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Life's Greatest Secret is the story of the discovery and cracking of the genetic code. This great scientific breakthrough has had far-reaching consequences for how we understand ourselves and our place in the natural world. The code forms the most striking proof of Darwin's hypothesis that all organisms are related, holds tremendous promise for improving human well-being, and has transformed the way we think about life. Matthew Cobb interweaves science, biography and anecdote in a book that mixes remarkable insights, theoretical dead-ends and ingenious experiments with the pace of a thriller. He describes cooperation and competition among some of the twentieth-century's most outstanding and eccentric minds, moves between biology, physics and chemistry, and shows the part played by computing and cybernetics. The story spans the globe, from Cambridge MA to Cambridge UK, New York to Paris, London to Moscow. It is both thrilling science and a fascinating story about how science is done.

The encoded biosynthesis of proteins provides the ultimate paradigm for high-fidelity synthesis of long polymers of defined sequence and composition, but it is limited to polymerizing the canonical amino acids. Recent advances have built on genetic code expansion — which commonly permits the cellular incorporation of one type of non-canonical amino acid into a protein — to enable the encoded incorporation of several distinct non-canonical amino acids. Developments include strategies to read quadruplet codons, use non-natural DNA base pairs, synthesize completely recoded genomes and create orthogonal translational components with reprogrammed specificities. These advances may enable the genetically encoded synthesis of non-canonical biopolymers and provide a platform for transforming the discovery and evolution of new materials and therapeutics.The ability to reprogramme cellular translation and genomes to produce non-canonical biopolymers has wide-ranging applications, including in therapeutics, but has yet to be fully realized. In this Review, de la Torre and Chin discuss recent advances towards achieving this goal.

This book tracks the key experiments and discoveries that set in motion efforts to crack the code and explores the many ways humans have applied knowledge of the genetic code to alter gene activity.

Engineering the genetic code of an organism has been proposed to provide a firewall from natural ecosystems by preventing viral infections and gene transfer 1 – 6 . However, numerous viruses and mobile genetic elements encode parts of the translational apparatus 7 – 9 , potentially rendering a genetic-code-based firewall ineffective. Here we show that such mobile transfer RNAs (tRNAs) enable gene transfer and allow viral replication in Escherichia coli despite the genome-wide removal of 3 of the 64 codons and the previously essential cognate tRNA and release factor genes. We then establish a genetic firewall by discovering viral tRNAs that provide exceptionally efficient codon reassignment allowing us to develop cells bearing an amino acid-swapped genetic code that reassigns two of the six serine codons to leucine during translation. This amino acid-swapped genetic code renders cells resistant to viral infections by mistranslating viral proteomes and prevents the escape of synthetic genetic information by engineered reliance on serine codons to produce leucine-requiring proteins. As these cells may have a selective advantage over wild organisms due to virus resistance, we also repurpose a third codon to biocontain this virus-resistant host through dependence on an amino acid not found in nature 10 . Our results may provide the basis for a general strategy to make any organism safely resistant to all natural viruses and prevent genetic information flow into and out of genetically modified organisms. A study details the creation of an Escherichia coli genetically recoded organism that is resistant to viral infection, and describes a further modification that keeps the organism and its genetic information biocontained.

Construction of a series of genomically recoded organisms whose growth is restricted by the expression of essential genes dependent on exogenously supplied synthetic amino acids introduces novel orthogonal barriers between these engineered organisms and the environment, thereby creating safer genetically modified organisms. Two routes to safer GMOs Two manuscripts published in this issue of Nature describe independent approaches towards generating an organism dependent on unnatural amino acids, a development which could find applications for biocontainment and exploration of previously unsampled fitness landscapes. George Church and colleagues redesigned essential enzymes in an organism ( Escherichia coli ) with an altered genetic code to make it metabolically dependent on non-standard amino acids for survival. The resulting genetically modified organisms (GMOs) cannot metabolically circumvent their biocontainment mechanisms and show unprecedented resistance to evolutionary escape. The few escapees are rapidly outcompeted by unmodified organisms. Using multiplex automated genome engineering, Farren Isaacs and colleagues construct a series of genomically recoded organisms whose growth is restricted by the expression of essential genes that depend on exogenously supplied synthetic amino acids. They constructed synthetic auxotrophs with advanced orthogonal barriers between engineered organisms and the environment, thereby creating safer GMOs. Genetically modified organisms (GMOs) are increasingly used in research and industrial systems to produce high-value pharmaceuticals, fuels and chemicals 1 . Genetic isolation and intrinsic biocontainment would provide essential biosafety measures to secure these closed systems and enable safe applications of GMOs in open systems 2 , 3 , which include bioremediation 4 and probiotics 5 . Although safeguards have been designed to control cell growth by essential gene regulation 6 , inducible toxin switches 7 and engineered auxotrophies 8 , these approaches are compromised by cross-feeding of essential metabolites, leaked expression of essential genes, or genetic mutations 9 , 10 . Here we describe the construction of a series of genomically recoded organisms (GROs) 11 whose growth is restricted by the expression of multiple essential genes that depend on exogenously supplied synthetic amino acids (sAAs). We introduced a Methanocaldococcus jannaschii tRNA:aminoacyl-tRNA synthetase pair into the chromosome of a GRO derived from Escherichia coli that lacks all TAG codons and release factor 1, endowing this organism with the orthogonal translational components to convert TAG into a dedicated sense codon for sAAs. Using multiplex automated genome engineering 12 , we introduced in-frame TAG codons into 22 essential genes, linking their expression to the incorporation of synthetic phenylalanine-derived amino acids. Of the 60 sAA-dependent variants isolated, a notable strain harbouring three TAG codons in conserved functional residues 13 of MurG, DnaA and SerS and containing targeted tRNA deletions maintained robust growth and exhibited undetectable escape frequencies upon culturing ∼10 11 cells on solid media for 7 days or in liquid media for 20 days. This is a significant improvement over existing biocontainment approaches 2 , 3 , 6 , 7 , 8 , 9 , 10 . We constructed synthetic auxotrophs dependent on sAAs that were not rescued by cross-feeding in environmental growth assays. These auxotrophic GROs possess alternative genetic codes that impart genetic isolation by impeding horizontal gene transfer 11 and now depend on the use of synthetic biochemical building blocks, advancing orthogonal barriers between engineered organisms and the environment.

Recoding–the repurposing of genetic codons–is a powerful strategy for enhancing genomes with functions not commonly found in nature. Here, we report computational design, synthesis, and progress toward assembly of a 3.97-megabase, 57-codon Escherichia coli genome in which all 62,214 instances of seven codons were replaced with synonymous alternatives across all protein-coding genes. We have validated 63% of recoded genes by individually testing 55 segments of 50 kilobases each. We observed that 91% of tested essential genes retained functionality with limited fitness effect. We demonstrate identification and correction of lethal design exceptions, only 13 of which were found in 2229 genes. This work underscores the feasibility of rewriting genomes and establishes a framework for large-scale design, assembly, troubleshooting, and phenotypic analysis of synthetic organisms.

Nature uses a limited, conservative set of amino acids to synthesize proteins. The ability to genetically encode an expanded set of building blocks with new chemical and physical properties is transforming the study, manipulation and evolution of proteins, and is enabling diverse applications, including approaches to probe, image and control protein function, and to precisely engineer therapeutics. Underpinning this transformation are strategies to engineer and rewire translation. Emerging strategies aim to reprogram the genetic code so that noncanonical biopolymers can be synthesized and evolved, and to test the limits of our ability to engineer the translational machinery and systematically recode genomes. A review of the recent developments in reprogramming the genetic code of cells and organisms to include non-canonical amino acids in precisely engineered proteins. Rewriting genetic guidelines In living organisms, proteins that are encoded by DNA are composed of 20 canonical amino acids, with some organisms using up to two additional derivatives. Theoretically, other molecules that are related to these amino acids could form a similar protein backbone and might confer new properties on the proteins. Jason Chin reviews the most recent studies on reprogramming the genetic code, where progress is being made in incorporating these non-canonical (that is, not naturally occurring) amino acids into proteins, even using the cell's own machinery to do so, and without disrupting overall protein function.

Genetically modified organisms (GMOs) are increasingly deployed at large scales and in open environments. Genetic biocontainment strategies are needed to prevent unintended proliferation of GMOs in natural ecosystems. Existing biocontainment methods are insufficient because they impose evolutionary pressure on the organism to eject the safeguard by spontaneous mutagenesis or horizontal gene transfer, or because they can be circumvented by environmentally available compounds. Here we computationally redesign essential enzymes in the first organism possessing an altered genetic code ( Escherichia coli strain C321.ΔA) to confer metabolic dependence on non-standard amino acids for survival. The resulting GMOs cannot metabolically bypass their biocontainment mechanisms using known environmental compounds, and they exhibit unprecedented resistance to evolutionary escape through mutagenesis and horizontal gene transfer. This work provides a foundation for safer GMOs that are isolated from natural ecosystems by a reliance on synthetic metabolites. Essential enzymes in genetically modified organisms are computationally redesigned to functionally depend on non-standard amino acids, thereby achieving biocontainment with unprecedented resistance to escape by evolution or by supplementation with environmental metabolites. Two routes to safer GMOs Two manuscripts published in this issue of Nature describe independent approaches towards generating an organism dependent on unnatural amino acids, a development which could find applications for biocontainment and exploration of previously unsampled fitness landscapes. George Church and colleagues redesigned essential enzymes in an organism ( Escherichia coli ) with an altered genetic code to make it metabolically dependent on non-standard amino acids for survival. The resulting genetically modified organisms (GMOs) cannot metabolically circumvent their biocontainment mechanisms and show unprecedented resistance to evolutionary escape. The few escapees are rapidly outcompeted by unmodified organisms. Using multiplex automated genome engineering, Farren Isaacs and colleagues construct a series of genomically recoded organisms whose growth is restricted by the expression of essential genes that depend on exogenously supplied synthetic amino acids. They constructed synthetic auxotrophs with advanced orthogonal barriers between engineered organisms and the environment, thereby creating safer GMOs.

Ascomycete yeasts are metabolically diverse, with great potential for biotechnology. Here, we report the comparative genome analysis of 29 taxonomically and biotechnologically important yeasts, including 16 newly sequenced. We identify a genetic code change, CUG-Ala, in Pachysolen tannophilus in the clade sister to the known CUG-Ser clade. Our well-resolved yeast phylogeny shows that some traits, such as methylotrophy, are restricted to single clades, whereas others, such as L-rhamnose utilization, have patchy phylogenetic distributions. Gene clusters, with variable organization and distribution, encode many pathways of interest. Genomics can predict some biochemical traits precisely, but the genomic basis of others, such as xylose utilization, remains unresolved. Our data also provide insight into early evolution of ascomycetes. We document the loss of H3K9me2/3 heterochromatin, the origin of ascomycete mating-type switching, and panascomycete synteny at the MAT locus. These data and analyses will facilitate the engineering of efficient biosynthetic and degradative pathways and gateways for genomic manipulation.