all these compounds, and in the case of 1-chlorobutane, there are 2-deallogenesis that have been identified that can be used to degrade the compound. So what we did was to construct a synthetic operon using this 2-deallogenesis, and what we did next
35:23
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 with the enzymes from Pseudomonas pabonacea, and what we did was to grow it in multi-well plates,
35:42
and after the cells establish a biofilm, we expose them to 1-chlorobutane. And after 48 hours, we remove the planktonic cells and the biofilm cells, and we measure the deallogenesis activity and the removal of 1-chlorobutane. And these are the results.
36:00
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 deallogenesis activity was recovered in cells that come from biofilm. So the cells in the biofilm were not only
36:25
catalytically active, but they were more active than planktonic cells in such a way that more than 65% of the total deallogenesis activity was recovered in the biofilm cells. Moreover, after 48 hours, all the chlorobutane, 0.5 millimolar, was completely removed in this
36:47
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
37:05
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 designed a microbial chassis that can be used for biodegradation. We also contributed new synthetic biology tools that can be used for
37:25
manipulation of the chassis. But a part of the metabolic engineering of 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
37:45
cells in a biodegradation process in order to get a superbug. 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.
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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
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have these preliminary results, let's say. We would like to use stronger cyclase enzymes. We know that the dewannylate 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
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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.
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Pputida Kt2440 is an obligate arrow. So we are trying to identify the elements needed to change that lifestyle completely dependent on oxygen to an arrow. That will be very useful in biofilm
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setups in which oxygen can get a limiting factor. With that, I would like to thank the financial support by the Marie Curie actions and the European molecular biology organizations. And of course, I would like to thank the people who actually conducted the experiments, Ilaria and Angeles Hueso in the lab. With this, I will end up here.
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And thank you very much for your attention.