Molecular gastronomy

Every successful serial includes an episode that tells the story of how it all began, and the story of life itself is no exception. It too contains a chapter about origins. Prior to the inception of biological evolution as we now know it, there must have a phase of chemical evolution that gave rise to the molecular precursors of cells. What did this prequel look like? How were the first building blocks formed out of which living systems were later assembled? What conditions were necessary to bring molecules together and interact to form complex informational units that had the ability to replicate themselves – the precursors of what we now call the hereditary material of cells?

Research on the origin of life has become an autonomous field with its own specific methodologies – and Munich and LMU have become one of its internationally acknowledged centers. Several dozen researchers are now working on a whole series of linked projects in a dedicated Collaborative Research Center, and as part of the ORIGINS Cluster of Excellence (both funded by the DFG), with further support in the form of generous grants awarded to individuals by the European Research Council.

Putting together a compelling plot for this story turns out to be a difficult challenge. Because there are many possible theories and scenarios, and still more details to be considered, researchers must first develop plausible storylines. What were the physical conditions that prevailed on the early Earth? What types of chemical reactions could have taken place? Could the resulting products have included substances like those found in the informational molecules now found in cells? And, if so, might these compounds have been able to trigger an evolutionary process at the chemical level? The methods available to modern chemists, integrated into an interdisciplinary context, provide a means of reconstructing such a scenario. The aim of these efforts is to construct a plausible and comprehensive description of such a pathway based on rigorous experimental evidence.

LMU chemists Thomas Carell and Oliver Trapp, together with Dieter Braun, a biophysicist, have long been involved with these questions. In the following conversation, they outline their approaches, which combine the design of model reactions with their verification by experimental reconstruction. So the tale oscillates between reflections on plausible settings on the early Earth and the acid test of reactions in the test tube.

What was in the beginning: words about the origin of life

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If you were asked to narrate the story of life on Earth, a natural scientist’s version of the Book of Genesis, where would you begin?

Braun: With the evolution of stars and planets. Under the influence of gravity, the particles in a rotating cloud of gas and dust evolve into a central star surrounded by a flat disk, within which they clump into discrete bodies which ultimately form planets.

Trapp: In this process, chemistry is already at work. In the gas phase, highly reactive substances called radicals form. Organic chemistry – the chemistry of carbon compounds, which will later serve as the material basis of all living organisms – has not yet begun in earnest. These reactions require metals, especially iron, as catalysts. In my laboratory, we try to work out what sorts of organic compounds could have been synthesized under such conditions. It turns out to be a whole plethora of molecules, which give rise to more and more organic substances. Over the course of hundreds of millions of years, this menagerie comes to include the set of prebiotic molecules from which living systems subsequently evolved.

When did the Earth form?

Braun: About 4.43 billion years ago – and it was the product of a very turbulent process.

Carell: Shortly after the Earth formed, it collided with a body about the size of Mars, the protoplanet Theia. The impact was so immense that it melted the whole of Earth‘s surface, converting it into a fireball and consuming all of the existing organic molecules. The surviving fragments of Theia eventually gave rise to the Moon. This whole episode acted as a reset.

Braun: It’s still not clear exactly how the Moon was formed. There are several competing models. Some scientists argue that there were several later bombardments of the Earth. But the simulations are getting more realistic, and with them the calculations of what the early atmosphere looked like.

Trapp: Geologists now agree that the atmosphere of the early Earth contained very little oxygen and was probably rich in hydrogen, which is logical. After all, the water that condensed on the Earth reacted with metals such as iron, which produced iron oxides and lots of molecular hydrogen. The hydrogen-rich atmosphere in turn provided a favorable environment for the formation of organic compounds.

Carell: After its encounter with Theia, the Earth was molten. Elements such as gold, silver and iridium are now found in the Earth’s crust, but at that time they will have been dissolved in the molten iron. There are no native metals on the Moon.

So where did Earth’s gold and silver come from?

Carell: There was probably a second impact, which was more of a glancing blow, most likely a meteorite with an iron core that then broke up. The Earth melted for a second time, and the fragments of the meteorite collected around it. These included a large fraction of molten iron, which rained down on the Earth and fell on water that had already cooled, with which the metal reacted. For the next few hundred million years, Earth had a hydrogen-rich atmosphere – what chemists call a strongly reducing atmosphere, in which the first compounds needed for life formed.

Is the timing right?

Carell: Yes, about 4 billion years ago.

Braun: All this can be deduced from analyses of the earliest Earth-derived rocks, such as those collected on the Moon by Apollo-17 mission. Rocks from the early Earth can be found on the surface of the Moon, because plate tectonics never got started there – while Earth’s surface was repeatedly reworked by subduction. These “moon rocks” tell us what was going on when the Earth was young.

What happened after that?

Carell: The hydrogen atmosphere was replaced by one that was dominated by nitrogen, and contained far more carbon dioxide than it does today, as well as a few noble gases and water vapor, but no molecular oxygen. In an atmosphere comprised largely of nitrogen and carbon dioxide, frequent lightning discharges can be expected, which makes chemistry possible.

However, this doesn’t tell us where prebiotic chemistry might have taken place. A recent piece in the journal Science cited evidence suggesting that continents first emerged about 3.5 billion years ago. Before that, the Earth was a water world. Do you think that’s plausible?

Carell: That’s what some people believe. But it would make chemistry difficult. It’s difficult to envisage synthesis of the bases found in nucleic acids in an aqueous environment, as their chemical precursors would be highly diluted.

Braun: It’s certainly true that there was lots of water about. But that doesn’t rule out the presence of crustal fragments the size of Iceland or Hawaii. Geologists have yet to agree on the size of the first continents, when temperatures began to fall below 100°C.

Carell: Many chemical processes can only get started if the precursors are sufficiently concentrated. That’s why we believe that terrestrial settings with geothermal fields and volcanic activity, like those found on Iceland or in Yellowstone Park today, or shallow ponds, offer the necessary conditions.

So you favor the pond hypothesis?

Carell: Yes, because ponds can dry out, and then it rains again. They are exposed to periods of cold, drought and dampness, circadian and seasonal cycles – and these fluctuations make interesting chemistry possible. We routinely test the influence of these cycles in our experiments over three or four periods, and we have found intriguing processes that operate without further intervention on our part. They promote the synthesis of interesting molecules. Some look more like amino acids, the basic subunits of proteins, others resemble nucleobases.

Darwin himself proposed that life might have originated in small, warm ponds. Might life have emerged from mud, so to speak?

Carell: Mud is humus, organic material, the product of decaying leaves and myriads of bacteria – and all that was missing. The setting we have in mind is more like the tree line in the Alps, with lots of eroding rock debris.

Trapp: Or the vicinity of hot springs such as those on Iceland.

Braun: In volcanic regions there is lots of ash and dust, both of which readily react with water to form mud-like molecules.

Carell: Talking of ponds, we have found a setting in which all four components of RNA can be synthesized under very similar conditions – all ingredients of the soup can be prepared in the same pot, as it were. Prior to that, no one believed that was possible. It is highly likely that electrical discharges took place in the nitrogen-hydrogen atmosphere of the early Earth, as well as volcanic activity that produced large amounts of sulfur dioxide.

And you used these agents in your experiments?

Carell: In our experiments we use simple chemicals like ammonia, urea and formic acid, together with nitrite and carbonate salts, and metals such as iron and zinc. That’s enough to initiate a reaction cascade that leads to the synthesis of the four bases found in RNA. Though I should add that we needed three ‘ponds’ in our scenario. We assumed that each was underlain by different types of rocks. It’s not necessary for them to be located adjacent to each other, but they must be in contact. What’s important is that they are exposed to episodes of flooding and drought. We are now reconstructing such a pond scenario in the laboratory, based on a landscape constructed from glass reaction vessels in which we simulate the original conditions. Our goal is to perform a Miller-2.0 experiment, an improved version of Stanley Miller’s classical Ursuppe experiments of 1953.

Trapp: We have the experiment set up in the laboratory, with electrical discharge and everything else. Our focus differs from that of Thomas Carell. He concentrates on the synthesis of RNA, while my primary interest is in DNA synthesis. These two genetic materials are closely related. We begin with meteoritic rock in the form of nanoparticles. These particles are highly reactive catalytic agents that promote the synthesis of small and simple molecules, starting from carbon dioxide and hydrogen. The products include formaldehyde, acetaldehyde, alcohols, alkanes and short fatty acids. And we have shown that these chemical reactions proceed in a reproducible fashion, even under very different conditions.

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What do these experiments tell you?

Trapp: Our experiments connect two processes: the formation of sugars and the synthesis of the bases in nucleic acids – the two fundamental components of the nucleotides from which DNA is built. We have made a number of interesting observations. Other researchers often ask us why nature selected ribose as the sugar in RNA. We have shown that this choice is a consequence of the primordial chemical processes that gave rise to it. The sugar and the bases are natural partners. – The important thing is that the finished sugar is not attached to the base, as previously thought. Instead, it is assembled onto the finished base. This in turn suggests that precursors of DNA may have emerged about 400 million years earlier than hitherto assumed.

Researchers have been invoking an earlier RNA world for decades.

Carell: The ‘RNA world’ is simply shorthand for a nucleic-acid world. Whether RNA or DNA came first is immaterial for the concept. The concept of an RNA world arose because, biosynthetically speaking, RNA is the precursor of DNA. But DNA may well have originated first. And many scientists contend that other sugars, such as tetroses, could have played a role early on. When I connect bases with a 4-carbon sugar instead of the 5-carbon ribose, what I get is neither DNA nor RNA, but TNA, and the bases pair up very well. So we can get double-stranded molecules analogous to DNA. Indeed, that’s the feature that enables the hereditary material to be replicated. What we seek is a nucleic-acid world that gives rise to self-replicating molecules which have properties that permit the emergence of selection at the molecular level, and allow one to postulate a molecular version of Darwinian selection. In this context, it probably doesn’t matter which sugar is chosen.

Braun: It’s also conceivable that a division of labor between RNA and DNA emerged early on, with DNA serving as a form of long-term information storage, while RNAs had shorter half-lives.

Carell: Perhaps many systems were in competition with each other, and the system that won out is the one we have today. But I wouldn’t stick my neck out for any of these theories.

No matter how they work in detail, evolutionary systems require some form of selection: What favors survival and what doesn’t? Some of your experiments have explored whether non-equilibrium conditions and temperature differences can initiate selection processes in populations of RNA molecules.

Braun: Living systems as we know them can’t be put in an empty glass and continue to grow. They must be fed regularly, they need nutrients or – as we physicists would say – they must be kept out of equilibrium. These systems interact with their environment and exchange energy and matter with it. So the crucial question is what types of non-equilibrium states can we imagine on the early Earth, and what impact might they have had on the replicating molecules we are interested in? We are experimenting with temperature differences and allow the conditions to oscillate between hot and cold, which enables molecules to accumulate. We are also testing whether differences in levels of acidity, i.e. pH, emerge under such conditions and whether that can initiate reactions. The question is whether fluctuations in non-equilibrium systems have a positive effect on the the extension of strands of DNA or RNA?

In other words, you are looking for the most favorable environment for such systems?

Braun: Exactly. A hike through a volcanic landscape shows that much of the rock is highly porous, and many are full of water. If the volcano is active, hot steam will often waft over these pores, creating temperature gradients. We analyze how, in such non-equilibrium systems, molecules can be accumulate and be selectively replicated depending on their length. That scenario provides a mechanism by which molecules can be subjected to selection on the basis of length.

An essential feature of every evolutionary system?

Braun: Yes, even a rudimentary form of molecular evolution needed longer molecules, sequences with more information – and a copying mechanism that enables them to be replicated. As our experiments have shown, after a few replication cycles, a random base sequence is not quite as random as it was at first. We want to know how nature eventually or rather, inevitably, hit upon this form of Darwinian evolution. Only because nature discovered tricks for replicating nucleotide sequences and conserving the information they contained was it possible in the long run for something like a ribosome – the molecular machine that translates this information into the proteins which are the basis of all modern organisms – to evolve. Admittedly, we know very little about what actually happened along the way.

Is your work based on the assumption that the prebiotic synthesis of nucleotides and their polymeric elongation took place under essentially the same conditions, in the same environment?

Braun: We try to develop theories, but we are mainly interested in finding experimental ways of reproducing the origin of prebiotic evolution. The hope is that conditions that are permissive for replication and Darwinian evolution are also appropriate for the synthesis of nucleotides. In collaboration with Oliver Trapp, we are trying to find experimental conditions that make this linkage possible. For example, chemical reactions proceed much faster in pore systems, and temperature oscillations and dry-wet cycles are also much more effective on the micrometer scale. In my view, in order to establish an evolutionary process, it’s important that the system be driven into a non-equilibrium state, and that the molecules involved are able to cooperate with each other in the same setting for long periods.

How well can such conditions be recreated in an Ursuppe experiment 2.0?

Trapp: It will be difficult, and we have a long way to go. But we have had some success. We’ve been able to synthesize fatty-acid-like molecules called phospholipids, which assemble into cell-like structures in which organic molecules are accumulated and concentrated. If a reaction network is initiated with the right ingredients, metabolic pathways should emerge in which molecules are transformed into new ones. That in turn can give rise to concentration gradients that create non-equilibrium conditions.

How might primordial cells actually have originated?

Braun: Perhaps at boundary surfaces, at an interface between water and air. If you heat such a system and add lipids and DNA, both constituents accumulate on the warmer side. If you wait a little longer, you get a vesicular system consisting of many lipids. Interestingly, the concentration of RNA in the interior of these vesicles is 20 times higher than that in the external medium, and it’s properly folded and retains its catalytic activity. So this sort of non-equilibrium system could resemble the ancestors of biological cells.

The Ursuppe experiment 2.0

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At what point do such precursors appear?

Braun: In the early developmental phase of life on Earth, cells like those we now know are likely to have been a hindrance. Cells need to take up nutrients, and they generate waste products that have to be excreted. These processes are complex, and they are mediated in modern cells by complex proteins. That’s why I believe that the first cells emerged only after the ribosome and the genetic code had evolved.

Over the course of evolution, processes such as cell division, energy conversion and photosynthesis develop. Does all this have to be kept in mind when designing experiments to explore the earlier stages?

Trapp: We draw our inspiration from nature, and we try to break these systems down into their fundamental steps. In the case of photosynthesis, we would like to know how energy could have been stored under prebiotic conditions.

Braun: One shouldn’t take too much for granted. There are no logical grounds for assuming that what proteins are now do or how cells now synthesize DNA is directly connected with the conditions in the early prebiotic world. There is so much time, so much opportunity for evolution in between.

To get back to the very beginning for a moment, according to its own mission statement, the goal of the Cluster of Excellence on Origins is to test the hypothesis “that the origin of life was a natural product of the initial conditions established by the Big Bang”.

Carell: The fundamental question is whether life is deterministic or not? Is life itself a law of nature that emerged in the context of conditions that were predetermined in the immediate aftermath of the Big Bang? Or was life an unpredictable phenomenon that happened to emerge on Earth, and nowhere else in the Universe?

Cosmologists are convinced that within 100 seconds of the Big Bang all the ingredients are there, the whole particle zoo is in existence. And from then on, the development of the Universe proceeds like clockwork. Is it at all possible to prove such a degree of determinism?

Carell: Maybe not. But one can construct logically coherent scenarios. Having thought out complicated schemes, we are often surprised to discover how simple many of the processes that contributed to the emergence of life have turn out to be. But we still can’t prove that the small fraction of matter which the Big Bang produced made the evolution of life inevitable.

Braun: Perhaps starting with the Big Bang takes us a bit too far back. There is a logical gulf between what astrophysicists know about the formation of planets and what geologists have found out about the Earth.

Trapp: With respect to their organic composition, meteorites or asteroids are in principle no different from the Earth. The chemical processes that take place are the same everywhere, and the components involved can be found everywhere.

The search for signs of organic life on Mars has been underway for decades, and the moons of Saturn or Jupiter’s Enceladus and Europa are other possible candidates. If signs of life were found on any of them, would that clarify the issue?

Trapp: Undoubtedly.

Carell: If life were found in our immediate neighborhood, it would prove that the Earth is not unique in this respect

So how would you rate the chances that there is life ‘out there’?

Braun: Close to 100%.

Trapp: Yes, there is.

Carell: I think so too.

Braun: You only have to look at the numbers. On average, every Sun-like star probably has one planet. Now, just consider how many Suns there are in our galaxy – and how many galaxies you have!

Would you expect it to follow the same principles as life on Earth?

Carell: Carbon is an element that’s unlike any other in some respects. It‘s very flexible and highly dynamic, and it is likely to serve as the basis for all life. But I doubt that it looks at all like us – maybe more like giant worms.

The participants

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Prof. Dr. Dieter Braun

Professor of Systems Biophysics at LMU since 2007. Braun (b. 1970) studied Physics at Ulm University and the Technical University of Munich. He did his PhD at the Max Planck Institute for Biochemistry in Martinsried, and was a postdoc at Rockefeller University in New York. He subsequently led an Emmy Noether Junior Reserach Group funded by the DFG. In 2010 he received a Starting Grant from the European Research Council (ERC), which was followed by an Advanced Grant in 2018. Since then he has served as Spokesperson for the DFG-funded Collaborative Research Center (CRC) on the Emergence of Life and as Coordinator of research on the origin of life in the Cluster of Excellence “Origins”.

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Prof. Dr. Thomas Carell

Professor of Organic Chemistry with a focus on Nucleic Acid Research at LMU. Carell (b.1966) studied Chemistry in Münster and Heidelberg, and received his PhD for work done at the Max Planck Institute for Medical Research in the latter city. After his postdoc at the Massachusetts Institute of Technology (MIT) in Cambridge, he completed his Habilitation at the ETH Zürich and was appointed to a professorship in Münster. He moved to LMU in 2004, and won the Leibniz Prize in the same year. Carell was Spokesperson for the Center for Integrated Protein Science Munich (CIPSM) and the CRC on Dynamics of Intermediate Molecular Transformations. In 2019, he was nominated as Spokesperson for the CRC on “Chemical Biology of Epigenetic Modifications”.

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Prof. Dr. Oliver Trapp

Professor of Organic Chemistry at LMU and a Max Planck Fellow at the Max Planck Institute for Astronomy in Heidelberg. Trapp (b.1973) studied Chemistry at Tübingen University, where he received his PhD. Following a stint as a postdoc at Stanford University, he led a DFG-funded Emmy Noether Group at the Max Planck Institut für Kohlenforschung in Mülheim an der Ruhr. He completed his Habilitation at Bochum University and held a professorship at Heidelberg University, before taking up his present position at LMU in 2016. In 2010 he received an ERC Starting Grant, followed by a Proof-of-Concept Grant in 2012.

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