Developing an enzyme selection tool supporting multiple hosts contexts

Engineering biological organisms that allow the integration of alternative metabolic pathways to natural ones is one of the goals of synthetic biology. Based on this, some of the most attractive applications in terms of synthetic organisms manufacture include the production of a wide range of pharmacologically useful metabolites produced in a sustainable and environmentally friendly way. Also, the biostable molecules green-production involves different types of therapeutic processes, e.g. prostheses and grafts stabilisation. Regarding the viability of genetically modified organisms, metabolic pathways must be first properly designed, taking into consideration the type of host organism that will be involved in metabolic production, as well as its biochemical and environmental conditions. To ensure the correct growth of these synthetic organisms, the enzyme selection must guarantee both the organism survival (and proliferation) and the optimal production of the desired metabolite. Developing enzyme selection tools is essential to enhance and make cost-effective the metabolic pathways design. This technical note presents the update of Selenzyme, the enzyme selection tool which is based on organisms taxonomic compatibility and allows appropriate enzyme selection considering its amino acid sequence. The purpose of the update is to allow multiple host input, in order to perform an affinity comparison between target organisms and each host. The affinity differences will depend on which host to be considered, allowing the user to select the optimal host for the enzyme concerned.

An engineered IL-2 reprogrammed for anti-tumor therapy using a semi-synthetic organism

The implementation of applied engineering principles to create synthetic biological systems promises to revolutionize medicine, but application of fundamentally redesigned organisms has thus far not impacted practical drug development. Here we utilize an engineered microbial organism with a six-letter semi-synthetic DNA code to generate a library of site-specific, click chemistry compatible amino acid substitutions in the human cytokine IL-2. Targeted covalent modification of IL-2 variants with PEG polymers and screening identifies compounds with distinct IL-2 receptor specificities and improved pharmacological properties. One variant, termed THOR-707, selectively engages the IL-2 receptor beta/gamma complex without engagement of the IL-2 receptor alpha. In mice, administration of THOR-707 results in large-scale activation and amplification of CD8+ T cells and NK cells, without Treg expansion characteristic of IL-2. In syngeneic B16-F10 tumor-bearing mice, THOR-707 enhances drug accumulation in the tumor tissue, stimulates tumor-infiltrating CD8+ T and NK cells, and leads to a dose-dependent reduction of tumor growth. These results support further characterization of the immune modulatory, anti-tumor properties of THOR-707 and represent a fundamental advance in the application of synthetic biology to medicine, leveraging engineered semi-synthetic organisms as cellular factories to facilitate discovery and production of differentiated classes of chemically modified biologics.

Characterization of a triad of genes in cyanophage S-2L sufficient to replace adenine by 2-aminoadenine in bacterial DNA

Cyanophage S-2L is known to profoundly alter the biophysical properties of its DNA by replacing all adenines (A) with 2-aminoadenines (Z), which still pair with thymines but with a triple hydrogen bond. It was recently demonstrated that a homologue of adenylosuccinate synthetase (PurZ) and a dATP triphosphohydrolase (DatZ) are two important pieces of the metabolism of 2-aminoadenine, participating in the synthesis of ZTGC-DNA. Here, we determine that S-2L PurZ can use either dATP or ATP as a source of energy, thereby also depleting the pool of nucleotides in dATP. Furthermore, we identify a conserved gene (mazZ) located between purZ and datZ genes in S-2L and related phage genomes. We show that it encodes a (d)GTP-specific diphosphohydrolase, thereby providing the substrate of PurZ in the 2-aminoadenine synthesis pathway. High-resolution crystal structures of S-2L PurZ and MazZ with their respective substrates provide a rationale for their specificities. The Z-cluster made of these three genes – datZ, mazZ and purZ – was expressed in E. coli, resulting in a successful incorporation of 2-aminoadenine in the bacterial chromosomal and plasmidic DNA. This work opens the possibility to study synthetic organisms containing ZTGC-DNA.

8. Biotechnology and synthetic biology

This chapter explores the fields of biotechnology and synthetic biology. The micro-scale of synthetic biology clearly indicates the use of knowledge drawn from biochemistry. But its philosophy is more closely aligned to the principles of engineering than those of pure science. The chapter then looks at some examples of synthetic biochemistry, and reflects on what this new field might herald. It studies the creation of synthetic organisms and genomes. The chapter also considers gene editing and the emergence of a powerful gene editing technique, known as CRISPR (from clustered, regularly interspaced, short palindromic repeats). Finally, it addresses ethical issues and the darker applications of these technologies.

Characterization of a triad of genes in cyanophage S-2L sufficient to replace adenine by 2-aminoadenine in bacterial DNA

Cyanophage S-2L is known to profoundly alter the biophysical properties of its DNA by replacing all adenines (A) with 2-aminoadenines (Z), which still pair with thymines but with a triple hydrogen bond. It was recently demonstrated that a homologue of adenylosuccinate synthase (PurZ) and a dATP triphosphohydrolase (DatZ) are two important pieces of the metabolism of 2-aminoadenine, participating in the synthesis of ZTGC-DNA. Here, we determine that S-2L PurZ can use either dATP or ATP as a source of energy, thereby also depleting the pool of nucleotides in dATP. Furthermore, we identify a conserved gene (mazZ) located between purZ and datZ genes in Siphoviridae phage genomes, and show that it encodes a (d)GTP-specific diphosphohydrolase, thereby providing the substrate of PurZ in the 2-aminoadenine synthesis pathway. High-resolution crystal structures of S-2L PurZ and MazZ with their respective substrates provide a rationale for their specificities. The Z-cluster made of these three genes – datZ, mazZ and purZ – was expressed in E. coli, resulting in a successful incorporation of 2-aminoadenine in the bacterial chromosomal and plasmidic DNA. This work opens the possibility to study synthetic organisms containing ZTGC-DNA.

Synthetic threads through the web of life

CRISPR-Cas gene editing tools have brought us to an era of synthetic biology that will change the world. Excitement over the breakthroughs these tools have enabled in biology and medicine is balanced, justifiably, by concern over how their applications might go wrong in open environments. We do not know how genomic processes (including regulatory and epigenetic processes), evolutionary change, ecosystem interactions, and other higher order processes will affect traits, fitness, and impacts of edited organisms in nature. However, anticipating the spread, change, and impacts of edited traits or organisms in heterogeneous, changing environments is particularly important with “gene drives on the horizon.” To anticipate how “synthetic threads” will affect the web of life on Earth, scientists must confront complex system interactions across many levels of biological organization. Currently, we lack plans, infrastructure, and funding for field science and scientists to track new synthetic organisms, with or without gene drives, as they move through open environments.

Labs, Lives, Technoscience

This chapter looks at Sharon Traweek's classic study of physicists, which tracks the way the experimental particle physics community reproduces itself through the training of novices. It identifies the patterns through which education and inculcation occur, and by which particle physicists learn the criteria for a successful career. It also examines the images Traweek conveys of community, stability, and gendered reproduction that can be discerned only within a sufficiently entrenched discipline. The chapter describes synthetic biology as an unstable and ambiguously bounded field in which idiosyncratic individual paths are figured prominently, especially for members of the first generation of practitioners whose training took place within the reproductive mechanisms of established disciplines. It explores paths that are embedded with different concepts and logics within the synthetic organisms that were made in different labs.