ACS Synthetic Biology最新文献

Label-free detection of microRNAs (miRNAs) has emerged as a pivotal approach in molecular diagnostics, integrating the programmability of CRISPR systems with the high sensitivity of nanomaterial-based transduction. miRNAs are short, noncoding RNAs that play central roles in gene regulation and disease pathogenesis, serving as valuable biomarkers for early diagnosis and prognosis. Conventional miRNA detection methods rely on labeling and multistep amplification, which hinder their adaptability for rapid and point-of-care applications. In contrast, label-free biosensing translates molecular recognition into intrinsic optical, electrochemical, or mechanical signals, enabling real-time, amplification-free analysis. This review summarizes recent advances in label-free miRNA biosensing, with emphasis on CRISPR/Cas12a, Cas13a, and Cas14a systems that couple target recognition with signal transduction, and nanomaterial-assisted platforms including gold and silver nanoparticles, carbon nanotubes, quantum dots, silica nanostructures, and magnetic composites. Particular attention is given to innovations that achieve attomolar-level sensitivity, single-nucleotide discrimination, and multiplex detection. We also discuss integration into microfluidic and wearable platforms, addressing persistent challenges in repeatability and stability, antifouling performance, and clinical translation. Emerging trends in artificial intelligence-assisted data processing, molecular logic circuits, and digital single-molecule biosensing are highlighted. These advances collectively outline the pathway toward intelligent, amplification-free, and portable miRNA diagnostics, bridging molecular biology and synthetic bioengineering for next-generation healthcare applications.

Extraintestinal pathogenic Escherichia coli O45 is an emerging multidrug-resistant serotype. Herein, we developed a glycoengineered E. coli strain for efficient biosynthesis of O45 O-polysaccharide (OPS45) and the OPS45-based glycoconjugate vaccine. Systematic optimization enhanced the production of OPS45, a polysaccharide containing the rare sugars 6-deoxy-l-talose (6d-l-Tal) and N-acetylfucosamine (FucNAc), with structure confirmed by NMR. Using oligosaccharyltransferase-mediated conjugation in this glyco-optimized chassis strain, we generated a homogeneous cholera toxin B subunit (CTB)-OPS45 glycoconjugate with enhanced antigen loading yield (11.31 ± 0.59 mg/L vs 8.24 ± 0.075 mg/L). LC-MS/MS verified site-specific glycosylation on CTB. Immunization in mice elicited strong O45-specific IgG responses and conferred 90% protection against lethal neonatal meningitis-causing Escherichia coli (NMEC) infection, with a nearly 80% reduction in bacterial burden. These results demonstrate that our integrated biosynthesis and conjugation approach enables rapid and efficient production of a well-defined glycoconjugate vaccine, showing strong potential for combatting resistant NMEC and O45 infections.

Genetic programs can direct living systems to perform diverse, prespecified functions. As the library of parts available for building such programs continues to expand, computation-guided design is increasingly helpful and necessary. Predictive models aid the challenging design process, but iterative simulation and experimentation are intractable for complex functions. Computer-aided design accelerates this process, but existing tools do not yet capture the behavior of mammalian-specific parts and population-level effects needed by mammalian synthetic biologists. To address these needs, we developed a framework for mammalian genetic program computer-aided design. Starting with a user-defined design specification to quantify circuit performance, the framework uses a genetic algorithm to search through possible designs. Circuit space is defined by a library of experimentally characterized parts and dynamical systems models for gene expression in a heterogeneous cell population. We developed this genetic algorithm using a directed graph-based formulation with biologically constrained rules to explore regulatory connections and parts. We evaluated the framework for design problems of varying complexity, including programs we describe as an amplifier, signal conditioner, and pulse generator, demonstrating that the algorithm can successfully find optimal circuit designs. Finally, we experimentally evaluated selected circuits, demonstrating the path from a predicted circuit design to experimental testing and highlighting the importance of characterization in enabling predictive design. Overall, this framework establishes general approaches that can be refined and expanded, accelerating the design and implementation of mammalian genetic programs.

Clopidogrel is a widely used antiplatelet prodrug to treat acute coronary syndromes. However, its clinical efficacy is hampered by ineffective bioactivation to produce the pharmacologically active metabolite (AM), leading to variability in the antiplatelet response among different ethnic groups. To overcome the shortcomings of clopidogrel, DT-678 was developed by conjugating AM to 3-nitropyridine-2-thiol via a mixed disulfide bond. It has been challenging to produce the conjugate in a high yield by chemical synthesis. Here, we report the first de novo biosynthesis of DT-678 using engineered CYP102A1 variants. We applied structure-based computational design using UniDesign to generate three variants (UD4, UD5, and UD6) that enhanced the catalytic activity and selectivity toward DT-678 synthesis. Among them, UD6 demonstrated the highest total turnover number and DT-678-specific productivity under the optimized conditions. Mechanistic analysis revealed that rapid enzyme inactivation, driven by reactive oxygen species (ROS) such as superoxide and hydrogen peroxide, limited the overall yield. Remarkably, we found that ascorbic acid significantly protected CYP102A1 variants from inactivation and, hence, increased production yield. This work establishes a scalable enzymatic strategy for DT-678 biosynthesis and highlights the importance of combining protein engineering with redox control to overcome limitations in CYP-catalyzed reactions.

Protein circuits organize cell biology, but synthetic dynamics are challenging to engineer due to stochastic genetic and biochemical variation. Genetically encoded oscillators (GEOs) built from bacterial MinDE-family ATPases and activators generate synthetic protein waves that act as novel frequency-domain imaging barcodes in eukaryotic cells, providing a platform for understanding, engineering, and applying synthetic protein dynamics. Using budding yeast, we disentangle how expression levels and expression noise govern the GEO waveform and encodability. While the GEO amplitude is sensitive to extrinsic noise, the GEO frequency is stably encoded by the activator:ATPase ratio. By integrating GEO components into the yeast modular cloning toolkit, we developed different noise-guided expression strategies that act like filters on the GEO waveform. We paired these filters with hundreds of biochemically distinct GEO variants to engineer clonal populations that oscillate at distinct frequencies and to design waveform libraries with customizable spectral features and tunable waveform variation. Our work establishes a robust platform for precision genetic encoding of synthetic GEO oscillations and highlights the utility of noise-guided strategies for dynamic protein circuit design.

Maintaining proper redox conditions is essential for protein stability and function. In cell-free protein synthesis, reducing agents, such as dithiothreitol and reduced glutathione, are commonly added to mimic the cytosolic environment and prevent unwanted oxidation. The PURE system, which is a fully reconstituted protein synthesis system, also contains reducing agents. Here, we systematically examined how reducing agents affect the protein synthesis in the PURE system. We found that the reducing activity of dithiothreitol decreased during prolonged reactions, leading to the formation of disulfide bonds in synthesized proteins. Dissolved oxygen and contaminating metal ions were identified as major factors causing this loss of activity. Based on these findings, we developed a method to maintain reducing conditions throughout the reaction, ensuring consistent protein quality. Our results provide new insights into redox regulation in cell-free systems and offer a practical strategy for the efficient synthesis of functional proteins, with potential applications in biotechnology and therapeutic protein production.

Bacillus subtilis is a widely used microbial host for both fundamental research and the industrial production of enzymes and biopharmaceuticals. However, its four known signal peptide-dependent secretion pathways impose inherent limitations on the efficient extracellular expression of heterologous proteins. Here, we identify fructose 1,6-bisphosphate aldolase (FbaA) as a nonclassically secreted protein that achieves high-level expression and efficient extracellular export in B. subtilis. Structural and biochemical data indicate tetrameric FbaA as the predominant secreted form. Site-directed mutagenesis shows that the hydrophobic residue methionine 66 (M66) is critical for tetramer formation; substituting M66 disrupts oligomerization, abolishes secretion, and eliminates FbaA's ability to mediate the export of heterologous proteins. Together, these results establish that FbaA oligomerization is essential for its nonclassical secretion and that FbaA functions as a modular export element capable of facilitating the secretion of fused heterologous proteins. This work provides mechanistic insight into oligomerization-dependent protein export and offers a promising strategy for engineering efficient secretion systems in B. subtilis.

The benzylisoquinoline alkaloid (BIA) family of tetrahydroisoquinolines (THIQs) comprises >2,500 members, including the pharmaceuticals morphine, codeine, and papaverine, as well as the antibiotics sanguinarine and chelerythrine. Agricultural cultivation can supply the demand for the BIAs that accumulate in plants, but broader access to the BIA family would facilitate additional research and commercialization. Microbial synthesis presents an attractive option due to cheap feedstock, genetic tractability, and ease of scale-up. Previously, we reported titers of the branch-point BIA (S)-reticuline of 4.6 g/L in yeast, which was achieved through leveraging the Ehrlich pathway 2-oxoacid decarboxylase Aro10 to generate the intermediate 4-hydroxyphenylacetaldehyde (4-HPAA). Here, we establish a superior route to (S)-reticuline by switching the pathway intermediate from 4-HPAA to 3,4-dihydroxyphenylacetaldehyde (3,4-dHPAA) using monoamine oxidase A (MAO). The resulting (S)-norlaudanosoline route to (S)-reticuline synthesis is more selective, resolving prior issues with off-pathway THIQs synthesized due to cascading enzyme promiscuity, and more efficient, enabling titers of 4.8 g/L (S)-reticuline while improving yields by over 40%, from 17 to 24 mg/g sucrose in fed-batch fermentations. Finally, we extend de novo (S)-reticuline synthesis to dihydrosanguinarine, achieving 635 mg/L dihydrosanguinarine and sanguinarine in fed-batch fermentation, the highest reported titer of these BIAs by a factor of 40.

Synthetic mRNA provides a powerful platform to transfer genetic information encoding therapeutic proteins in vivo. However, their applications are limited by their intrinsic instability and insufficient protein yield. Here we report a 40-nt RNA sequence (ARF6.40) that markedly improves mRNA stability and protein bioavailability of synthetic mRNA. ARF6.40, identified from the 3' UTR of the human ARF6 mRNA, contains a novel CU-rich element that interacts with the protein U2AF2. Compared with the broadly used globin 3' UTR, fusing ARF6.40 to the 3' UTR of EGFP mRNA results in markedly increased mRNA half-life and protein level; transfection of cells with SARS-CoV-2 RBD-encoding mRNA or administration of mice with LNP-encapsulated firefly luciferase mRNA fused with ARF6.40 leads to significantly higher and more sustained RBD secretion or luciferase expression in vivo. Together, our study demonstrates the potential of ARF6.40 in mRNA therapeutics and provides new insights into how the expression of ARF6 is regulated.

Clostridium tyrobutyricum Δcat1::adhE2 is a promising cell factory for butanol production because of its robustness, high butanol tolerance, and minimal butyrate production. However, excessive acetate and ethanol production remains a major bottleneck limiting its butanol yield. Coexpressing an exogenous hbd(Ck) encoding the NADPH-dependent 3-hydroxybutyryl-CoA dehydrogenase (HBD) from Clostridium kluyveri with adhE2 could increase the C4 carbon flux, resulting in increased butanol and decreased acetate and ethanol production. However, constitutively overexpressing hbd(Ck) in Δcat1::adhE2 shows little improvement in butanol yield, productivity, and selectivity, which might be caused by redox imbalance and growth inhibition. To alleviate this problem, C. tyrobutyricum MΔcat1::adhE2-Pbgal-hbd(Ck) with a dynamic expression of hbd(Ck) controlled by an inducible promoter was developed. In serum bottle fermentation at 37 °C, when the hbd(Ck) expression was induced at 12 h or in the early exponential phase, butanol production increased ∼20% in yield (from 0.22 to 0.27 g/g glucose), 87.5% in productivity (from 0.16 to 0.30 g/L·h), and 52% in selectivity (from 0.46 to 0.70 g/g total products) compared to the control strain without expressing any hbd(Ck), whereas hbd(Ck) expression induced at 0 or 24 h in MΔcat1::adhE2-Pbgal-hbd(Ck) or constitutively in MΔcat1::adhE2-Pcat1-hbd(Ck) showed significantly lower butanol yield and productivity. At 25 °C, MΔcat1::adhE2-Pbgal-hbd(Ck) with 12 h induction produced the highest butanol titer of 23 g/L with 0.32 g/g yield, 0.16 g/L·h productivity, and 0.83 g/g product selectivity due to much reduced acetate formation. Subsequent scale-up to a stirred-tank bioreactor at 37 °C increased productivity to 0.39 g/L·h while also achieving high butanol titer (21.8 g/L), yield (0.30 g/g), and selectivity (0.67 g/g). The optimized induction timing resulted in a balanced NAD(P)H pool, effectively channeling substrates toward butanol biosynthesis. It was concluded that the timing for hbd(Ck) expression was critical as it affected glucose catabolism, cell growth, redox balance, and carbon flux distribution. These findings underscore the potential of dynamic metabolic regulation to overcome bottlenecks in biobutanol production, providing a scalable and economically viable bioprocess for industrial application.