Bioremediation: Smart Degradation of Environmental Pollutants

00:00:12.14 Hi.
00:00:14.03 My name is Victor de Lorenzo, and in this video I am going to tell you
00:00:17.17 a little bit of the work we have been running in my laboratory
00:00:21.01 to domesticate environmental bacteria for different environmental applications.
00:00:25.21 Let me show you where I come from.
00:00:28.02 I work in the National Center for Biotechnology in Madrid.
00:00:30.24 And before I forget, let me show you the faces of my collaborators.
00:00:34.21 In particular, those who have made most of the work I will share with you today,
00:00:39.01 Pablo Nikel and Esteban Martinez.
00:00:41.19 Now, our interest for a long time has to do with
00:00:46.14 using bacteria as whole-cell catalysts.
00:00:50.16 Well, what is that?
00:00:52.04 Well, basically, it's what is in the cartoon.
00:00:55.01 You have an input, that is, a substrate,
00:00:57.21 and an output, that is, a product.
00:00:59.11 And there's a bacterium that acts as a biocatalyst,
00:01:02.18 in many cases with a very high efficiency.
00:01:05.21 You can wonder, well, where do we find all these catalysts
00:01:08.22 that could be of interest for the industry or environmental applications?
00:01:13.08 Well, unlike other biologists that look for samples
00:01:16.20 in very beautiful places,
00:01:19.03 we in fact go to sites with a history of pollution
00:01:23.01 by chemical industry and by human activities,
00:01:26.24 and places, like the one shown in the picture,
00:01:29.05 that, for most of you are completely disgusting...
00:01:32.14 for us, are a gold mine to find interesting bacteria and interesting genes
00:01:36.18 that, when we manipulate them with the tools of synthetic biology
00:01:40.16 and the tools of genetic engineering,
00:01:43.01 then do wonderful things.
00:01:45.12 Why are we looking for these catalysts in such places?
00:01:49.12 Well, in these places, in fact,
00:01:51.21 there's a kind of battle between a chemical landscape
00:01:55.02 that is brought about by the presence of a large number of environmental pollutants,
00:02:00.12 and in this picture you have a small number of them.
00:02:03.15 Some of them come from the petroleum industry, like toluene, alkanes.
00:02:06.13 In other cases, they are genuinely xenobiotic compounds,
00:02:10.19 namely compounds that are there only because
00:02:13.17 our chemical industry put them there.
00:02:15.09 Otherwise, they would have never existed in the biosphere.
00:02:18.24 But then here comes our heroes,
00:02:21.12 namely bacteria that, by using or by exploiting
00:02:25.12 different mechanisms of genetic and biochemical adaptation,
00:02:27.19 end up being able to use these compounds as carbon sources.
00:02:32.16 And therefore, what for us are bad environmental pollutants
00:02:35.23 for many of them are gourmet food.
00:02:38.20 This is just extraordinary because, you see,
00:02:42.06 the chemical industry used to have very strong, environmentally damaging in some cases,
00:02:48.00 catalysts for executing reactions that bacteria, in other cases,
00:02:52.04 can do at room temperature and with a minimal environmental impact.
00:02:56.02 And this is why we are so interested in environmental bacteria,
00:03:00.00 because we argue that they are pre-endowed
00:03:03.22 with a number of properties that make them ideal platforms for biocatalysis
00:03:07.03 in industrial and environmental settings.
00:03:10.22 Now, what are the names and the surnames of these bacteria?
00:03:14.08 Well, there are many, many of them.
00:03:16.04 In this picture, I just show you some of what you may call
00:03:19.05 the top 10 stains that have been isolated
00:03:22.20 and that do extraordinary things.
00:03:25.03 The names may sound very weird,
00:03:26.22 like Pseudomonas putida,
00:03:28.07 Burkholderia, Acinetobacter, you name it,
00:03:31.01 and this is just a small fraction of the entire wealth of isolates
00:03:33.19 that have been obtained over the years,
00:03:36.19 that do things as extraordinary as growing on toluene, on phenol,
00:03:41.05 on phenantrene, 2,4D,
00:03:43.12 typical environmental pollutants that are the consequence,
00:03:45.19 as I said before, of our industrial and urban activities.
00:03:50.04 And what happens is that one of these bacteria,
00:03:53.21 that bacteria that is on the top of the list,
00:03:56.24 is this one called Pseudomonas putida.
00:03:59.12 The name sounds a little strange.
00:04:01.01 Taxonomists once in a while
00:04:03.21 provide very funny designations to bacteria.
00:04:06.08 Now, this bacterium was isolated from a polluted soil,
00:04:09.11 and it is a really, really extraordinary vehicle,
00:04:12.16 as a sort of container for running extraordinary reactions
00:04:17.14 of tremendous environmental and industrial capacity.
00:04:21.13 The reason for that can be found in the genome
00:04:24.06 that was, say, first identified entirely in the early 2000s.
00:04:29.13 And when you look in detail through the genes,
00:04:31.21 you find some of the reasons that are behind these properties
00:04:35.09 I was mentioning before.
00:04:36.24 As a matter of fact, you may make a list of beneficial traits
00:04:41.13 that one can find in Pseudomonas putida,
00:04:43.24 and other traits that are not so good.
00:04:46.02 But in the beneficial traits,
00:04:48.00 you have for instance the fact that Pseudomonas putida
00:04:50.04 is very, very highly solvent tolerant,
00:04:53.09 and therefore it can run reactions under conditions
00:04:55.23 in which other bacteria perhaps could not do it.
00:04:58.14 It has some interesting metabolic features,
00:05:01.09 for instance the entire metabolism of it
00:05:03.21 seems to be geared for production of NADPH,
00:05:07.06 and this is something that is important for bringing about
00:05:10.22 a high resistance to environmental stress,
00:05:13.12 in particular oxidative stress.
00:05:16.06 It also has a diverse metabolism.
00:05:18.02 It can grow in many different substrates.
00:05:19.19 And it also -- and this is important for biotechnology --
00:05:22.02 is a bacterium that is generally regarded as "safe",
00:05:25.15 and therefore it has the so-called GRAS status
00:05:28.08 that makes our life easier in terms of future applications.
00:05:31.19 But it has other bad traits.
00:05:33.12 In many cases, or in some cases,
00:05:35.03 it displays a high level of resistance to various antibiotics.
00:05:40.02 It has a complex surface in the cell.
00:05:42.12 It has a number of prophages in the genomes.
00:05:45.07 It doesn't have a really good glycolysis,
00:05:47.22 and this something that for metabolic engineering
00:05:50.18 sometimes makes our life a little difficult,
00:05:53.05 because in this case, instead of having the typical textbook glycolysis,
00:05:56.08 we have an alternative glycolytic pathway
00:05:58.24 that is called the Entner-Doudoroff pathway.
00:06:01.05 I will return to that in a minute.
00:06:03.00 And also, well, it's an obligate aerobe.
00:06:06.00 So that means that some reactions
00:06:08.01 that we could run in anaerobiosis
00:06:10.14 are not possible with the wild type strain, as it is.
00:06:13.12 And finally, it's what you might call a strain or a species
00:06:17.04 that has a little bad reputation,
00:06:19.07 because there are other members of the same genus,
00:06:21.09 in particular, Pseudomonas aeruginosa,
00:06:23.05 that are pathogens that are involved in various diseases.
00:06:26.24 However, and here's the interest of synthetic biology,
00:06:29.19 we can do much better than nature,
00:06:32.02 go to the genome as nature gives it to us
00:06:34.24 and then look for traits that we want to enhance
00:06:38.18 and other traits that we want to suppress.
00:06:41.01 And this is what we have called
00:06:44.01 the process of complete domestication.
00:06:48.06 So, while in nature you find some biological entities and...
00:06:54.22 well, in the cartoon, it's a kind of wolf,
00:06:56.22 a very beautiful animal, but very dangerous and unpredictable to use.
00:06:59.10 And at the end, we have something that could be
00:07:02.22 completely predictable, like a robotic dog.
00:07:05.21 So, eventually, for biotechnology and for the applications,
00:07:08.10 we have to have some biological systems
00:07:10.12 with a predictability that would be similar
00:07:13.03 to what one has, for instance, in robots.
00:07:16.04 But we are not there yet.
00:07:17.13 So, I would argue that we have to go through various statuses,
00:07:21.10 in which you start with a naturally existing biological system
00:07:25.21 -- the wolf, in this case --
00:07:27.13 and then you go first to domestication
00:07:30.04 -- that means that you enhance some properties and you suppress others --
00:07:33.05 and there's this other state, that I will argue that is precisely
00:07:36.09 where we are at the moment,
00:07:38.04 in which we combine traits that are natural
00:07:41.01 with traits that we knock in the system
00:07:43.09 just to increase the predictability and the usefulness of the biological system.
00:07:47.01 And eventually, maybe in the future, in a few years,
00:07:49.13 we will be able to design altogether, from first principles,
00:07:52.14 biological systems with biotechnological potential.
00:07:55.04 But I don't think we are there immediately.
00:07:57.18 But we have to do something in between,
00:07:59.16 and this is exactly what we call cyborg-ization.
00:08:03.03 It's this idea of combining artificial things with natural things.
00:08:07.09 What is cyborg-ization?
00:08:09.19 Well, there are at least four aspects of it.
00:08:11.22 One of them is enhancement of innate traits,
00:08:15.15 then also replacement of traits that are there by better ones,
00:08:20.01 but with the same functionalities.
00:08:22.02 Then, you can knock in entirely new traits.
00:08:25.03 And importantly, you can eliminate drawbacks.
00:08:27.14 Well, this is exactly what we have been doing with Pseudomonas.
00:08:32.00 But there is a little detail
00:08:34.14 that it's really worth it to spend one minute discussing.
00:08:37.23 And it's that to get into this massive cyborg-ization, reprogramming,
00:08:42.24 or improvement of an existing biological system
00:08:45.07 -- in particular, if it comes straight from the environment,
00:08:47.13 like Pseudomonas putida --
00:08:49.07 you need tools.
00:08:51.03 And the quality of the tools will determine
00:08:54.06 the quality of your final product.
00:08:55.23 So, if you have what you may call primitive tools,
00:08:58.02 then you can do nice things,
00:09:00.00 but primitive at the end of it.
00:09:01.22 You can improve the tools, and you'll get a better engineered system.
00:09:06.08 And if at the end you're able to follow the track
00:09:08.20 that has been followed before by electrical engineers and industrial engineers,
00:09:12.16 namely standards and standard tools,
00:09:16.00 then you can do much, much better,
00:09:18.00 and really push the engineering of biological systems
00:09:20.14 much, much better than traditional engineering
00:09:23.19 has been doing for a long time.
00:09:26.14 So, in the case of Pseudomonas putida,
00:09:28.18 we have made our contribution to the field of standardization and development of tools
00:09:33.08 by putting together a large collection of molecular vectors
00:09:36.14 for doing all types of operations
00:09:39.19 in terms of genetics with Pseudomonas putida
00:09:42.14 and other Gram-negative environmental bacteria.
00:09:45.17 Namely, we have this long list of vectors
00:09:48.21 that can be plasmids, that can be transposons,
00:09:51.09 and a large number of antibiotic resistances,
00:09:55.07 a large number of replication origins,
00:09:56.22 and we have made sure that they are all compatible, interchangeable, reusable,
00:10:01.24 and can be easy to use for potential,
00:10:06.04 say, applications.
00:10:08.00 And, well, with these tools in our hands,
00:10:09.13 we have been able to do things that, so far,
00:10:12.07 we were unable to do.
00:10:13.20 All this standardization has been inspired
00:10:17.16 by many of the tenets of contemporary synthetic biology
00:10:21.00 and the emphasis in standards, rigorous description of systems,
00:10:24.12 description of boundaries, and all the rest of it.
00:10:27.15 And this has been a big change,
00:10:30.04 in contrast with the type of engineering and biotechnology
00:10:32.10 that we used with this...
00:10:34.06 that we used to do with this bacterium before that.
00:10:36.23 Well, I'm going to give you an example of the power
00:10:40.00 of applying synthetic biology and standardized tools
00:10:43.07 for knocking in to Pseudomonas putida interesting properties.
00:10:48.04 As I said before, Pseudomonas putida is a natural bacterium.
00:10:51.09 You can find them in many places.
00:10:52.23 And there are things that it can do,
00:10:54.21 and there are things that it cannot do.
00:10:56.17 And for instance, one of the things that it cannot do
00:10:59.06 is to grow, or to be active, in the absence of oxygen.
00:11:03.19 And this is something that for some environmental applications
00:11:06.08 may give you trouble.
00:11:08.23 Why?
00:11:10.02 Because let's imagine that you want to make a strain
00:11:12.10 that is able to degrade a typical soil pollutant, a chemical.
00:11:15.11 Well, if you engineer some type of inoculation procedure
00:11:18.09 in which you have your pathway for degradation
00:11:21.04 of that component in soil,
00:11:22.18 you have to be aware that soil
00:11:25.11 is such that the oxygen is present
00:11:28.22 only in the very, very upper layers of soil.
00:11:31.17 The moment you go a little down, then oxygen is depleted.
00:11:35.01 And for the most part, when you a little farther down,
00:11:37.13 then you end up in an anaerobic niche.
00:11:41.05 Well, you have a single catalyst,
00:11:43.17 and you want the catalyst to work in a soil column, for instance,
00:11:46.23 then it is evident that you need something
00:11:49.05 that works as well in the presence or in the absence of oxygen.
00:11:52.03 And this is something that...
00:11:54.02 nature had not really invented a procedure, a trick,
00:11:57.19 to make the same catalyst to work under these two conditions.
00:12:01.11 At that point, we said, well,
00:12:03.13 can we look at the metabolic network that is present in Pseudomonas putida,
00:12:09.17 and, by using these standardized tools,
00:12:12.18 see what type of cyborg-ization we can make with them
00:12:17.07 so that enter some new genes,
00:12:19.11 perhaps delete some other genes,
00:12:20.24 and at the end we may be able to reprogram the metabolism
00:12:23.05 in such a way that bacteria that were formerly
00:12:26.12 only able to grow in the presence of oxygen,
00:12:29.18 now we make them to be metabolically active in the absence of oxygen as well.
00:12:35.08 Well, the first step is to take a look to the metabolic network
00:12:38.20 that one finds in this type of microorganism.
00:12:43.01 And fortunately, it is possible now to develop metabolic models
00:12:47.05 on the basis of the genome sequence that one has
00:12:50.23 plus simple parameters that one can measure.
00:12:52.23 There are various metabolic models
00:12:54.24 that have been proposed by various authors
00:12:57.01 to explain how P putida, Pseudomonas putida,
00:13:01.04 has the lifestyle that I have just referred to.
00:13:04.20 And I will just quote the three that are in the screen,
00:13:09.05 and there are others coming.
00:13:11.01 Every time, a more and more refined...
00:13:13.05 because this comes from systems biology,
00:13:15.10 and the advances in that field are immense as well.
00:13:17.20 Okay.
00:13:19.07 So, by looking at the three models and other tools
00:13:22.10 that we have to model metabolism,
00:13:24.16 well, we observed some interesting features
00:13:27.23 that ultimately explain why Pseudomonas putida
00:13:31.09 is unable to grow in the absence of oxygen.
00:13:34.21 Well, there's no surprise that...
00:13:38.15 you know, the phenotype, namely lack of growth when oxygen is absent...
00:13:45.05 and therefore that means that many or some of the genes that are typically present in anaerobic bacteria,
00:13:49.13 for instance, anaerobic respiration,
00:13:52.02 are altogether absent.
00:13:54.10 However -- and this something that I will tell you how important it is --
00:13:57.12 there are some genes related to fermentation.
00:13:59.21 And this is a kind of entry in this manipulation
00:14:02.19 that I will tell you in a minute.
00:14:05.09 But, one way or the other, as nature gives the metabolic network to us,
00:14:08.18 Pseudomonas cannot grow in the absence of oxygen.
00:14:11.15 That's something that we knew already.
00:14:13.05 Well, you can take a look to the various steps
00:14:15.14 that are involved in the degradation of glucose, for instance.
00:14:19.23 And then, well, you can make a classification of the enzymes
00:14:23.05 and see which of them would be...
00:14:26.08 could be improved could be knocked in, could be knocked out.
00:14:29.15 And well, at the end, one comes to the conclusion that
00:14:35.03 two of the reasons -- maybe there are more --
00:14:37.10 for why P putida is unable to grow in the absence of oxygen
00:14:41.11 are because you don't have a lot of ATP
00:14:45.13 and that you have an excess of reductive power.
00:14:49.13 And let me tell you in one second why that is.
00:14:51.23 Well, that you don't have ATP can be traced...
00:14:54.18 or that you don't have enough ATP to support anaerobic growth
00:14:57.07 can be traced to various origins,
00:15:01.12 but one of them that we found to be quite important
00:15:04.02 is the fact that having the typical glycolysis, as I mentioned before,
00:15:08.12 Pseudomonas putida has this other type of glucose pathway
00:15:14.13 that is called the Entner-Doudoroff pathway.
00:15:17.11 And if you measure, you quantify, the number of ATPs
00:15:20.04 that are produced per molecule of glucose, etc,
00:15:23.11 then you find that the outcome of ATP
00:15:28.00 is worse than if you have the entire classical glycolytic pathway.
00:15:33.05 And therefore one possibility to increase the ATP levels
00:15:39.17 could be to trick it a little bit,
00:15:44.04 with a production of ATP through, for instance, substrate-level phosphorylation.
00:15:46.22 So, this is something that we have explored,
00:15:49.12 and you will see that it makes a big difference.
00:15:51.16 But then we have this other problem,
00:15:53.19 and it's the problem that when you grow cells in the absence of oxygen
00:15:56.23 then you get a big excess of NADH.
00:16:00.21 That is the reduced form of this important cofactor of many enzymes.
00:16:05.22 So, one way or the other,
00:16:07.19 we have to get rid of an excess of NADH
00:16:11.05 and recreate, reproduce, enough of NAD,
00:16:14.14 that is, the oxidized form of this cofactor.
00:16:18.04 Well, and... what I'm going to tell you in the next few minutes
00:16:20.16 is how we were able to tackle, by separating, these two problems.
00:16:24.22 And then how, ultimately, we were able to overcome the limitations
00:16:28.08 that are connected to the dearth of ATP
00:16:31.01 and the dearth of NAD.
00:16:33.23 Okay.
00:16:35.13 Again, let's take a closer look at some of the pathways
00:16:38.03 that one can find in the metabolism,
00:16:40.01 and it goes as follows.
00:16:41.13 There's a way in many bacteria to generate ATP.
00:16:45.01 It starts with pyruvate.
00:16:47.03 Then the pyruvate is transformed into acetyl-CoA
00:16:49.09 by an enzyme that is called pyruvate dehydrogenase.
00:16:52.20 Then acetyl-CoA is converted into acetyl-phosphate
00:16:56.18 by another enzyme called phosphotransacetylase.
00:16:59.24 And then eventually, there's another enzyme called acetate kinase
00:17:03.19 that can regenerate, or can form, ATP
00:17:06.21 by taking the phosphorus out of the acetyl and thus produce acetate.
00:17:11.01 Now, when one looks at the metabolism of P putida,
00:17:14.23 one finds something that is very, very weird.
00:17:17.08 The number... the number one...
00:17:21.07 well, that you have pyruvate dehydrogenase,
00:17:23.09 you have phosphotransacetylase,
00:17:26.06 but -- and this comes as a complete surprise --
00:17:28.10 you don't have acetate kinase.
00:17:30.16 So, it seems that, for whatever reason,
00:17:32.20 the pathway is incomplete.
00:17:34.14 So, one pathway that in anaerobic bacteria,
00:17:36.20 or in bacteria that can grow in the absence of oxygen
00:17:39.15 or be metabolically active in the absence of oxygen,
00:17:42.02 it's a must,
00:17:44.23 happens to be missing in this particular strain.
00:17:47.00 Well, that the previous enzyme is there is a reality,
00:17:51.23 because we made the effort to make an enzymatic analysis
00:17:55.12 and see whether the activity was there or not.
00:17:57.22 There's a range of function.
00:17:59.06 Sometimes one finds a gene in a genome,
00:18:00.24 and you may believe that it's there and the sequence is there.
00:18:02.24 But it may not be expressed, or perhaps the protein formed, that it encodes,
00:18:08.16 may not be completely active.
00:18:10.09 So it's always important to really check that the activity
00:18:13.03 that is predicted for a gene happens to occur in reality.
00:18:16.09 So, we made a simple assay for this enzyme,
00:18:19.05 for the phosphotransacetylase,
00:18:21.01 that is, the key...
00:18:23.17 one of the two key activities in the process of phospho...
00:18:26.15 getting, at the end, ATP and acetate.
00:18:28.00 And, well, the enzyme is there.
00:18:29.23 It's not only there, but it's present at high levels.
00:18:32.11 So, that means that in principle one can anticipate
00:18:35.13 that in P putida the basal metabolism that is already there
00:18:39.20 is able to produce enough amount of acetyl-phosphate.
00:18:44.00 The acetyl-phosphate is there, it stays there,
00:18:46.08 and it cannot be further processed into acetate.
00:18:49.07 So, that was a very, very good hint of what the strategy...
00:18:51.24 what strategy we would follow to overcome
00:18:54.20 this problem of anaerobiosis, as I told you before.
00:18:56.18 So, the kind of take that we figured out is that,
00:19:02.08 well, if we recruit an acet...
00:19:05.01 the same enzyme, acetate kinase,
00:19:08.10 from other... other... let's say, microbes,
00:19:12.19 then we should be able to complete this phosphorylation
00:19:17.02 at the level of substrate,
00:19:19.10 and therefore knock in a new reaction
00:19:22.10 that will end up in the production of acetate,
00:19:24.18 and would end up in the production of ATP.
00:19:26.21 And at the end, this is exactly what we wanted -- to have ATP.
00:19:29.22 Okay, so this remained as a kind of possibility.
00:19:34.02 Now we have the other problem that I mentioned before,
00:19:36.22 namely how to get rid of NADH,
00:19:40.16 this compound, this cofactor, that gets overaccumulated in bacteria
00:19:45.03 that are unable to grow under anaerobic conditions,
00:19:48.18 and you put them in the absence of oxygen.
00:19:51.22 Well, again, if you take a look to the pathway
00:19:54.00 that is present already in Pseudomonas putida
00:19:56.12 and you observe that you can start at pyruvate.
00:19:59.21 There's an enzyme called pyruvate decarboxylase
00:20:02.00 that will convert pyruvate into acetaldehyde.
00:20:05.01 And, well, there's another enzyme,
00:20:08.06 that is, alcohol dehydrogenase,
00:20:10.24 that could end up in ethanol.
00:20:12.18 But, as a matter of fact, when you look at the
00:20:16.02 presence of these possible enzymes in the genome,
00:20:18.14 you don't find them.
00:20:20.04 So, it is clear that you have, again,
00:20:23.24 to go out hunting for enzymes that do the job properly,
00:20:26.04 and knock them in, and see what happens.
00:20:28.24 So, the search of those two wonderful enzymes
00:20:32.10 that can start with pyruvate, end in ethanol,
00:20:35.09 and in the meantime can get rid of an excess of NADH,
00:20:38.16 can be found in various bacteria.
00:20:41.11 The ones that we were... that we were lacking,
00:20:44.16 and that work very well, was the one that comes from Zymomonas mobilis.
00:20:47.09 That is a bacterium that is an obligate anaerobe,
00:20:50.21 used in Mexican biotechnology, traditional biotechnology,
00:20:52.14 for production of alcoholic drinks.
00:20:55.09 And these two enzymes have been characterized,
00:20:57.07 and we were able to get them from this bacteria, from Zymomonas.
00:21:01.17 And then extract the sequence,
00:21:04.08 format it following the rules of synthetic biology
00:21:07.20 using our vectors and our standardized approaches,
00:21:11.13 and at the end we were able to construct an operon,
00:21:14.11 a synthetic operon,
00:21:17.03 that contained all of the activities that were necessary for meeting these two demands
00:21:22.05 that we observed out of the metabolic model
00:21:24.16 that were impeding these strains to grow in the absence of oxygen.
00:21:29.17 So, on the one hand,
00:21:31.19 we had this acetate kinase from E coli,
00:21:35.07 and then we put that together with the other two genes
00:21:39.05 that came from Zymomonas to get rid of NADH.
00:21:42.16 And we constructed a polycistronic operon
00:21:45.19 in which we had, first,
00:21:48.00 the gene from E coli and then the two other genes.
00:21:50.12 We formatted them by putting them in nice intergenic regions
00:21:54.00 with Shine-Dalgarnos,
00:21:56.08 and... well, all the, say, additions that you can get out of the growing knowledge
00:22:00.07 of how to build pathways in synthetic biology.
00:22:03.15 And, well, at the end we put that in Pseudomonas putida
00:22:06.24 and we observed that all the activities
00:22:09.15 that we expected to be expressed
00:22:11.13 were indeed expressed,
00:22:13.05 as is demonstrated in the table
00:22:15.00 that you have in the lower part of the slide.
00:22:17.12 But, that was not sufficient, of course.
00:22:19.21 Then we wondered what would be the physiological effect
00:22:24.00 of expressing these genes in Pseudomonas putida.
00:22:27.17 And these are our cells, what we observed.
00:22:29.23 And these were... the very, very good news is that when you enter...
00:22:32.21 you knock in this artificial operon in Pseudomonas putida,
00:22:36.19 then the cells survive the absence of oxygen.
00:22:40.15 So, in this graph, as you go through it,
00:22:42.22 the graph will tell you that wild type bacteria
00:22:46.01 are very, very sensitive to the lack of oxygen,
00:22:50.06 and they die off very, very quickly in the moment that oxygen is over.
00:22:54.20 However, if you knock in this artificial operon,
00:22:57.17 then you gain survival to levels
00:23:01.13 that are nearly the same as the wild type bacteria in the presence of oxygen.
00:23:05.13 So, that was very good news,
00:23:07.05 and that was an indication that one can...
00:23:09.11 one can change the physiology of the bacteria
00:23:12.01 by knocking in genes that are recruited
00:23:14.15 from very, very different places.
00:23:16.15 Okay.
00:23:17.08 So, we have our favorite bacterium growing or surviving in anaerobiosis.
00:23:23.19 Another question would be, well,
00:23:27.06 can we measure what you may call its metabolic vitality?
00:23:30.23 And to do that, what we did was to knock in
00:23:33.18 a reporter system that produces a fluorescent protein
00:23:38.02 that depends in its functioning on the levels of FMN.
00:23:42.16 That is a metabolite that is an indicator of this metabolic...
00:23:47.13 metabolic vitality that we were after.
00:23:49.19 And in these type of experiments,
00:23:51.23 we observed that whereas in the case of the wild type bacteria
00:23:54.24 the presence and absence of oxygen made a big difference
00:23:58.14 in terms of the vitality that we will observe in the cells.
00:24:01.21 Namely, in the presence of oxygen, the cells were very happy,
00:24:04.24 they had a lot of signal.
00:24:06.23 In the absence, it went down to near zero.
00:24:09.01 When you knock in this artificial operon,
00:24:10.22 then you see that the difference is decreased,
00:24:13.03 and they are very, very similar -- in the same range.
00:24:16.08 So, that means that cells now have changed their physiology, their metabolism,
00:24:20.02 and they are metabolically active in the absence of oxygen,
00:24:23.03 something that was not possible before.
00:24:26.00 Okay.
00:24:27.02 So, in that way, we already have part of the story,
00:24:29.17 that is, to have a bacterium that is able to be metabolically active
00:24:33.15 in the absence of oxygen.
00:24:36.04 But now, here comes the interesting part.
00:24:38.12 Can we use this bacterium to knock in a pathway
00:24:42.04 for degradation of a real environmental pollutant,
00:24:44.20 and make it work under anaerobic... anaerobiosis conditions?
00:24:48.20 Well, that was the next step.
00:24:50.21 And then... what you have there is a chlorinated compound.
00:24:55.03 And you see it's a dichloro compound.
00:24:58.21 And it has been put in the environment, in the soil,
00:25:02.07 as a pesticide,
00:25:05.00 as something that you use for treating various types of agricultural problems.
00:25:11.23 And it goes into the soil and it's very, very recalcitrant,
00:25:16.00 and it's really difficult to degrade.
00:25:17.22 Now, this compound is very difficult
00:25:22.04 because the extremes that are at the end of the carbon chain
00:25:26.08 are occupied by chlorides, and enzymes are really...
00:25:28.10 they have difficulties to really get into the final degradation.
00:25:32.18 However, there are strains that have been described in the past
00:25:35.13 that can take this compound, dichloropropene,
00:25:38.10 and start its degradation
00:25:42.08 by means of two key enzymes
00:25:45.10 that are encoded by other Pseudomonas strains
00:25:47.19 that are also aerobic and that manage to get this compound, dichloropropene,
00:25:52.01 from the very, very beginning all the way to CO2 and water.
00:25:54.14 And then, in this case,
00:25:56.18 the challenge was to make yet another artificial operon
00:25:59.13 in which we could clone...
00:26:02.11 we could put together in a single transcriptional unit
00:26:05.21 the various activities that were coming from another Pseudomonas
00:26:09.03 under the control of a single promoter
00:26:10.15 that could be regulated and could be controlled
00:26:13.02 at will from an external promoter.
00:26:14.23 And then we were able to find variants
00:26:17.15 that really worked very well in Pseudomonas putida,
00:26:20.00 and we could trace the activity of these variants,
00:26:22.20 because their reaction produces chloride,
00:26:25.10 and the chloride is released and can be measured...
00:26:28.14 and can be measured very accurately through analytical procedures.
00:26:31.19 So we were able to find a combination of genes
00:26:34.08 that were very efficient at degradation of these...
00:26:37.05 of these compounds.
00:26:39.15 So, at the end what we made was to put everything together.
00:26:42.15 So, we put the genes from E coli
00:26:46.01 and from Pseudomonas
00:26:49.00 for bringing about resistance to the lack of oxygen.
00:26:52.06 And then, at the same time,
00:26:54.21 we enter into the bacterium this other set of genes
00:26:57.13 for degradation of dichloropropene.
00:27:00.13 And at the end, what we did was
00:27:02.16 to put Pseudomonas putida
00:27:06.00 in the presence or in the absence of oxygen with dichloropropene
00:27:07.23 and... bingo.
00:27:09.03 It worked.
00:27:10.11 So, at the end, what we obtained was precisely
00:27:12.11 a bacterium that was able to grow
00:27:15.10 -- or at least to be metabolically active --
00:27:19.10 under anaerobic conditions,
00:27:22.02 and that was able to degrade, very efficiently,
00:27:24.24 these environmental pollutants.
00:27:27.15 And to me, this is one example of how biosynthetic technologies
00:27:30.14 and the tricks of synthetic biology...
00:27:33.02 one can really create new-to-nature properties.
00:27:36.00 Because obviously...
00:27:38.02 well, not obviously, but certainly to this point,
00:27:40.00 it's very difficult if not impossible to find bacteria of the type...
00:27:44.08 the Pseudomonas type that can degrade these compounds
00:27:46.15 when you don't have oxygen around.
00:27:48.16 Well, this is one of the examples that I wanted to share with you.
00:27:51.03 We have many others.
00:27:52.14 And I really encourage and advocate synthetic biologists
00:27:56.23 to consider not only E coli and other model systems
00:27:59.23 but also environmental bacteria
00:28:01.20 as wonderful chassis and platform genomes
00:28:05.23 for engineering all types of interesting properties
00:28:08.07 with tremendous environmental and industrial applications.
00:28:12.00 So, the final take that I want to share with you
00:28:15.01 is this concept that you can start with something
00:28:18.02 that nature gives to us
00:28:20.00 -- for instance, a tree --
00:28:21.15 and then, by using very basic techniques,
00:28:23.19 there are many things that you can do.
00:28:25.05 And you have that raft that you see in the picture.
00:28:27.11 You can go through a river.
00:28:28.24 And, well, you know, by using trial and error,
00:28:30.14 by using different homemade approaches,
00:28:33.09 more or less sophisticated,
00:28:34.22 there are many things that you can do.
00:28:36.14 But what makes a big difference is the adoption of engineering principles,
00:28:40.04 such as those that have been running in other fields for a long time,
00:28:44.12 and only in that way you can really go much, much farther
00:28:48.08 than simple trial and error could do,
00:28:50.19 as has been the case for many years in genetic engineering.
00:28:54.06 So, with this message, I leave you,
00:28:56.19 not without thanking you for your attention.
00:28:58.09 Thank you.

This Talk

Speaker: Victor de Lorenzo

Audience:

Educators of Adv. Undergrad / Grad
Researcher
Educators

Recorded: June 2015

Talk Overview

Dr. Victor de Lorenzo discusses applications of bacteria as whole-cell catalysts for decontamination and bioremediation. Dr. de Lorenzo shows that many bacteria can use pollutants as carbon sources, allowing them to decontaminate dangerous chemicals in the environment. He highlights one example of engineering the bacterium Pseudomonas putida for bioremediation, using a set of standardized tools, to metabolize 1,3-dichloropropene under anaerobic conditions. This project resulted in both enhanced natural capabilities and introduced novel functions to P. putida.

Speaker Bio

Victor de Lorenzo

Although trained as a chemist, Víctor de Lorenzo is now a Professor of Molecular Environmental Microbiology at the Centro Nacional de Biotecnología-CSIC. His lab uses Pseudomonas putida to recreate and build circuits for the sake of new-to-nature biological activities that will have an environmental impact by interacting with chemical waste. Dr. de Lorenzo is a… Continue Reading