ACS Synthetic Biology最新文献

Plastids represent promising targets in plant genetic engineering for many biotech applications, ranging from their use as bioreactors for the overproduction of valuable molecules to the installation of transgenes for improving plant traits. For over 30 years, routine methods of plastid transformation have relied on homologous recombination integrating vectors. However, nonintegrating episomal plasmids have recently received more attention as an innovative tool for the plastid genetic engineering of plant cells. One of these novel technologies is the mini-synplastome, an episomal plasmid with a chloroplast-specific origin of replication (ori) used to express transgenes in plastids. In order to improve episome sequence stability overtime by reducing the frequency of spurious recombination events, an optimized version of mini-synplastome (Gen3) was designed. The innovation in the Gen3 design was to substantially reduce the size of the plastomic sequence containing oris to include only domains involved in replication and to reduce the sequence homology of the whole episome with the endogenous plastome. In this work, we have demonstrated that Gen3 can be used to install a multigene pathway in Solanum tuberosum (potato) chloroplasts, and the episome is stable in a full-length circular form at high copy number throughout all plant developmental stages to anthesis in plants with normal phenotypic parameters. It is anticipated that in the next decade the mini-synplastome will be a valuable tool for installing complex genetic circuits in plastids.

Benzylisoquinoline alkaloids (BIAs) are a class of natural compounds found in plants of the Ranunculaceae family, known for their diverse pharmacological activities. However, the extraction yields of BIAs from plants are limited, and the cost of chemical synthesis is prohibitively high. Recent advancements in systems metabolic engineering and genomics have made it feasible to use microbes as bioreactors for BIAs production. This review explores recent progress in enhancing the production and yields of BIAs in two microbial systems: Escherichia coli and Saccharomyces cerevisiae. It covers various BIAs, including (S)-reticuline, morphinane, protoberberine, and aporphine alkaloids. The review provides strategies and technologies for BIAs synthesis, analyzes current challenges in BIAs research, and offers recommendations for future research directions.

RNA-encoded viral nucleic acid analyte reporter (REVEALR) is a rapid and highly sensitive point-of-care diagnostic developed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection, genotyping, and quantification. Here, we extend the breadth of this nucleic acid technology to include a viral respiratory panel that can detect low attomolar levels of influenza A (IAV), influenza B (IBV), SARS-CoV-2 (CoV2), and the respiratory syncytial virus (RSV). Of 39 clinical samples collected at the UCI Medical Center in Orange, California, the extended REVEALR panel showed a positive predictive agreement and negative predictive agreement of 100% for IAV, CoV2, and RSV in sequence-verified clinical samples, with 0 false positive results. Additionally, REVEALR was able to detect a synthetic IBV genome and precisely identify the viruses from clinical samples that were combined to simulate a patient infected with more than one virus. Together, these results demonstrate that the REVEALR respiratory panel provides a valuable diagnostic for identifying the pathogens responsible for causing lower respiratory infections that pose serious healthcare challenges in the United States.

Biological systems can directly upgrade carbon dioxide (CO₂) into chemicals. The CO₂ fixation rate of autotrophic organisms, however, is too slow for industrial utility, and the breadth of engineered metabolic pathways for the synthesis of value-added chemicals is too limited. Biotechnology workhorse organisms with extensively engineered metabolic pathways have recently been engineered for CO₂ fixation. Yet, their low carbon fixation rate, compounded by the fact that living organisms split their carbon between cell growth and chemical synthesis, has led to only cell growth with no chemical synthesis achieved to date. Here, we engineer a lysate-based cell-free expression (CFE)-based multienzyme biocatalyst for the carbon negative synthesis of the industrially relevant amino acids glycine and serine from CO₂ equivalents─formate and bicarbonate─and ammonia. The formate-to-serine biocatalyst leverages tetrahydrofolate (THF)-dependent formate fixation, reductive glycine synthesis, serine synthesis, and phosphite dehydrogenase-dependent NAD(P)H regeneration to convert 30% of formate into serine and glycine, surpassing the previous 22% conversion using a purified enzyme system. We find that (1) the CFE-based biocatalyst is active even after 200-fold dilution, enabling higher substrate loading and product synthesis without incurring additional cell lysate cost, (2) NAD(P)H regeneration is pivotal to driving forward reactions close to thermodynamic equilibrium, (3) balancing the ratio of the formate-to-serine pathway genes added to the CFE is key to improving amino acid synthesis, and (4) efficient THF recycling enables lowering the loading of this cofactor, reducing the cost of the CFE-based biocatalyst. To our knowledge, this is the first synthesis of amino acids that can capture CO₂ equivalents for the carbon negative synthesis of amino acids using a CFE-based biocatalyst. Looking ahead, the CFE-based biocatalyst process could be extended beyond serine to pyruvate, a key intermediate, to access a variety of chemicals from aromatics and terpenes to alcohols and polymers.

Precise, stringent, post-translational activation of enzymes is essential for many synthetic biology applications. For example, even a few intracellular molecules of unregulated T7 RNA polymerase can result in growth cessation in a bacterium. We sought to mimic the properties of natural enzymes, where activity is regulated ubiquitously by endogenous metabolites. Here we demonstrate that full-length, single subunit T7-derived RNA polymerases (T7 RNAP) can be activated by physiologically relevant concentrations of indoles. We used rational design and directed evolution to identify T7 RNAP variants with minimal transcriptional activity in the absence of indole, and a 29-fold increase in activity with an EC₅₀ of 344 μM. Indoles control T7-dependent gene expression exogenously, endogenously, and between cells. We also demonstrate indole-dependent bacteriophage viability and propagation in trans. Specificity of different indoles, T7 promoter specificities, and portability to different bacteria are shown. Our ligand activated RNA polymerases (LARPs) represent a new chemically inducible "stop and go" platform immediately deployable for novel synthetic biology applications, including for modulation of synthetic cocultures.

Transmembrane receptors that endow mammalian cells with the ability to sense and respond to biomaterial-bound ligands will prove instrumental in bridging the fields of synthetic biology and biomaterials. Materials formed with thiol-norbornene chemistry are amenable to thiol-peptide patterning, and this study reports the rational design of synthetic receptors that reversibly activate cellular responses based on peptide-ligand recognition. This transmembrane receptor platform, termed Extracellular Peptide-ligand Dimerization Actuator (EPDA), consists of stimulatory or inhibitory receptor pairs that come together upon extracellular peptide dimer binding with corresponding monobody receptors. Intracellularly, Stimulatory EPDAs phosphorylate a substrate that merges two protein halves, whereas Inhibitory EPDAs revert split proteins back to their unmerged, inactive state via substrate dephosphorylation. To identify ligand-receptor pairs, over 2000 candidate monobodies were built in silico using PETEI, a novel computational algorithm we developed. The top 30 monobodies based on predicted peptide binding affinity were tested experimentally, and monobodies that induced the highest change in protein merging (green fluorescent protein, GFP) were incorporated in the final EPDA receptor design. In soluble form, stimulatory peptides induce intracellular GFP merging in a time- and concentration-dependent manner, and varying levels of green fluorescence were observed based on stimulatory and inhibitory peptide-ligand dosing. EPDA-programmed cells encapsulated in thiol-norbornene hydrogels patterned with stimulatory and inhibitory domains exhibited 3D activation or deactivation based on their location within peptide-patterned hydrogels. EPDA receptors can recognize a myriad of peptide-ligands bound to 3D materials, can reversibly induce cellular responses beyond fluorescence, and are widely applicable in biological research and regenerative medicine.

Base editors, e.g., cytosine deaminases, are powerful tools for precise DNA editing in vivo, enabling both targeted nucleotide conversions and segment-specific diversification of bacterial genomes. Yet, regulation of their spatiotemporal activity is crucial to avoid off-target effects and enabling controlled evolution of specific genes and pathways. This work reports a strategy for tight control of base-editing devices through subjecting their expression to a genetic AND logic gate in which two chemical inducer inputs are strictly required for cognate activity. The case study involves an archetypal genetic device consisting of a cytosine deaminase (pmCDA1) fused to a T7 RNA polymerase (RNAP^T7), which cause intensive diversification of DNA portions bordered by a T7 promoter and a T7 terminator─but whose activity in vivo has been shown unattainable to govern with standard conditional expression systems. By encoding up to three UAG stop codons into the DNA sequence of the pmCDA1-RNAP^T7 fusion, which is transcribed by the 3-methylbenzoate inducible promoter Pm, we first broke the structure of the hybrid protein. Then, to overcome the interruptions caused by UAG codons, we placed transcription of a supF tRNA under the control of a cyclohexanone-dependent system. When tested in the soil bacterium and metabolic engineering chassis Pseudomonas putida KT2440, these modifications changed the performance of the sliding base editor from a flawed YES logic to a precise AND logic. We also showed that such a 2-layer control brings about a minimal background activity as compared to a single-input digitalizer circuit. These results show the ability of suppressor tRNA-based logic gates for achieving stringent expression of otherwise difficult to control devices.

Engineered bacteria offer a novel approach to targeted cancer therapy, but challenges remain in delivering enough bacteria safely for effective treatment. Previous efforts have used either a native or synthetic coating to achieve better control over the half-life of bacteria in the body but have limitations in delivery or versatility. In this work, we optimized and evaluated a synthetic coating for probiotic Escherichia coli Nissle 1917 to increase its half-life in blood and thereby increase the bioavailability of intravenous doses of bacteria to colonize and treat tumors. Using a simple one-pot chemical process, we coated bacteria with iron and tannic acid (FeTA) to form a temporary adhesive protective coating surrounding the bacterial cell surface. The iron to tannic acid ratio of the coating was optimized for intravenous use, and FeTA-coated bacteria of several different genetic backgrounds showed 15-fold higher survival in blood survival assays for up to 4 hours. We found that the FeTA coating reduced both complement-mediated bacterial killing and phagocyte-mediated bacterial killing in vitro. As a result, systemic delivery of attenuated bacteria had up to 60% colonization efficiency of FeTA-coated bacteria in an orthotopic breast cancer mouse model compared to 10% for the non-coated control, all the while maintaining a two-fold decrease in weight loss of attenuated bacteria compared to wild-type. Altogether, we show that an optimized FeTA coating significantly extends the half-life and colonization efficiency of engineered bacteria, overcoming a key limitation of their application in cancer therapy.

The fusion of synthetic biology and materials science offers exciting opportunities to produce sustainable materials that can perform programmed biological functions such as sensing and responding or enhance material properties through biological means. Bacterial cellulose (BC) is a unique material for this challenge due to its high-performance material properties and ease of production from culturable microbes. Research in the past decade has focused on expanding the benefits and applications of BC through many approaches. Here, we explore how the current landscape of BC-based biomaterials is being shaped by progress in synthetic biology. As well as discussing how it can aid production of more BC and BC with tailored material properties, we place special emphasis on the potential of using BC for engineered living materials (ELMs); materials of a biological nature designed to carry out specific tasks. We also explore the role of 3D bioprinting being used for BC-based ELMs and highlight specific opportunities that this can bring. As synthetic biology continues to advance, it will drive further innovation in BC-based materials and ELMs, enabling many new applications that can help address problems in the modern world, in both biomedicine and many other application fields.

Immune cells play a pivotal role in the establishment, growth, and progression of tumors at primary and metastatic sites. Macrophages, in particular, play a critical role in suppressing immune responses and promoting an anti-inflammatory environment through both direct and indirect cell-cell interactions. However, our understanding of the mechanisms underlying such interactions is limited due to a lack of reliable tools for studying transient interactions between cancer cells and macrophages within the tumor microenvironment. Recent advances in mammalian synthetic biology have introduced a wide range of synthetic receptors that have been used in diverse biosensing applications. One such synthetic receptor is the synNotch receptor, which can be tailored to sense specific ligands displayed on the surface of target cells. With this study, we aimed at developing a novel αCD206-synNotch receptor, targeting CD206⁺ macrophages, a population of macrophages that play a crucial role in promoting metastatic seeding and persistent growth. Engineered in cancer cells and used in mouse metastasis models, such a tool could help monitor─and provide an understanding of─the effects that cell-cell interactions between macrophages and cancer cells have on metastasis establishment. Here, we report the development of cancer landing-pad cells for versatile applications and the engineering of αCD206-synNotch cancer cells in particular. We report the measurement of their activity and specificity, and discuss unexpected caveats regarding their in vivo applications.