Reconstituting alternative life using the test-bed of cell-free systems

The current form of life on Earth is the outcome of a series of biochemical events that led to the formation of early life with a specific molecular composition encoding its many functions. However, other trajectories of events with widely different outcomes could have led to alternative forms of life with different compositions. Although these ‘roads not taken’ have been hypothesized in Origins of Life research for long, only recently have advances in several life technologies enabled us to experimentally explore them. Here, we discuss how one such technology, cell-free systems (CFS), which offers a promising avenue for reconstitution of specific biological functions in a controlled in vitro environment. Breaking free of the complex biochemical interactions of the closed cellular compartment facilitates testing of engineered alternatives of these functions, in whole or part, reconstituted using both natural and synthetic orthogonal components. In this perspective, we focus on how CFS has enabled characterization and reconstitution of several steps of biological information transfer using alternative machinery (xeno nucleic acids, RNA polymerases, ribosomes, non-canonical amino acids, transfer RNA synthetases, mirror-image biomolecules), including in alternative biochemistries and compartments, identifying the applicable constraints. Once these alternative functions have been characterized and co-optimized, how do we foresee their reintegration into alternative life forms of the future? This article is part of the theme issue ‘Origins of life: the possible and the actual’.