ACS Synthetic Biology最新文献

Chitin biomass is the second most abundant natural polysaccharide after cellulose on the earth, yet its recalcitrance to degrade and utilize severely limits its application. However, many microorganisms, such as Serratia marcescen, can secrete a range of free chitinases to degrade chitin, though their activity is typically insufficient to meet industrial demands. In this study, we employed self-assembly systems, named SpyTag/SpyCatcher and SnoopTag/SnoopCatcher, to modularize the molecular design of CHB, ChiB, ChiC, and CBP21 derived from S. marcescens ATCC14756, and we successfully constructed a variety of chitinase complexes. The assembled complexes showed higher chitinolytic activity and stability, compared to free chitinase mixture. Moreover, the distinct arrangements and combinations of chitinases within these complexes led to varied activities, suggesting that the spatial proximity and substrate channeling effects contribute to the synergy of chitinase complexes. The findings lay a solid technical foundation for the application of chitinosome in the industrial production of N-acetylglucosamine and chitooligosaccharides.

Cell-free transcription-translation (TX-TL) systems have been used for diverse applications, but their performance and scope are limited by variability and poor predictability. To understand the drivers of this variability, we explored the effects of metabolic perturbations to anEscherichia coli (E. coli) Rosetta2 TX-TL system. We targeted three classes of molecules: energy molecules, in the form of nucleotide triphosphates (NTPs); central carbon "fuel" molecules, which regenerate NTPs; and magnesium ions (Mg²⁺). Using malachite green mRNA aptamer (MG aptamer) and destabilized enhanced green fluorescent protein (deGFP) as transcriptional and translational readouts, respectively, we report the presence of a trade-off between optimizing total protein yield and optimizing total mRNA yield, as measured by integrating the area under the curve for mRNA time-course dynamics. We found that a system's position along the trade-off curve is strongly determined by Mg²⁺ concentration, fuel type and concentration, and cell lysate preparation and that variability can be reduced by modulating these components. Our results further suggest that the trade-off arises from limitations in translation regulation and inefficient energy regeneration. This work advances our understanding of the effects of fuel and energy metabolism on TX-TL in cell-free systems and lays a foundation for improving TX-TL performance, lifetime, standardization, and prediction.

The field of engineering living materials (ELMs) seeks to engineer cells to form macroscopic materials with tailorable structures and properties. While the rheological properties of ELMs have been altered using synthetic biology methodology, the relationships connecting their sequence, structural, and rheological properties remain to be elucidated. Recently, our lab created centimeter-scale ELMs from Caulobacter crescentus that offer a platform to investigate this paradigm. Here, we explore how changing the elastin-like polypeptide (ELP) length within the protein matrix of this ELM impacts its microstructure and viscoelastic behavior. We demonstrate that shortening ELP produces fibers almost 2× thicker than other variants, resulting in a stiffer material at rest. Interestingly, the midlength ELP forms a complex structure with globules and multidirectional fibers with increased yield stress under flow conditions. Lengthening ELP creates thinner strands between cells with similar storage and loss moduli to those of the midlength ELP. This study begins to elucidate sequence-structure-property relationships in these ELMs and shows that they are complex with few parallels to other biocomposite models. Furthermore, it highlights that fine-tuning genetic sequences can create significant differences in rheological properties, uncovering new design principles of ELMs.

Hand, foot, and mouth disease (HFMD), caused by enteroviruses, mostly including EV71, CVA6, CVA10, and CVA16, is an acute infectious disease commonly found in children. Due to no approved antiviral therapies and available vaccines, except for EV71, developing accurate diagnostic methods of HFMD is essential for controlling its spread and mitigating its impact on public health. Here, we create a MIRA-HEV-PAND multiple nucleic acid typing method that utilizes PfAgo to identify enterovirus type A pathogens (EV71, CVA6, CVA10, and CVA16) and universal type EVU. The MDC (minimum detection concentration) level of MIRA-HEV-PAND is within the range of 1.66 aM (1.0 copy/μL), which was matched to that of qPCR assays and even more sensitive up to 10%. Importantly, the MIRA-HEV-PAND method exhibits higher sensitivity and less time-consuming efficiency compared to the approach that combines PCR amplification instead of MIRA amplification. Meanwhile, though the quintuple and single-tube multiple MIRA-HEV-PAND detection system can be used for one viral target or multiple viral target detection, the single-tube detection system detects more efficiently and rapidly than the quintuple-tube multiple detection system. Moreover, the diagnostic results obtained by evaluating clinical samples using MIRA-HEV-PAND show a complete consistency of 100% with qPCR assays. The MIRA-HEV-PAND method can screen a wider range of target regions using low-cost guide DNA without being limited to PAM sequences, compared to the MARPLES based on the CRISPR-Cas12a. The utilization of this correlation can be beneficial for the application of molecular testing for clinical diagnoses and the study of human enteroviruses A infection and virus typing on an epidemiological scale.

Nature has evolved to exclusively use a genetic code consisting of triplet nucleotide codons. The translation system, however, is known to be compatible with 4-nucleotide frameshift or quadruplet codons. In this study, we begin to explore the possibility of a genome made up entirely of quadruplet codons using the minimal mitochondrial genome of Saccharomyces cerevisiae as a model system. We demonstrate that mitochondrial tryptophanyl- and tyrosyl-tRNAs with modified anticodons effectively suppress mutant cox3 genes containing a TAG stop or TAGA quadruplet codon, leading to the production of full-length COX3 and a respiratory-competent phenotype. This work provides a method for introducing heterologous tRNAs into the yeast mitochondria for genetic engineering applications and serves as a starting point for the development of a quadruplet codon genetic code.

Protein phase transitions are gaining traction among biologists for their wide-ranging roles in biological regulation. However, achieving precise control over these phenomena in vivo remains a formidable task. Optogenetic techniques present us with a potential means to control protein phase behavior with spatiotemporal precision. This review delves into the design of optogenetic tools, particularly those aimed at manipulating protein phase transitions in complex biological systems. We begin by discussing the pivotal roles of subcellular phase transitions in physiological and pathological processes. Subsequently, we offer a thorough examination of the evolution of optogenetic tools and their applications in regulating these protein phase behaviors. Furthermore, we highlight the tailored design of optogenetic tools for controlling protein phase transitions and the construction of synthetic condensates using these innovative techniques. In the long run, the development of optogenetic tools not only holds the potential to elucidate the roles of protein phase transitions in various physiological processes but also to antagonize pathological ones to reinstate cellular homeostasis, thus bringing about novel therapeutic strategies. The integration of optogenetic techniques into the study of protein phase transitions represents a significant step forward in our understanding and manipulation of biology at the subcellular level.

Designing de novo enzymes is complex and challenging, especially to maintain the activity. This research focused on motif design to identify the crucial domain in the enzyme and uncovered the protein structure by molecular docking. Therefore, we developed a Generative Redesign in Artificial Computational Enzymology (GRACE), which is an automated workflow for reformation and creation of the de novo enzymes for the first time. GRACE integrated RFdiffusion for structure generation, ProteinMPNN for sequence interpretation, CLEAN for enzyme classification, and followed by solubility analysis and molecular dynamic simulation. As a result, we selected two gene sequences associated with carbonic anhydrase from among 10,000 protein candidates. Experimental validation confirmed that these two novel enzymes, i.e., dCA12_2 and dCA23_1, exhibited favorable solubility, promising substrate-active site interactions, and achieved activity of 400 WAU/mL. This workflow has the potential to greatly streamline experimental efforts in enzyme engineering and unlock new avenues for rational protein design.

Repeat expansion disorders, exemplified by myotonic dystrophy type 1 (DM1), present challenges in diagnostic quantification because of the variability and complexity of repeat lengths. Traditional diagnostic methods, including PCR and Southern blotting, exhibit limitations in sensitivity and specificity, necessitating the development of innovative approaches for precise and rapid diagnosis. Here, we introduce a CRISPR-based diagnostic method, REPLICA (repeat-primed locating of inherited disease by Cas3), for the quantification and rapid diagnosis of DM1. This method, using in vitro-assembled CRISPR-Cas3, demonstrates superior sensitivity and specificity in quantifying CTG repeat expansion lengths, correlated with disease severity. We also validate the robustness and accuracy of CRISPR diagnostics in quantitatively diagnosing DM1 using patient genomes. Furthermore, we optimize a REPLICA-based assay for point-of-care-testing using lateral flow test strips, facilitating rapid screening and detection. In summary, REPLICA-based CRISPR diagnostics offer precise and rapid detection of repeat expansion disorders, promising personalized treatment strategies.

Cell-free systems are advancing synthetic biology through fast prototyping and modularity. Complex regulatory networks can now be implemented in cell-free systems enabling various applications, such as diagnostic tool development, gene circuit prototyping, and metabolic engineering. As functional complexity increases, the need for regulatory components also grows. This review provides a comprehensive overview of native as well as engineered regulatory components and their use in bacterial cell-free systems.

Oligopool synthesis and next-generation sequencing enable the construction and characterization of large libraries of designed genetic parts and systems. As library sizes grow, it becomes computationally challenging to optimally design large numbers of primer binding sites, barcode sequences, and overlap regions to obtain efficient assemblies and precise measurements. We present the Oligopool Calculator, an end-to-end suite of algorithms and data structures that rapidly designs many thousands of oligonucleotides within an oligopool and rapidly analyzes many billions of barcoded sequencing reads. We introduce several novel concepts that greatly increase the design and analysis throughput, including orthogonally symmetric barcode design, adaptive decision trees for primer design, a Scry barcode classifier, and efficient read packing. We demonstrate the Oligopool Calculator's capabilities across computational benchmarks and real-data projects, including the design of over four million highly unique and compact barcodes in 1.2 h, the design of universal primer binding sites for one million 200-mer oligos in 15 min, and the analysis of about 500 million deep sequencing reads per hour, all on an 8-core desktop computer. Overall, the Oligopool Calculator accelerates the creative use of massively parallel experiments by eliminating the computational complexity of their design and analysis.