Conceptual foundations for minimal cells Eörs Szathmáry Collegium BudapestEötvös University Budapest

Quest for a biological minimal system Chemical supersystem Should be conceptually as simple as possible Must not necessarily be realizable in its simplest form Comparison with other elementary units (such as the elementary cell in crystallography) FORMAL AND EXACT

The eukaryotic cell is very complex—too complex! These cells have endosymbiont-derived organelles

The simplest cells are bacterial THUS we want to explain the origin of some primitive bacterium-like cell Even present-day bacteria are far too complex The main problem is the genetic code

The top-down approach Compare genomes of reduced bacterial cells (such as endosymbiotic parasites) Look for genes that are common to all such genomes Define the intersection of gene sets as the minimal set for prokaryotic organism

A problem with the top-down approach Suppose that –Gene a is only in organism A –Gene b is only in organism B –Gene c is common to both From this it DOES NOT follow that you can afford to miss gene a AND b at the same time! Maybe (EITHER a OR b) is ESSENTIAL Compare it with animal locomotion

Another problem with the top-down approach Even if you can reduce the set of essential genes to between 200 and 300, it still does include the genes for the genetic code Evolution usually does not solve two diffcult problems at the same time (low probablity x low probability is a very small number!) Origin of life per se and origin of the genetic code should preferably by separated If they cannot be solved sequentially, the situation is hopeless

We have to try the bottom-up approach! It must start in chemistry Prebiotic evolution also started in chemistry In vitro construction of minimal cells is an exercise in the design and assembly of chemical supersystems

A lot of confusion arises when units of evolution and units of life are taken to be identical The problem of the virus Gánti’s analogy: the virus is the living cells as a self-replicating programme is to the computer Neither the virus nor the programme do not do anything alone

Units of evolution (JMS) hereditary traits affecting survival and/or reproduction 1.multiplication 2.heredity 3.variation

Units of evolution Units of evolution and units of life viruses memes mules sterilized workers nondividing cells bacteria, protists, etc. Units of life

Tibor Gánti Born in 1933 A chemical engineer Patents in industrial biochemistry Syntheses using the controlled operation of enzymatic reaction networks First book on molecular biology in Hungary (1966)

The first edition of the Principles A serious book in a popular science disguise (1971) There was no other way to publish Proposal included the term “chemoton” ‘reductionist’ and ‘vitalist’ at the same time!

The latest edition: OUP 2003 After several editions in Hungarian Two previous books (the Principles and Contra Crick) plus one essay Essays appreciating the biological and philosophical importance

The investigation of life criteria: absolute criteria 1.Inherent unity 2.Metabolism 3.Inherent stability 4.Information carrying subsystem 5.Processes regulated and controlled by a programme VERBAL AND PHENOMENOLOGICAL, BUT RIGOROUS

Gánti’s chemoton model ALL THREE SUBSYSTEMS ARE AUTOCATALYTIC template copying metabolism membrane growth

Organisation of chemical systems into a supersystem (1974) CHEMOTON: a particular supersystem which is also a biological minimal system

Chemical cycles are metabolic engines (1971) The cycle as a whole is a catalyst The Noble prize of Szent-Györgyi was awarded for catalysis by fumaric acid Krebs has recognized the whole cycle Enzymes are superimposed on the metabolic cycle

Enzymes and cycle stoichiometry Very important: the cyclic process sign with the number of turns

A self-reproducing vesicle (1978) Metabolism and reproduction No genetic subsystem

Compartments are important, because They prohibit constituents diffusing away Can increase local concentration Create a special microenvironment, e.g. by keeping many molecules out For example, imagine when a poison cannot enter the compartment The problem of the origin of life is essentially that of metabolite channelling! Last but not least: a higher-level unit of evolution

The fission of the chemoton Membrane surface doubled Quantity of internal materials doubled Assume spherical shape Concentration cannot be kept with a growing sphere: volume increases with the cubic of the radius Volume of sphere with a surface are doubled would be more than doubled

Chemoton fission II More detailed calculations based on continuum mechanics Continuous distortion of the spherical shape Final resolution: two new spheres with size identical to that of the parental sphere STRICT STOICHIOMETRIC COUPLING BETWEEN THE GROWTH OF THE SUBSYSTEMS

The informational subsystem The pV n molecule consists of n molecules of V Result of template polycondensation R is the by-product, necessary for the formation of the membranogenic molecule T Information carried by quantity, frequency (composition), or sequence of signs Importance of sequence increases in evolution

Von Kiedrowski’s replicators

Replication of short templates is possible By the non-enzymatic scheme of von Kiedrowski (1986) If bound to a surface, exponential growth is also possible But the latter reaction does require intervention!

SPREAD for replication (von Kiedrowski)

How are we to substitute evolved agents (such as chemists and enzymes?) A robust replication must be found NOTE: template-directed synthesis is necessary but not sufficient for replication Should be preferably non-enzymatic Should be plausible under prebiotic conditions The idea should be testable

At the heart of the chemoton… …there is a metabolic cycle It is autocatalytic Produces the raw materials for the functioning of all subsystems at the expense of the difference between nutrients and waste Has homeostatic capacity The Calvin cycle and the reductive citric acid cycle are such core systems (controlled by enzymes) today

Most biological reactions are catalyzed by protein enzymes Without catalysis the reaction is slow S  P The catalyzed reaction is fast S + E  ES ES  EP EP  E + P

Structures form in 3 dimensions... …and are suggestive of enzymatic capability

Some RNA molecules act as enzymes (ribozymes) today

Test-tube selection experiments yield novel ribozymes and show that Catalysis of C-C bonds was feasible Even hydrophobic molecules can be specifically recognized The RNA world is likely to have been metabolically complex

The assembly of RNA structures Combinatorial assembly of RNA structures Submitted to selection for function between chemotons 1979

The channelled assembly of ribozymes (1983) The presence of substrates gives guidance to ribozyme assembly Good enzymes and bad enzymes will affect the fitness of the chemotons

The major open issue Is the chemoton viable without some form of enzymatic catalysis? Does membrane confinement provide enough metabolite channelling? Is non-enzymatic replication feasible at all? EVEN IF THE CHEMOTON IS NOT FEASIBLE WITHOUT ENZYMES, IT REMAINS THE ABSTRACTION OF THE ESSENTIAL SYSTEM THAT THE ENZYMES REGULATE

All present-day living systems have an autocatalytic metabolic network This applies even to heterotrophs Although heterotrophs do not have an autocatalytic core cycle It is a distributed autocatalytic network A set of small molecules is NECESSARY to kick-start the life processes These compounds cannot be taken up from the medium

The energetic logic of catalysis Without catalysis

The formose ‘reaction’ formaldehyd e glycolaldehyde autocatalysi s Butlerow, 1861

Classification of replicators Limited heredity Unlimited heredity Holisticformose ModularVon Kiedrowski genes Limited(# of individuals)  (# of types) Unlimited(# of individuals) << (# of types)

Is this just logical or also historical order? How much evolution did take place (presumably on surfaces) before protocells appeared?

Pathways of supersystem evolution boundary template metabolism M BM B B TB T M TM T M B TM B T INFRABIOLOGICAL SYSTEMS

An aim of the COST D27 action (President von Kiedrowski) is to facilitate collaborative emprirical and theoretical research on infrabiological systems