Synthetic biology (Oxford, England)最新文献

Data science is playing an increasingly important role in the design and analysis of engineered biology. This has been fueled by the development of high-throughput methods like massively parallel reporter assays, data-rich microscopy techniques, computational protein structure prediction and design, and the development of whole-cell models able to generate huge volumes of data. Although the ability to apply data-centric analyses in these contexts is appealing and increasingly simple to do, it comes with potential risks. For example, how might biases in the underlying data affect the validity of a result and what might the environmental impact of large-scale data analyses be? Here, we present a community-developed framework for assessing data hazards to help address these concerns and demonstrate its application to two synthetic biology case studies. We show the diversity of considerations that arise in common types of bioengineering projects and provide some guidelines and mitigating steps. Understanding potential issues and dangers when working with data and proactively addressing them will be essential for ensuring the appropriate use of emerging data-intensive AI methods and help increase the trustworthiness of their applications in synthetic biology.

Synthetic biology has immense potential to ameliorate widespread environmental damage. The promise of such technology could, however, be argued to potentially risk the public, industry or governments not curtailing their environmentally damaging behavior or even worse exploit the possibility of this technology to do further damage. In such cases, there is the risk of a worse outcome than if the technology was not deployed. This risk is often couched as an objection to new technologies, that the technology produces a moral hazard. This paper describes how to navigate a moral hazard argument and mitigate the possibility of a moral hazard. Navigating moral hazard arguments and mitigating the possibility of a moral hazard will improve the public and environmental impact of synthetic biology.

Cell-free expression (CFE) has shown recent utility in prototyping enzymes for discovery efforts. In this work, CFE is demonstrated as an effective tool to screen putative polyester polyurethane degrading enzyme sequences sourced from metagenomic analysis of biofilms prospected on aircraft and vehicles. An automated fluid handler with a controlled temperature block is used to assemble the numerous 30 µL CFE reactions to provide more consistent results over human assembly. In sum, 13 putative hydrolase enzymes from the biofilm organisms as well as a previously verified, polyester-degrading cutinase were expressed using in-house E. coli extract and minimal linear templates. The enzymes were then tested for esterase activity directly in extract using nitrophenyl conjugated substrates, showing highest sensitivity to shorter substrates (4-nitrophenyl hexanoate and 4-nNitrophenyl valerate). This screen identified 10 enzymes with statistically significant activities against these substrates; however, all were lower in measured relative activity, on a CFE volume basis, to the established cutinase control. This approach portends the use of CFE and reporter probes to rapidly prototype, screen and design for synthetic polymer degrading enzymes from environmental consortia. Graphical Abstract.

Synthetic cells are a novel class of cell-like bioreactors, offering the potential for unique advancements in synthetic biology and biomedicine. To realize the potential of those technologies, synthetic cell-based drugs need to go through the drug approval pipeline. Here, we discussed several regulatory challenges, both unique to synthetic cells, as well as challenges typical for any new biomedical technology. Overcoming those difficulties could bring transformative therapies to the market and will create a path to the development and approval of cutting-edge synthetic biology therapies. Graphical Abstract.

The FdeR regulator has been reported as a transcriptional activator dependent on the interaction with naringenin. Previously, FdeR and its cognate promoter were used to construct naringenin-sensitive sensors, though no correlation was associated between the FdeR level of expression and outputs. Therefore, to understand this correlation, we constructed a circuit with FdeR expression adjusted by the arabinose concentration through an AraC-P_BAD system and the FdeR-regulated promoter controlling the expression of GFP. We observed a significant reduction in the activity of the target promoter by increasing FdeR expression, indicating that although FdeR has been primarily classified as a transcriptional activator, it also represses transcription. Leveraging the bifunctional feature of FdeR, acting as both transcriptional activator and repressor, we demonstrated that this genetic circuit, when previously switched on by naringenin, can be switched off by inducing an increased FdeR expression level. This engineered system functioned as a NIMPLY gate, effectively decreasing GFP expression by 50% when arabinose was added without removing naringenin from the medium. Exploiting FdeR versatility, this study demonstrates an innovative application of this transcriptional factor for developing novel NIMPLY gates activated by a molecule with low toxicity and nutraceutical properties that may be important for several applications. Graphical Abstract.

Recent advances in bottom-up synthetic biology have made it possible to reconstitute cellular systems from non-living components, yielding artificial cells with potential applications in industry, medicine and basic research. Although a variety of cellular functions and components have been reconstituted in previous studies, sustained biological energy production remains a challenge. ATP synthesis via ribulose-1,5-diphosphate carboxylase/oxygenase (RuBisCO), a central enzyme in biological CO₂ fixation, holds potential as an energy production system, but its feasibility in a cell-free expression system has not yet been tested. In this study, we test RuBisCO expression and its activity-mediated ATP synthesis in a reconstituted Escherichia coli-based cell-free translation system. We then construct a system in which ATP is synthesized by RuBisCO activity in giant vesicles and used as energy for translation reactions. These results represent an advance toward independent energy production in artificial cells. Graphical Abstract.

The engineering of pre-defined functions in living cells requires increasingly accurate tools as synthetic biology efforts become more ambitious. Moreover, the characterization of the phenotypic performance of genetic constructs demands meticulous measurements and extensive data acquisition for the sake of feeding mathematical models and matching predictions along the design-build-test lifecycle. Here, we developed a genetic tool that eases high-throughput transposon insertion sequencing (TnSeq): the pBLAM1-x plasmid vectors carrying the Himar1 Mariner transposase system. These plasmids were derived from the mini-Tn5 transposon vector pBAMD1-2 and built following modular criteria of the Standard European Vector Architecture (SEVA) format. To showcase their function, we analyzed sequencing results of 60 clones of the soil bacterium Pseudomonas putida KT2440. The new pBLAM1-x tool has already been included in the latest SEVA database release, and here we describe its performance using laboratory automation workflows. Graphical Abstract.

Cannabinoids are a therapeutically valuable class of secondary metabolites with a vast number of substituents. The native cannabinoid biosynthetic pathway of Cannabis sativa generates cannabigerolic acid (CBGA), the common substrate to multiple cannabinoid synthases. The bioactive decarboxylated analog of this compound, cannabigerol (CBG), represents an alternate gateway into the cannabinoid space as a substrate either to non-canonical cannabinoid synthase homologs or to synthetic chemical reactions. Herein, we describe the identification and repurposing of aromatic prenyltransferase (AtaPT), which when coupled with native enzymes of C. sativa can form an Escherichia coli production system for CBGA in cell lysates and CBG in whole cells. Engineering of AtaPT, guided by structural analysis, was performed to enhance its kinetics toward CBGA production for subsequent use in a proof-of-concept lysate system. For the first time, we show a synthetic biology platform for CBG biosynthesis in E. coli cells by employing AtaPT under an optimized microbial system. Our results have therefore set the foundation for sustainable production of well-researched and rarer cannabinoids in an E. coli chassis. Graphical Abstract.

Cell-free systems can expedite the design and implementation of biomanufacturing processes by bypassing troublesome requirements associated with the use of live cells. In particular, the lack of survival objectives and the open nature of cell-free reactions afford engineering approaches that allow purposeful direction of metabolic flux. The use of lysate-based systems to produce desired small molecules can result in competitive titers and productivities when compared to their cell-based counterparts. However, pathway crosstalk within endogenous lysate metabolism can compromise conversion yields by diverting carbon flow away from desired products. Here, the 'block-push-pull' concept of conventional cell-based metabolic engineering was adapted to develop a cell-free approach that efficiently directs carbon flow in lysates from glucose and toward endogenous ethanol synthesis. The approach is readily adaptable, is relatively rapid and allows for the manipulation of central metabolism in cell extracts. In implementing this approach, a block strategy is first optimized, enabling selective enzyme removal from the lysate to the point of eliminating by-product-forming activity while channeling flux through the target pathway. This is complemented with cell-free metabolic engineering methods that manipulate the lysate proteome and reaction environment to push through bottlenecks and pull flux toward ethanol. The approach incorporating these block, push and pull strategies maximized the glucose-to-ethanol conversion in an Escherichia coli lysate that initially had low ethanologenic potential. A 10-fold improvement in the percent yield is demonstrated. To our knowledge, this is the first report of successfully rewiring lysate carbon flux without source strain optimization and completely transforming the consumed input substrate to a desired output product in a lysate-based, cell-free system.