ACS Synthetic Biology最新文献

Mycomaterials, materials made from filamentous fungi, have several advantages over traditional materials such as their genetic programmability and self-healing properties. However, their lack of mechanical strength and cost of production often constrain the applications in which they can be used in. In this work, we take inspiration from natural systems to overcome these challenges by elucidating design principles for mineralization-based enhancement of mechanical strength and synthetic lichen-based low-cost growth. We demonstrate that surface display of an enzyme from sea sponges, silicatein α, on the hyphae of the filamentous fungus Aspergillus niger enables mineralization of polysilicate and that this does not impact fungal growth. We also show that this strategy can be extended to other silicatein α variants and characterize how the degree of mineralization can be modulated. We then demonstrate that mineralization enhances the mechanical properties of the mycelium including its tensile strength, modulus, and toughness. Finally, we show how these reinforced mycelia can be grown without external carbon sources using a synthetic lichen-based coculture to facilitate low-cost biomanufacturing. Together, our results lay the groundwork for the sustainable production of mineralized mycomaterials and create a new model system to study how mineralization impacts growth and mechanical properties.

Phage genome engineering methods accelerate the study of phage biology, the discovery of new functions, and the development of innovative genetic engineering tools. Here, we present QuickPhage, a rapid, technically accessible, precise, and cost-effective method for engineering Bacillus subtilis phages. Our approach uses CRISPR-Cas9 as a counter-selection system to isolate mutants of the model lytic siphovirus phage, SPP1. Efficient genome editing was achieved using homologous repair patches as short as 40 nucleotides, enabling streamlined patch construction and parallel engineering, resulting in highly accurate genome edits within a day. We applied QuickPhage to delete both essential and nonessential phage genes and to insert reporter genes. Protein production, such as GFP, was synthetically regulated using inducible systems without significantly affecting phage fitness, achieving induction levels of up to 400-fold. Time-series coinfection experiments with fluorescent protein expressing phages also revealed a highly efficient superinfection arrest mechanism that prevents reinfection as early as 13 min after initial infection. These findings highlight the potential of phages for protein production, opening new opportunities for metabolic engineering. This work also lays the foundation for systematic phage genome refactoring workflows and the development of phage-based tools for efficient DNA delivery, thereby expanding the synthetic biology toolbox for B. subtilis.

Temporal transcriptional modulation of immune-related genes offers powerful therapeutic potential for treating inflammatory diseases. Here we introduce an enhanced zinc finger (ZF)-based transcriptional repressor delivered via lipid nanoparticles for controlling immune signaling pathways in vivo. By targeting Myd88, an essential adaptor molecule involved in immunity, our system demonstrates therapeutic efficacy against septicemia in C57BL/6J mice and improves repeated AAV administration by reducing antibody responses. This epigenetic engineering approach provides a platform for safe and efficient immunomodulation applicable across diseases caused by imbalanced inflammatory responses.

Metabolite-responsive, protein-based biosensors are powerful tools for monitoring cellular metabolite dynamics in vivo and accelerating strain engineering workflows in microorganisms. In this study, we introduced a previously developed protein-based biosensor, computationally designed to detect farnesyl diphosphate (FPP), in the marine diatom Phaeodactylum tricornutum. We expressed two versions of the biosensor constitutively, under a strong promoter-terminator pair using extrachromosomal episomes, and we parametrized the capacity of both designs in detecting intracellular metabolite levels. Initial assays revealed that the two versions of the biosensor we investigated, S3–2D and S3–3A, had specificity not only for FPP but also for other exogenously supplied prenyl phosphates such as geranyl diphosphate (GPP) and geranylgeranyl diphosphate (GGPP) in a dose-dependent manner, showcasing broader specificity for multiple prenyl phosphates. We further demonstrated the capacity of S3–3A to track perturbations in the endogenous prenyl phosphate pools by testing it in the presence of pharmacological inhibition of the mevalonate pathway. Moreover, S3–3A generated dot-like, fluorescent signal “hotspots” in the cytosol of diatoms, suggesting a complex subcellular organization of the isoprenoid biosynthesis in P. tricornutum. These findings lay the groundwork for developing metabolite-responsive biosensors as useful tools for monitoring and investigating prenyl phosphate dynamics in diatoms, providing a foundation for advanced metabolic engineering of microalgae.

R6K plasmids are commonly used for a wide range of genome engineering applications due to their ability to support transient delivery of genetic cargos in many hosts. The maintenance of R6K plasmids requires specific strains. Unfortunately, many of these have obscure backgrounds, limited availability and were not built for efficient cloning. To address this issue, we present the construction and characterization of a series of Pir E. coli strains called SHARK that are built from the DH10B derivative, Marionette-Clo. All SHARK strains have a genome encoded pir gene for stable R6K plasmid maintenance and a λCI gene for tight unconditional repression of specific genes on plasmids. We show that SHARK strains are >100-fold more efficient than a commercial Pir strain when transformed with large and complex cloning reactions. SHARK is intended to help facilitate the cloning of R6K plasmids for challenging genome engineering projects, with all strains and genetic tools for their assembly being made publicly available.

Advances in synthetic biology continue to potentiate bacterial cancer therapy. Here, we constructed biosensor-driven encapsulation systems for autonomous control of capsular polysaccharides of Escherichia coli Nissle 1917 to improve pharmacokinetic profiles. The engineered bacteria were programmed to express capsular polysaccharides for immune evasion upon intravenous administration to reach tumors and then turn off gene expression upon colonizing the tumors based on quorum-sensing or acid-sensing to prevent dissemination of bacteria into the systemic circulation. Because a classical pharmacokinetic model could not capture the dynamic nature of living therapeutics, a two-state pharmacokinetic model was developed to simulate the autonomous control of capsular polysaccharides in different biological compartments and their impact on biodistribution. Using this model, we identified parameters in gene circuit dynamics and immune clearance that influence tumor colonization and systemic bacterial persistence. In a “humanized” pharmacokinetic model with an increased rate of complement-mediated lysis of bacteria, biosensor-driven systems achieved tumor seeding densities comparable to wild-type bacteria while reducing bacterial loads in blood and liver by several orders of magnitude, highlighting their potential for safe systemic delivery. The biosensor-driven systems represent a more effective strategy to control living drugs than inducible systems, and the two-state pharmacokinetic model is a first step to capture the autonomous nature of this new class of therapeutics for clinical translation.

Biosensors have been widely applied for high-throughput strain screening and dynamic regulation of metabolic networks. However, existing tryptophan sensors based on transcription factors or riboswitches often suffer from a narrow dynamic range and limited response threshold. In this study, we developed a series of tryptophan-responsive biosensors in Escherichia coli using the tryptophan-activated RNA-binding attenuation protein (TRAP) as the sensing module. First, we validated TRAP functionality and engineered a functional biosensor by fine-tuning its expression. Subsequently, screening of TRAP variants and optimization of TRAP–leader sequence interactions yielded two biosensors that exhibited distinct dynamic ranges (up to 22.1-fold) and response thresholds of 0–2.2 g/L, respectively. Using these biosensors, we screened two beneficial variants of key rate-limiting enzymes in the tryptophan biosynthetic pathway and further investigated their catalytic mechanisms through molecular dynamics simulations. Collectively, this study provides tools for engineering high tryptophan-producing strains and new strategies for biosensor design.

Hyperlysinemia is a life-threatening metabolic disorder that requires the continuous clearance of lysine. Engineered probiotics capable of degrading lysine in the gut represent a promising therapeutic strategy. However, the introduction of heterologous metabolic pathways can impose a substantial fitness burden on the bacterial host, potentially compromising the therapeutic efficacy. Current screening methods fail to adequately assess this pathway-induced stress. Therefore, optimizing methods to evaluate bacterial fitness after pathway modification is essential for developing effective bacterial therapies. Here, we present a label-free phenotypic screening approach using Fourier transform infrared (FTIR) spectroscopy to evaluate the physiological burden imposed by two distinct lysine catabolism pathways engineered Escherichia coli Nissle 1917 (EcN): the plant-derived bifunctional enzyme LKR-SDR and the yeast-derived two-enzyme cascade Lys2-Lys5. Employing FTIR under lysine stress mimicking pathological concentrations, decoded pathway-specific stress signatures, and molecular resilience. Probiotics expressing LKR-SDR exhibited severe multisystem damage, including proteotoxicity, lipid peroxidation, and significant nucleic acid stress. In contrast, the Lys2-Lys5 strain demonstrated superior resilience, maintained structural integrity, and exhibited adaptive metabolic changes, primarily through lipid membrane remodeling. This study establishes FTIR spectroscopy as a rapid screening platform that identifies the Lys2-Lys5 pathway as optimal for probiotic therapies. By directly linking spectroscopic signatures to cellular fitness, FTIR spectroscopy accelerates the rational development of durable microbial therapeutics for inborn metabolic disorders.

Lipoic acid (LA) is a sulfur-containing cofactor with significant antioxidant and metabolism-regulating functions, which is widely used in the pharmaceutical and nutraceutical industries. However, current microbial production of LA relies on exogenous octanoic acid and synthesizes the product in a protein-bound form, requiring a subsequent dissociation step to obtain free LA. In this study, we constructed an Escherichia coli strain capable of de novo synthesizing free LA by deleting the compensatory pathway gene lplA and introducing eflpA, a lipoamidase that hydrolyzes protein-bound LA. A high-efficiency LA synthase (sllipA) was subsequently screened from Serratia liquefaciens and optimized at the gene copy-number level, resulting in a 44% increase in LA production. Furthermore, by enhancing the carbon flux from acetyl-CoA to the precursor octanoic acid and improving the intracellular supply of the key cofactors S-adenosylmethionine (SAM) and [4Fe-4S] iron–sulfur clusters, the LA titer was further increased by 184%. Finally, under controlled microaerobic production conditions, the optimized strain achieved an LA titer of 138.32 mg/L, representing the highest level of microbial LA production reported to date.

The pathogenic bacterium Vibrio parahaemolyticus represents a substantial economic and public health concern; however, elucidating its virulence mechanisms has been significantly impeded by its inherent resistant to genetic manipulation, primarily attributed to sophisticated immune defense systems including restriction-modification (R-M) modules, CRISPR-Cas systems, standalone DNases, and DdmDE systems. Paradoxically, while genetic modification is essential for overcoming these barriers, the very barriers themselves obstruct DNA introduction. Our investigation focused on the V. parahaemolyticus X1 strain, where initial plasmid transformation attempts proved unsuccessful. However, low-efficiency conjugation allowed knockout of defense genes, thereby silencing the host’s defense mechanisms. Our findings revealed a standalone DNase, Vpn, as the predominant obstacle to foreign DNA entry in the X1 strain, while a DdmDE system executes elimination of invaded plasmids. Leveraging these insights, we created the V. parahaemolyticus X2 strain via sequential depletion of the Vpn nuclease and the DdmDE system. Capitalizing on the bacterium’s exceptional growth rate, characterized by a generation time of approximately 10.5 min, we established a highly efficient molecular cloning platform capable of creating a new plasmid construct within a single day. This work not only presents a strategic framework for genetic manipulation of previously recalcitrant bacterial species but also underscores the potential of fast-growing marine bacteria as promising candidates for next-generation biotechnological applications.