FOCUS ON SYNTHETIC BIOLOGY COMMENTARY A synthetic approach to abiogenesis James Attwater & Philipp Holliger Synthetic biology seeks to probe fundamental aspects of biological form and function by construction (resynthesis) rather than deconstruction (analysis). Here we discuss how such an approach could be applied to assemble synthetic quasibiological systems able to replicate and evolve, illuminating universal properties of life and the search for its origins. Four billion years of evolution on Earth new metabolites3 or carry out computa- activity within model membranes13–16 but has yielded a biosphere packed with tion4. Advances in DNA solid-phase syn- cannot yet regenerate the proteins or trans- exquisitely optimized molecular compo- thesis and assembly have culminated in the lation factors needed to support replication. nents that enable life to evolve and thrive. first synthesis of a genome of a unicellular To achieve this, a minimal cell would need Traditionally, molecular biologists seek to organism5. Such technologies can explore to encapsulate a sufficient set of cellular analyze their function and interconnect- the arrangement and context of genes in replication, transcription and translation edness in order to better understand the genomes on an unprecedented scale, open- machinery components together with their nature of living systems. Yet ultimately these ing up biotechnological opportunities encoding genome (Fig. 1b). This could components and systems are all representa- through our growing mastery over the cen- define the core set of natural components tives of a single interrelated biology deriving tral dogma (Fig. 1a). required for propagation and expression of from the last universal common ancestor This naturally leads to questions as to how genetic information, establishing heredity (LUCA), a breakthrough organism (or set far we can stray beyond life’s present molecu- and providing a stable chassis for bioengi- of genes1) already reminiscent of modern lar paradigms. New chemical functionalities neering17. However, although clearly much Nature America, Inc. All rights reserved. America, Inc. Nature prokaryotes. In the absence of the discov- such as unnatural amino acids and carbohy- simpler than any extant cellular organism, 4 ery of other biologies (on Earth or beyond2) drates can be added to both unicellular and such a semisynthetic cell is still estimated 6,7 © 201 not related to LUCA, it is challenging to multicellular organisms , and in one case, to require between 100 and 150 genes establish universal principles and laws of one of the bases of the genome has been expressed from a >100-kilobase genome. biology. To paraphrase Carl Sagan: our biol- entirely replaced by an unnatural analog8. Is such complexity a prerequisite of life, or ogy, although amazingly diverse, is ‘provin- Ribosome engineering9 or global recod- is it merely a consequence of using compo- npg cial’—in contrast, for example, to the laws of ing10 allows reassignment of the genetic nents firmly rooted in extant biology? In physics, whose generality can be observed code, and expansion of the genetic alphabet other words, is life more complicated than throughout the cosmos. However, by going itself might be possible11. The challenge of all it needs to be? beyond the simple analysis and deconstruc- these approaches in supporting stable aug- An appreciation of the minimal require- tion of extant life and building ‘new biolo- mentation is to fit the new functionalities ments of life as a self-perpetuating phenom- gies’ through modification, reconstruction into preexisting biological networks through enon may emerge from even more radical and de novo construction, synthetic biology either replacement or the establishment of strategies. Constructing systems exhibiting promises a fresh perspective and ultimately orthogonal pathways or chemistries12. Such key traits of life, using synthetic compo- a better understanding of the unifying prin- efforts promise to reveal the limits (if any) to nents not found in biology, will challenge ciples of living systems. life’s tolerance of expansion into new chemi- our preconceptions of what constitutes life. Our ability to modify existing biol- cal and informational space. Abandoning the superbly refined molecular ogy has grown rapidly since the advent of These approaches, however, remain machinery of extant biology may seem an genetic engineering. Biological tools allow defined by and embedded in preexisting inauspicious step to take but may prove both the insertion, deletion and modification of biology and thus are likely to reflect its instructive and liberating. Much of the cen- genes at will, enabling the rewiring of path- constraints. The underlying principles of tral complexity of biology arises from the ways to generate new phenotypes, produce our biology may be more clearly exposed need to support and integrate three sepa- by a fully synthetic approach: constructing rate biopolymer systems: protein, RNA and simple cells comprising a limited number of DNA. If we can step back from this para- Philipp Holliger and James Attwater are at the essential components derived from biology. digm of cooperating biopolymers, simple, Medical Research Council Laboratory of Molecular Biology, Cambridge, UK. A number of strategies have recapitulated more streamlined forms of life might be e-mail: [email protected] transcription, translation and metabolic feasible. NATURE METHODS | VOL.11 NO.5 | MAY 2014 | 495 COMMENTARY FOCUS ON SYNTHETIC BIOLOGY 26 abGenome: ~1 Mbp ~100 kbp, 150 genes c 2 genes template ), demonstrating the synthetic potential of such ribozymes. One might ask if relatively complicated Synthetic DNA DNA DNA ‘replicases’ are needed to implement simple Whole- forms of self-replication and evolution. genome RNA RNA RNA assembly Work on replication of RNA using a purely In silico Membrane Protein Protein non-enzymatic, chemical strategy was design 27 Metabolism initiated in the 1970s and has seen sig- nificant recent progress through the use of non-native linkage and base chemistries28, stepwise removal of hydrolysis products29 and discovery of cofactors enabling some Figure 1 | Synthetic biological systems of increasing simplicity. (a) DNA as software: a bacterium reprogrammed by transformation with a synthetic genome5. Changes are transmitted through the template-directed RNA synthesis inside a 30 central dogma, implementing a new phenotype by influencing informational systems, metabolism and model protocell . Non-enzymatic replica- the cell membrane (black arrows represent information transfer; orange arrows show catalysis). tion systems, though, remain limited in rate (b) A proposed minimal heterotrophic cell, lacking metabolism and comprising components dedicated and fidelity and, consequently, the genome solely to maintaining DNA replication, transcription and translation17. (c) A putative maximally simple size that they would support. RNA organism: a synthetic protocell founded on a single biopolymer (RNA) inhabiting a dynamic Furthermore, both non-enzymatic and membranous vesicle20. An RNA replicase copies both itself and a metabolic ribozyme (synthase) that provides building blocks (by activating or trapping permeable precursors). The synthase is dispensable enzymatic approaches must still overcome a if activated building blocks are membrane permeable; alternatively, the replicase is dispensable if set of formidable challenges before a robust activated building blocks are capable of non-enzymatic RNA synthesis. replicating and evolving RNA system can be achieved31. Among the most pressing issues are the inhibitory effects of template One might begin by asking which critical able to evolve. To access and explore such a secondary structures and the related need requirements these must satisfy. A unique scenario, synthetic strategies must gener- for strand separation. These require the property of life is its capacity for self- ate and integrate novel components: for definition of either enzymatic activities or replication and open-ended improvement, instance, a replicating RNA genome. physicochemical regimes able to unfold through evolution enabled by a heritable Can self-replication be implemented template RNA duplex structures while sup- molecular memory. In the search for plau- using RNA as the sole informational com- porting RNA folding and base-pairing for sible molecular strategies to implement ponent? Dramatic exponential growth has synthesis. A counterintuitive yet elegant this capability in a synthetic system, one already been observed for a pair of engi- concept that has emerged from recent work conceptually attractive approach is to take neered cross-catalytic RNA ligases wherein is that nucleic acid replication (as well as Nature America, Inc. All rights reserved. America, Inc. Nature inspiration from research into abiogenesis, each ribozyme catalyzes the other’s assembly assembly and division of protocellular 4 the emergence of life on the early Earth, from two pieces, allowing emergence of new membranes32) may be aided by a degree of 22 33 © 201 which provides useful constraints on both ligase phenotypes through recombination . ‘helpful heterogeneity’ (Fig. 2). Chemical chemical and biophysical parameters18,19. Cooperative networks of self-assembly have heterogeneity in the form of sporadic incor- The generation of a simple evolving system also been described developing from pools poration of transient 2′ modifications34 or guided by these parameters in turn would of