We are surrounded by systems of astonishing complexity - the immensity of the cosmos with stars, galaxies, black holes...- to the complexity of life in its various forms. And generally, we assume that this complexity has its roots in simpler forms which, in the course of time have accumulated material arriving up to the present form.

Going to the origin of life on Earth, we have Oparin's view: that life came from non-life, starting with the original simple components of the prebiotic biosphere, that combined with each other formed i the first amino acids and nitrogen -containing compounds. Then, these first molecules by combination with each other according to simple natural forces, gave origin to more and more complex molecular architectures till the formation of self-replicating cells- the basic condition for life-see fig.1.

Thus, the general approach to the origin of life and to the origin of cells is basically a bottom-up conceptual and operational procedure, according to which the present complexity has its origin from simpler, primitive forms. We can add material, one piece after another, and in this way we build the complex whole.

This is also the basic idea with the origin of life on Earth, following the seminal work of the Russian chemist Oparin on the early 20. ties of last century. Several years later, there was the famous 1953 demonstration by the young PhD Student Stanley Miller, who was able to demonstrate the synthesis of amino acids and other biologically complex molecules by simply reacting CH4, H20, NH3, and H2. Since then, several groups began to work at the aim of understanding the origin of life on Earth, and also with the idea of repeating in the lab the molecular evolution pathway to life. Teams of excellent scientists were at work all around the world, but it is fair to say that the origin of life on Earth is still unsolved; and so is the idea to make synthetic biology of cells, by way of the so-called synthetic biology, as shown in the illustration below, even in the simplest possible form of the "minimal cell".

I discuss "the raisons why" these basic problems are still unsolved, at length, in my last 2016 book on the emergence of life. There are of course hypotheses on how life on Earth might have originated, the most popular still being still the RNA world hypothesis, which I call for clarity "prebiotic RNA world" hypothesis.

I have a profound skepticism about this very idea, that a single molecule can start and do the all job of making life- the non-plus-ultra of reductionism. And again, I have to refer to my book where I believe to demonstrate that this idea has not a single solid base of chemistry.

But let us go back to the question of the bottom-up approach, and let us see in a little more detail the attempts, mentioned above, to make the minimal cell by synthetic biology, again a bottom-up approach. This is exemplified in the next illustration: basically, the idea to use a vesicle (generally a liposome, namely a vesicle formed by phospholipids) into which enzymes and nucleic acids can be incorporated. How many? The basic idea was to entrap the minimal and sufficient number of genes and enzymes so as to arrive at the minimal cell.

This work began in the 90. ties, mostly at the ETH in Zurich, with the work of Thomas Oberholzer in my group. Following that, several groups all around the world became active with the biochemistry of enzymes and nucleic acids entrapped in vesicles -see my mentioned book for a detailed review of data. However, in all these experiments, one could not entrap more than a couple of different enzymes in one compartment, and all these systems, although representing an important new concept in synthetic biology, are really too simple to be considered close to biological cells.

There was a qualitative jump in this field with the discovery and commercialization of the so-called Pure System by the group of Ueda in 2001. This is a minimal transcription-translation system, containing only 37 enzymes and a total of ca. 90 macromolecules, with a series of small molecules including ATP. It was possible to entrap the entire system in vesicles. This is very important, as it shows that even large systems can be easily incorporated.

Thanks to the Ueda' PureSystem, several groups around the world were then able to express the green fluorescence protein GFP (GFP for obvious detection reasons) inside vesicles under various conditions- see my book for an extensive review.

Still, it is fair to say that we are still far away from a living self-maintaining biological cell. In fact, the Pure System cannot reproduce itself, and therefore most of these studies are relative to one-batch reactions, and the perspective of a self-sustaining cell, or of a self-reproduction of the GFP-forming vesicle systems, does not appear as yet realistic. And there is another, quite different angle to consider: the studies on the minimal genome-which show that the minimal genome to sustain a modern type of biological cell cannot have less than 200 genes, and more likely should be around 250 genes.

In addition to these theoretical studies, there is the experimental work based on the technique of knock-out, more notably, in recent times, by the group of Craig Venter-again showing that you can simplify the genome of Mycoplasma genitalium but you cannot go below 200 genes. Now, if we compare the figure of hundred genes with the experimental results and possibilities of the bottom-up approach to the minimal cell, -see fig.2- the empirical conclusion is that the bottom up approach will never be able to reach such a threshold of 200-250 genes.

As already mentioned, the idea that the origin of complexity in nature and life itself is due to a stepwise increase of molecular architecture is something almost inborn in us, perhaps an archetypical Jungian form of thought. But maybe is not the right approach. But then, what is left?

The alternative to the bottom-up approach

Let us start from the top, namely from a large population of compartments which have been randomly filled with biopolymers. We have to assume of course that mixtures of nucleic acids and proteins are already existing; and still we do not know how they were formed in prebiotic time, but we know that they were certainly there in that time, and most likely not in tiny amounts; and we have also to assume that in the same place there is a natural, spontaneous source of surfactants, for example a hot spring ejecting a good concentration of fatty acids.

We also need an efficient method of entrapment of the biopolymers into the vesicles; and we need that the local concentration is high enough -as in our modern cells. And: it has been experimentally shown that precisely these two apparently so stringent conditions -high entrapment efficiency and high local concentration- can be realized in the laboratory.

To this aim, one should call to mind the reports on the spontaneous macromolecular overcrowding in vesicles, described first in 2010 at the University of Rome3, Italy, and then in a few following papers. This is the following: when in a diluted solution of macromolecules, vesicles are produced in situ, the solute distribution in the vesicles does not follow the expected classic (Poisson type of) distribution. Instead, one obtains a kind of all-or-nothing situation, with a lot of empty vesicles (i.e., not containing biopolymers) and a few over-filled vesicles, in which most of the solute is concentrated, with a concentration which can be up to 60 times higher than in the bulk solution.

And now the main assumption: that given this enormous number of overcrowded vesicles, there is a least one “good vesicle” – perhaps a small family – which has the right ingredients and the right concentration to start life. Or better, and more simply, to start the first dynamic steps towards life. The discussion of the possible steps for this mechanism cannot be told here in this very short contribution, and is the subject of specialized papers-see in the references. Here, just the main conceptual point: the possible reversal of the bottom -up approach.

This is then a systemic view: what is essential is the multiplicity, the great number of components that permits the reasonable assumption that at least one of them can be the right one – and following that, the mutual interaction of the components (the vesicles of the system) with each other. Quite a difference with respect to the bottom-up approach, in which, starting from one individual cell-like structure, one attempts has to increase its content so as to arrive at the complexity of the genome.

What is been proposed is a systemic view, in which complexity is there from the beginning. The fact that the entire Pure System can be easily incorporated into vesicles demonstrates the experimental reliability of the proposition. This is not a top-down approach, as the point is not to escalate down from complexity, but to adjust that starting complexity by way of selection and self-organized re-equilibration, so as to arrive at a quasi-homogeneous population – potentially a colony – of neighboring and eventually viable vesicles. It is until now a hypothesis, but one that provides indications about possible confirming experiments. As already mentioned, one of them is to find the conditions by which the overcrowded vesicles interact and fuse with each other, so as to arrive at an equilibrium homogeneous state – possibly in the form of a colony; the other, on a quite different direction, would be on the side of geology, to look for hot springs which may give rise to vesicles-producing surfactants.

More generally, the main message of this paper is an invitation to look beyond the simplistic bottom-up approach to complexity.

Oberholzer, T. and Luisi, P. L. (2002J. Biol. Phys., 28, 733–744.; Luisi, P. L., Ferri, F., and Stano, P. (2006). Naturwissenschaften, 93, 1–13; Luisi, P. L., and Stano, P., eds. (2011). The Minimal Cell, Springer.
Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K., and Ueda, T. (2001). Nat. Biotechnol., 19, 751–755
Luisi, P. L., Allegretti, M., de Souza, T. P., Steiniger, F., Fahr, A., and Stano, P. (2010). ChemBioChem, 11, 1989–1992; Souza, T., Stano, P., Steiniger, F., D’Aguanno, E., Altamura, E., Fahr, A., and Luisi, P. L. (2012). Orig. Life Evol. Biosph., 42, 421–428; Souza, T., Fahr, A., Luisi, P. L., Stano, P. (2014 Journal of Molecular Evolution, 79, 179–192; Stano, P., D’Aguanno, E., Bolz, J., Fahr, A., and Luisi, P. L. (2013) Angewandte Chemie International Edition, 52, 13397–13400; Souza, T., Volpe Bossa, G., Stano, P., Steiniger, F., May, S.,; Luisi, P. L., and Fahr, A. (2017 Physical Chemistry Chemical Physics, 19, 20082–20092.
Luisi, P. L. (2016). The Emergence of life, from chemical origin to synthetic biology, 2nd Edn., Cambridge University Press.
Luisi, P.L., (2018) Nature System Biology, submitted.