Realizing Synthetic Carbon Dioxide Fixation

Top

00:00:13;07 Hello.
00:00:14;07 My name is Tobias Erb.
00:00:15;10 I'm a Director at the Max Planck Institute for Terrestrial Microbiology in Marburg, Germany,
00:00:19;17 and I'm a member of the Center for Synthetic Microbiology at the same place.
00:00:24;14 And today I'm going to talk to you about synthetic metabolism.
00:00:26;16 I'm gonna start my lecture by showing a range of very different chemical compounds.
00:00:33;13 And even though these compounds look very different, they all share a common principle.
00:00:38;01 And this is that they've been synthesized by synthetic organic chemists.
00:00:42;17 And in fact, the very first molecule to be synthesized by chemists has been this molecule
00:00:46;11 up here, a very simple molecule called urea.
00:00:48;22 And ever since, chemists have actually synthesized many more different and more complex molecules.
00:00:56;08 And I think it is really true to state that synthetic chemistry has been one of the
00:01:01;08 driving forces of the 20th century.
00:01:02;20 Just even think how chemistry has changed our lives.
00:01:06;05 Think about the different textiles we are able to synthesize with synthetic chemistry.
00:01:10;21 Think about different drugs -- antibiotics -- we have been able to synthesize with synthetic chemistry.
00:01:16;00 And think about different materials, for instance, like plastics.
00:01:18;17 So, synthetic chemistry has changed our lives and has been one of the sciences of the 20th century.
00:01:24;23 But I also think that synthetic biology will become the science of the 21st century.
00:01:30;07 If you think for a moment about biological systems, they have a couple of advantages
00:01:35;19 over chemical synthesis.
00:01:37;07 And one thing is that biological systems are, for instance, self-optimizing, which means
00:01:43;09 that they really can improve themselves while they're operating, and that's a concept
00:01:47;00 we would not find in chemistry.
00:01:49;07 Biological systems are self-repairing.
00:01:52;14 Think about catalysts that would be able to repair themselves once they have broken.
00:01:57;06 And biological systems are self-propagating.
00:01:59;04 Again, this is a feature you would not find in classical chemical systems.
00:02:03;08 So, there are a couple of advantages biological systems bring with them.
00:02:06;13 And on top of that, biological systems operate in a sustainable fashion.
00:02:10;17 And biological systems are also environmentally friendly.
00:02:14;08 So, one of the tasks and challenges of synthetic biology is now to really use biological systems
00:02:22;18 to do chemistry, to synthesize new compounds and new products.
00:02:27;04 And today I'm going to show you how we actually can come and reprogram the chemistry of
00:02:31;18 living cells.
00:02:33;11 And we want to do this with a concept that we will call synthetic metabolism.
00:02:36;23 So, what is the concept of synthetic metabolism?
00:02:39;03 So, that would be that we'd start off by designing theoretical pathways, basically on the paper
00:02:45;11 or in silico.
00:02:46;11 Then we would go ahead, find and engineer the individual building blocks, the enzymes
00:02:50;22 you would need to build these pathways.
00:02:52;15 We would enter a cycle where we build and optimize the individual synthetic pathways.
00:02:58;21 And finally, we'd like to transplant these pathways into cells that would be mini-factories
00:03:03;12 that could produce products for our everyday lives.
00:03:10;07 So, the central question of today's lectures will be, how do we design such
00:03:15;13 synthetic metabolic networks from scratch?
00:03:17;21 So, what are the common design principles we have to use if we want to design metabolism
00:03:22;06 from scratch?
00:03:23;18 The second question will be, how can we realize synthetic metabolic networks in the lab?
00:03:28;11 So, how can we come from the in silico, from the design part, to the actual building part?
00:03:33;21 And finally, the last question is, how can we optimize synthetic metabolic networks
00:03:38;04 once we've built them in the lab?
00:03:40;07 So basically, improving these systems in the next step.
00:03:43;16 So, I'm going to exemplify these questions by talking about research in our lab, in which
00:03:49;04 we have tried to establish a process that will be called synthetic CO2 fixation.
00:03:53;12 So, what is synthetic CO2 fixation?
00:03:55;13 And why interest... and why are we interested in this process?
00:03:58;20 Well, the answer lies in the air surrounding us.
00:04:01;02 And as you all know, CO2 is a very potent greenhouse gas.
00:04:04;22 I think we also agree on the fact that the amount of CO2 in the atmosphere has been
00:04:09;01 steadily increasing over the last years, decades, even centuries.
00:04:12;10 And of course this has severe consequences:
00:04:14;23 just think about the phenomenon of global warming.
00:04:17;14 So, it's a typical view in looking at CO2 as being a challenge and a threat for science
00:04:21;08 and society.
00:04:23;02 At the same time, we can also look at CO2 being a chance.
00:04:26;10 And being a chance because, in principle, it's a very simple and ubiquitous carbon source
00:04:31;10 that sits out in the atmosphere.
00:04:33;18 And we as scientists, we as a society, should be able to harvest this molecule and
00:04:38;22 convert it into useful compounds.
00:04:41;04 Yet we cannot do that.
00:04:43;10 So, in fact, there is no chemical process, no catalysts, no simple synthetic process
00:04:48;00 in chemistry, that would allow us to harvest atmospheric CO2 and to convert CO2
00:04:53;04 in a sustainable fashion into a given multi-carbon compound.
00:04:57;11 And all our chemical efforts are still outcompeted by biology that fixes carbon dioxide on
00:05:03;05 a gigaton-scale per year.
00:05:04;11 And I think we all know very well the process of photosynthesis that takes, for instance,
00:05:08;18 place in plants, cyanobacteria, or algae.
00:05:12;06 Yet, biology is not perfect.
00:05:14;09 And it's not perfect because the systems have evolved over time, and they were not designed
00:05:18;21 a priori.
00:05:19;21 And one problem, for instance, is that these systems are not running under optimal conditions,
00:05:24;15 so they're not necessarily very fast.
00:05:26;16 And another problem is that, for instance, the product of biological CO2 fixation is biomass.
00:05:33;01 And biomass is not necessarily something that you can directly use in the chemical industry.
00:05:38;16 And this has inspired us, now, to really rethink biological CO2 fixation.
00:05:43;07 And we've come up with the question, so, what if... what if you were actually able to find
00:05:47;11 or design new CO2 fixation pathways, pathways that would allow us, for instance,
00:05:53;14 to convert CO2 directly into interesting products, or pathways that would operate faster than
00:06:00;08 naturally evolved pathways?
00:06:02;17 And this is then what we would consider for a name, synthetic CO2 fixation.
00:06:07;16 So, apparently, one critical step in fixing carbon dioxide is to identify enzymes
00:06:14;11 that are fixing carbon dioxide efficiently.
00:06:17;10 So the very first step in this project has been to identify enzymes that could fix CO2
00:06:22;02 very efficiently.
00:06:24;12 This slide reminds you of different... defining the efficiency of enzymes.
00:06:28;19 So, we want to actually have enzymes that have a very high kcat number, a very high turnover number,
00:06:35;13 which means that they can convert a given substrate molecule at a high speed.
00:06:42;00 So, that's one part.
00:06:43;19 The second part is the enzyme should actually have a very low Michaelis constant, a so-called
00:06:47;21 Km value.
00:06:48;21 So, this will tell you that these enzymes can actually also operate at very low substrate concentrations.
00:06:54;14 And what you want to have at the end is an enzyme that is operating at a high turnover rate
00:06:59;08 at very low substrate concentrations.
00:07:01;21 And this is what we would then call a catalytically efficient enzyme, so, kcat divided by Km.
00:07:06;18 And think about... for a moment about CO2 fixation.
00:07:09;19 So, carbon dioxide is at very low concentration in the atmosphere, only 0.04%.
00:07:14;00 And you want to have enzymes that can fix carbon dioxide very efficiently at these
00:07:18;08 low concentrations.
00:07:19;08 So, one of the first steps in this project has basically been to identify enzymes
00:07:25;05 that would fix carbon dioxide in an efficient manner under atmospheric concentrations.
00:07:28;22 And so what we have done is, in the first step... is going through biological databases
00:07:33;11 and identify the catalytic efficiency of different enzyme classes.
00:07:37;05 And what we see here is basically different enzyme classes plotted according to their
00:07:42;11 catalytic efficiencies.
00:07:44;15 And we were surprised to learn that the enzyme that is used in plants to fix carbon dioxide,
00:07:49;16 RubisCO, is not the most efficient enzyme.
00:07:52;03 In fact, there are other enzymes that can fix carbon dioxide more efficiently.
00:07:56;01 And among these enzymes are so-called enoyl-CoA carboxylases/reductases or, for short, ECRs.
00:08:01;15 And these enzymes are actually up to four times more efficient than RubisCO, the enzyme
00:08:05;21 that is used in plant photosynthesis.
00:08:07;19 So, what are these enzymes?
00:08:10;11 Well, the prime example is the so-called crotonyl-CoA carboxylase/reductase, that basically takes
00:08:17;01 a molecule, crotonyl-CoA, reduces this molecule of crotonyl-CoA with NADPH as cofactor,
00:08:21;20 and introduces a CO2 molecule to give rise to the carboxylated product, ethylmalonyl-CoA.
00:08:28;09 It's very nice in principle to be used, but it is only one reaction.
00:08:32;23 And ideally you want to have actually multiple reactions; you want have a set of enzymes
00:08:36;12 that can do very different carboxylation reactions.
00:08:39;16 And so one of our first efforts has actually been to open up and expand
00:08:43;17 the biosynthetic space of this class of enzymes.
00:08:45;22 So, we've asked the question, can we now fit other substrates into the active site of
00:08:52;14 these enzymes?
00:08:53;14 Can we expand the substrate scope, the substrate spectrum of these enzymes, to catalyze other reactions,
00:08:57;23 just besides crotonyl-CoA?
00:09:00;02 We've addressed this in two different approaches.
00:09:03;13 One way has been we've used enzyme engineering, so we've used crystal structures of the enzyme,
00:09:09;07 we've looked at the active-site pocket of these enzymes, and we've tried to open the
00:09:12;19 active-site pockets so that other substrates could fit in.
00:09:17;07 Another approach has been to use genomic mining, which means that you go ahead and you
00:09:22;02 just screen the biological diversity of this class of enzymes.
00:09:24;09 So in other words, we use the phylogenetic tree of these enzymes, then you synthesize
00:09:30;14 30/50/100 different variants, and you simply test the substrate spectrum
00:09:36;05 of these different enzymes.
00:09:38;15 And with these two approaches, we were actually able to identify a set of enzymes that catalyzes
00:09:44;10 very different carboxylation reactions.
00:09:47;02 So, we can now have enzymes that catalyze short substrates, and have enzymes that can
00:09:52;10 carboxylate longer substrates, branched substrates, bulky substrates, differently decorated substrates.
00:09:58;14 Or in other words, we have now a set of enzymes at hand that can carboxylate, with high efficiency,
00:10:04;13 very different reactions.
00:10:06;22 So, how can we now use these enzymes to build synthetic CO2 fixation pathways?
00:10:13;17 How can we come from an individual enzyme reaction to a complete pathway?
00:10:18;11 And this is now the second part of my talk, where I want to show you how we can
00:10:21;18 design synthetic metabolic networks from first principles.
00:10:25;15 So, what we do in the lab is we simply start with a blank piece of paper.
00:10:30;21 And we start to draw artificial synthetic theoretical pathways.
00:10:34;20 And all these pathways are very different, but all these pathways are centered on
00:10:40;10 an individual ECR reaction.
00:10:45;16 And even though all these pathways have different topologies, and even though these pathways
00:10:49;10 have different intermediates, they also share some common principles.
00:10:53;17 And one principle is that all these pathways are actually cycles.
00:10:58;04 What does it mean?
00:10:59;13 Well, basically, the product of a carboxylation reaction is turned back to become the substrate
00:11:06;10 of the carboxylation reaction.
00:11:08;09 And in this way, you can basically run, in a continuous fashion, carbon dioxide fixation,
00:11:14;02 which means you can really fix carbon dioxide continuously.
00:11:17;12 That's the principle of a metabolic cycle.
00:11:19;17 So, another common principle is that all these pathways are, again, based on an ECR reaction.
00:11:27;06 And a third principle is that all these pathways have a carbon-splitting reaction, for instance,
00:11:31;11 shown here, where we can take off some of the fixed carbon, and you can shuttle this carbon
00:11:36;11 into an interesting pattern, technological compound, or biological building block.
00:11:41;19 Again, these are theoretical cycles.
00:11:44;01 Don't forget that at this stage of the design we don't have any specific enzymes in mind
00:11:48;04 besides the ECR reaction.
00:11:49;17 We just think about potential transformations.
00:11:51;15 We just think about oxidations, potential oxidations.
00:11:54;18 We think about potential reduction reactions.
00:11:57;17 We think about potential CC-splitting reactions.
00:12:00;05 We think about potential hydration or dehydration reactions.
00:12:03;19 So, these are all theoretical enzymes that might exist.
00:12:07;12 So, from this design, we actually also have to decide which of these cycles is now
00:12:12;10 a really good cycle.
00:12:13;12 So, we have to evaluate the different cycles we have drawn.
00:12:16;20 And so we've used simple chemical criteria to decide which of these cycles is actually
00:12:20;21 the best cycle we realized.
00:12:22;03 And one of the important criteria is here: you need to have cycles that are kinetically favored.
00:12:28;13 This means you want to have cycles where you actually have fast and efficient enzyme reactions involved.
00:12:33;12 So all the individual reactions you would like to apply in your cycle should be fast and efficient,
00:12:37;15 which would allow us to run the cycle in an efficient manner.
00:12:42;16 A second criterion is actually a thermodynamic criterion.
00:12:45;16 So, all the cycles should be thermodynamically favored, which means you need to think
00:12:50;12 how much energy per CO2 molecule fixed do you have to spend.
00:12:55;22 And so we've done this calculation, for instance, for the CETCH cycle shown here.
00:12:59;08 So, the CETCH cycle can turn two times to produce one molecule of pyruvate.
00:13:04;10 And while turning two times, it actually has to spend four ATPs for one pyruvate molecule
00:13:09;19 fixed from CO2.
00:13:11;02 Now, you can compare this ATP efficiency with natural existing pathways.
00:13:17;02 And you can actually see that the Calvin cycle -- the pathway that is used by plants, bacteria,
00:13:22;14 and algae in photosynthesis -- uses seven ATP per CO2.
00:13:27;02 So, in other words, the synthetic solution from the lab is almost twice as energy-efficient
00:13:33;01 in terms of ATP usage.
00:13:34;05 So... which tells you already that you actually can come up with solutions which are
00:13:38;08 more ATP-efficient than naturally evolved systems.
00:13:41;08 And finally, the last criterion you want to actually apply is the criterion of thermodynamic feasibility.
00:13:49;04 So, we want to make sure that all the reactions you want to apply in your cycle have a favorable
00:13:54;20 thermodynamic equilibrium.
00:13:56;06 So, in other words, the concentration and the equilibrium of the... of the reaction
00:14:02;09 should lie on the side of the product formation.
00:14:04;06 In other words, the free-energy profile across the reaction should be negative.
00:14:09;12 And this will allow you to turn the cycle in a clockwise-fashion efficiently,
00:14:14;03 without any major humps.
00:14:16;03 So, having identified and... and evaluated the cycles, we came up with the idea that
00:14:23;09 the CETCH cycle, that fulfills all these three criteria, would be a very good starting point
00:14:27;23 to build, now, synthetic CO2 fixation in the lab.
00:14:30;09 So, this brings me into the second part, where I want to tell you how we can now realize
00:14:35;07 such synthetic metabolic networks from first principles.
00:14:38;16 So, on the one hand, we now have this theoretical cycle that we've drawn on the paper and that
00:14:46;10 we've evaluated.
00:14:47;10 On the other hand, we actually want to realize this cycle and build this cycle,
00:14:50;14 which means we need to find all the individual biological parts.
00:14:54;03 And a network... a metabolic network like the CETCH cycle, that features 15 different reactions,
00:14:58;13 means that you need to find 15 different enzymes that could do all the different transformations.
00:15:03;18 So what can you do?
00:15:05;12 You basically can go back and you can go through different databases, like BRENDA, KEGG, STRENDA,
00:15:12;17 or PubMed, to find enzymes that would do one or the other transformation.
00:15:19;05 So right now, we have more than 50 million annotated genes in databases, and we have
00:15:23;05 more than 40,000 characterized enzymes in databases, so the chances are very high
00:15:27;10 that you would find the enzyme reaction somewhere.
00:15:30;20 Of course, you still need to express the enzyme, you need to purify the enzyme, and you to
00:15:34;06 test the enzyme.
00:15:36;04 But in... ideally, you can find a lot of enzymes in these databases.
00:15:40;15 Yet, sometimes you find an enzyme in the database, but it does not have the properties it should have.
00:15:46;16 And this is shown here, where we were looking for an enzyme that could catalyze this reaction
00:15:51;00 up here, which is a dehydration reaction of a compound called through 4-hydroxybutyryl-CoA.
00:15:55;11 So, in fact, there has been an enzyme that was deposited in the database.
00:16:00;12 The problem is that this enzyme is highly oxygen-sensitive, and actually dies away.
00:16:04;23 Within 15 or 20 minutes, it loses all its activity.
00:16:08;21 So, how can you overcome this problem?
00:16:11;07 How can you find a better biological part?
00:16:13;06 Well, one strategy is that you simply go ahead and you screen for biological diversity.
00:16:18;21 So in other words, you try to express and test homologues of this enzyme.
00:16:22;06 And so, lo and behold, by using this strategy we identified a homologue, in a marine archaeon
00:16:28;17 called Nitrosopumilus maritimus, which shared 50 or 60% of sequence identity but was
00:16:34;22 approximately 100 times more stable in oxic conditions.
00:16:37;12 So, looking for biological parts and screening different enzyme homologues can help to identify enzymes.
00:16:44;08 But sometimes you have the problem that you would like to employ a certain enzyme reaction,
00:16:48;11 but you actually don't have an enzyme in the database.
00:16:51;18 And this is shown here, where you want to use a reaction in which a compound called
00:16:55;20 methylsuccinyl-CoA is oxidized to become a compound called mesaconyl-CoA.
00:17:00;18 Well, there had been an enzyme described that can do this chemistry.
00:17:04;01 The problem is that these enzymes are so-called dehydrogenases.
00:17:08;05 And these dehydrogenases... dehydrogenases... dehydrogenases are very complicated enzymes,
00:17:12;03 because they oxidize the substrate, put the electrons onto a cofactor, and from this cofactor
00:17:18;10 the electrons are put on a second protein, called electron-transfer flower protein,
00:17:23;00 and from there the electrons are put into a membrane-bound respiratory chain.
00:17:27;00 So in other words, if we want to use this kind of chemistry, we need not only the enzyme,
00:17:31;13 but you also need its interaction partners, and you need a membrane-bound respiratory chain,
00:17:35;06 which is awfully complicated.
00:17:36;17 So in this case, it might be helpful if you simply shortcut this whole electron transfer relay.
00:17:43;09 And what we intended to do is to convert this methylsuccinyl-CoA dehydrogenase into
00:17:49;10 a methylsuccinyl-CoA oxidase, an enzyme that could directly use oxygen instead of having this
00:17:54;20 complicated electron transfer chemistry.
00:17:57;13 So, what you need to do here is you need to look into the active site of the enzyme.
00:18:02;15 And if you think about ways how it, for instance, accommodates an oxygen molecule there.
00:18:05;21 So, you can open up the active site by creating a bit more space.
00:18:10;09 You can think about adopting and kind of allowing for a negative charge to be developed,
00:18:16;12 so putting more positively charged amino acids in the active site.
00:18:19;12 And then you simply can go ahead and can test different active site mutations.
00:18:23;08 And when we did this, we actually ended up and identified a triple-mutant that could,
00:18:30;01 indeed, catalyze the direct oxidation of methylsuccinyl-CoA in the presence of molecular oxygen.
00:18:36;14 Or, in other words, what we did here is... is we basically created our own enzyme
00:18:41;15 for our own pathway.
00:18:46;06 The question now is, at the end of the day, we had to identify 15 different enzymes from
00:18:50;22 for very different organisms.
00:18:52;14 So, we had enzymes in there for Methylobacterium extorquens.
00:18:56;03 It's a plant organism, a plant-associated organism.
00:18:59;22 We had enzymes that we would take from Rhodobacter sphaeroides, an organism we would find in ponds.
00:19:04;06 We would have enzymes that we've taken from Clostridium, which is a human pathogen.
00:19:08;21 We have enzymes that were from E. coli, a well-known lab organism.
00:19:13;20 And we would actually have enzymes from Nitrosopumilus maritimus, a marine archaeon.
00:19:19;13 And even an enzyme from the human liver.
00:19:21;12 And on top of it, our re-engineered enzyme that catalyzes the oxidation of methylsuccinyl-CoA.
00:19:29;04 The big question is, now, can you bring all these enzymes together?
00:19:31;21 And would they be able to fix carbon dioxide in an efficient manner?
00:19:34;16 So, we've done the experiment.
00:19:36;21 So, we've used an Eppendorf cup.
00:19:38;22 And we've simply pipetted all 15 enzymes into this Eppendorf.
00:19:42;17 We've added ATP and NADPH.
00:19:45;11 And we've also added this green compound down here, which is called propionyl-CoA.
00:19:50;04 The idea being, if the cycle is active, it would start to turn.
00:19:54;07 And for each time it starts to turn, it would actually accumulate a compound called malate,
00:19:59;15 which would be the direct carboxylation product.
00:20:01;17 So, in other words, if we follow malate concentration over time, we would be able to see if
00:20:06;10 the cycle is active or not.
00:20:08;00 And as you can see here, indeed, we could demonstrate that the CETCH cycle fixes carbon dioxide
00:20:13;00 over time, and actually at a rate of 0.2 CO2 molecules per acceptor and hour.
00:20:19;03 And this really demonstrates to you that you can come up with a theoretical pathway on the paper,
00:20:23;10 you can look for different enzymes, or even engineer your own enzymes,
00:20:28;06 you can bring these enzymes together, and such a cycle will be functional.
00:20:31;02 So, you can design biological systems just by using chemical criteria.
00:20:36;14 Yet, I hope you also realize that the cycle isn't turning perfectly.
00:20:41;03 So, the rate is actually per acceptor and hour, and we all know that biological systems
00:20:45;12 are operating at smaller timescales, so they should be operating per minute or per second.
00:20:50;07 So, the question here now is, why is the cycle not turning faster?
00:20:54;21 So in other words, which design principle did we miss?
00:20:58;15 And this brings me to the second part of my talk, where I want to tell you how we can
00:21:02;18 optimize such synthetic metabolic networks by including biological design principles.
00:21:07;17 Or, in other words, it's not enough to think about the biochemistry and the chemistry of
00:21:13;02 enzymes in metabolic networks, but it's also important to think about the biology.
00:21:18;14 So, revisiting the question, why is the cycle not turning faster?
00:21:22;00 What actually limits CO2 fixation efficiency of the cycle?
00:21:24;20 And the answer is very simple, and the answer isn't very surprising.
00:21:28;17 The point is that we've brought together very different enzymes from very different biological backgrounds.
00:21:34;12 And these enzymes do not have a common evolutionary history.
00:21:37;18 And these enzymes have not co-evolved together in this network.
00:21:40;21 And these enzymes are doing things they are not supposed to do.
00:21:42;23 In other words, these enzymes do side reactions.
00:21:47;09 And the whole metabolic network accumulates dead-end products like malyl-CoA, or for instance
00:21:52;12 like malonyl-CoA.
00:21:56;10 So, to optimize, now, CO2 fixation efficiency, what we have to do is we have to go back and
00:22:01;02 we have to debug the system.
00:22:02;14 So, we have to teach the system to be more specific.
00:22:04;21 We have to teach the enzymes to work together.
00:22:07;04 And we can apply very different principles.
00:22:09;22 So, one principle would, for instance, be that we can add more complexity to the system.
00:22:14;18 So remember, I talked about this compound called malyl-CoA, which is a dead-end metabolite,
00:22:19;18 and which holds the cycle back from turning more efficiently.
00:22:22;06 So, there might be way to actually remove this dead-end compound by another enzyme,
00:22:28;09 which is a so-called thioesterase.
00:22:30;14 And this thioesterase would cleave malyl-CoA into malate, as shown here, and it would
00:22:37;13 release the CoA residue, which could be fed back into the cycle.
00:22:41;13 So in other words, by increasing complexity of the cycle, by adding an additional enzyme
00:22:45;14 that would proofread and scavenge and recycle this dead-end metabolite, we would be able
00:22:50;02 to improve CO2 fixation efficiency.
00:22:53;08 And in fact, what you can see as soon as you add this thioesterase, you ramp up CO2 fixation efficiency.
00:22:59;07 And I think this is a very important principle, this metabolic proofreading, that we can use
00:23:04;07 in synthetic biology, I think it's also important to realize that in many biological systems
00:23:08;19 such metabolic proofreading principles are also operating.
00:23:14;00 So, that's one way -- metabolic proofreading -- to improve efficiency.
00:23:18;16 Another way we can... we can use is basically redesigning our original strategy.
00:23:22;23 And we have used this, for instance, to circumvent a reaction from propionyl-CoA to this compound,
00:23:28;08 here, called methylmalonyl-CoA.
00:23:29;20 This was not a reaction that we could use because it made too many problems.
00:23:34;10 So, we had to go back and detour the whole reaction by going through a compound
00:23:38;11 called acrylyl-CoA, and, from there, on then back to this compound, methylmalonyl-CoA.
00:23:42;19 So, pathway redesign was very useful to us to identify enzymes that could do the job.
00:23:48;20 And one other part of these design principles, of course, is to engineer enzymes to be more specific.
00:23:54;03 And we have used this, for instance, to improve and get rid of a side reaction up here.
00:23:58;08 So in other words, pathway redesign and enzyme engineering can help us also
00:24:05;18 to improve CO2 fixation efficiently, again.
00:24:07;11 And finally, playing around with the physical conditions, for instance improving and optimizing
00:24:12;20 buffer concentration and playing around with ionic strength, can also help us, then,
00:24:17;19 to finally speed up CO2 fixation efficiency.
00:24:21;02 So at the end of the day, without changing the chemistry of this cycle, just by
00:24:26;06 simply optimizing the enzymes, by adding metabolic proofreading, enzyme engineering, and pathway...
00:24:30;20 slight pathway redesign, we ended up with a cycle which was now turning
00:24:34;08 at a rate of 3.6 CO2 molecules per acceptor and hour.
00:24:38;13 In other words, implementing biological design principles has helped us to increase CO2 fixation
00:24:44;09 efficiency of the whole system by almost a factor of 20.
00:24:48;05 I guess what I want to say here is that you can really think about having really good individual players,
00:24:55;05 you can really think about getting the best enzymes together, and it's
00:24:58;10 a little bit like the Dutch national team.
00:25:00;02 They have really good individual players.
00:25:02;11 But at the end of the day, you need to form a team if you want to win championships.
00:25:05;13 So in other words, it's not only important to think about the chemistry and the biochemistry
00:25:08;18 of the enzymes, but it's also important to think about the enzymes working together efficiently.
00:25:16;02 So, this is where we're standing right now with the CETCH cycle.
00:25:18;20 It's in version 5.4.
00:25:20;07 The CETCH cycle now features 17 different enzymes from 9 different organisms, and includes,
00:25:24;23 now, 3 re-engineered enzymes.
00:25:26;15 And it fixes carbon dioxide at a rate of 5 nanomoles per minute and milligram of protein.
00:25:33;12 And this is actually something that you would be able to measure if you, for instance,
00:25:37;03 tried to measure other CO2-fixing... natural CO2-fixing organisms in vitro.
00:25:40;12 So, with this system, we are almost... very close to biological ability, at least in vitro.
00:25:45;19 Of course, the CETCH cycle is not, in its version, 5.4, the last version.
00:25:50;15 But I think it's... it's not the end.
00:25:52;09 I think it's a starting point for several research directions.
00:25:56;16 And one thing we now can do is entering the so-called design-build-test-and analyze cycle,
00:26:02;14 which is a classical improvement that you can use in synthetic biology.
00:26:07;04 So, the idea would be here, now, to test different principles.
00:26:10;19 So, where are the boundary conditions of our system?
00:26:14;08 How much can it be improved when we optimize enzyme concentrations and metabolite concentrations?
00:26:20;16 How can it be improved when you, for instance, add allosteric regulation?
00:26:24;06 Can it be improved if we try to implement metabolite channeling?
00:26:27;15 And will it actually be improved if you, for instance, tried to implement something
00:26:31;17 like compartmentalization?
00:26:32;17 And I think, through iterative rounds of reconstructing a system, and by changing parameters,
00:26:37;20 we will be able to improve the CETCH cycle even further.
00:26:42;15 What are the next steps once we have an improved CETCH cycle?
00:26:44;23 So, one idea would be to use the cycle in a true bottom-up fashion, so using this
00:26:50;04 as a metabolic module that we, for instance, could use to implement into cell or cell-like strutures
00:26:55;15 -- artificial cells, membranes, liposomes -- where we can, for instance,
00:27:00;22 feed in light, we can feed in hydrogen or electricity, where we can energize the system and use our
00:27:06;01 metabolic module to fix carbon dioxide.
00:27:07;15 If you want to say... so, for instance, developing an artificial organelle, an artificial chloroplast.
00:27:13;18 And of course, another thing we can do is we can try to implement and try to transplant
00:27:18;00 the CETCH circuit in a top-down fashion into living systems.
00:27:21;06 So, the in vivo transplantation has some of the big challenges that lie ahead of us.
00:27:25;16 I hope you can also understand that a complex network like the CETCH cycle,
00:27:29;20 that consists of 17 different enzymes, might not be very easily transplanted into a living system
00:27:35;00 that features 3000 different reactions.
00:27:37;00 And we expect this will be a challenge of synthetic biology.
00:27:41;06 Okay.
00:27:43;22 So, this is the end of my lecture.
00:27:45;22 I hope I could show you that synthetic metabolism is really something that we can achieve in
00:27:50;04 the future in synthetic biology.
00:27:52;15 And I hope I could show you that synthetic metabolism is built on the concept of being
00:27:56;08 freely able to choose and to design theoretical pathways on the paper.
00:28:00;14 I hope I could show you that synthetic biology involves finding and engineering enzymes to...
00:28:06;04 to realize this cycle in the lab.
00:28:07;23 But it also involves building and optimization of the pathway sequence, in iterative rounds.
00:28:13;21 I hope I could show you that one of the future steps in synthetic metabolism will be to
00:28:17;17 transplant such artificial cycles into living cells, rather it be natural or artificial cells.
00:28:24;10 That's the end of my talk.
00:28:25;21 And I would not be standing here without a great number of great scientists in my lab.
00:28:29;00 I just want to highlight Thomas Schwander, who actually build the CETCH cycle as
00:28:33;14 first proof of principle.
00:28:34;23 And I want to highlight those people that have been optimizing
00:28:37;16 the biochemistry of enoyl-CoA carboxylases/reductases.
00:28:42;01 Of course, I should not forget the funding agencies.
00:28:44;12 And I thank you for your attention.

This Talk

Speaker: Tobias Erb

Audience:

Educators of Adv. Undergrad / Grad
Researcher

Recorded: September 2017

Talk Overview

The conversion of atmospheric carbon dioxide (CO₂) to biomass via photosynthesis is the foundation for all of our food and energy. Tobias Erb explains how his lab is working to design, build and optimize pathways for synthetic CO₂ fixation. By combining enzymes from multiple organisms with “re-engineered” enzymes and optimizing the processes, Erb and his lab generated a synthetic cycle that fixes CO₂ more energy efficiently than photosynthesis. In the future, they plan to test the system in artificial cells and to transplant it into bacteria and chloroplasts. The video exemplifies the general rules and principles of building synthetic metabolism.

Speaker Bio

Tobias Erb

Tobias Erb studied biology and chemistry at the University of Freiburg and Ohio State University. He was a postdoctoral fellow at the University of Illinois before starting his own group at the Swiss Federal Institute of Technology (ETH) in Zurich, Switzerland. In 2014, Erb moved to the Max Planck Institute for Terrestrial Microbiology in Marburg,… Continue Reading