ACS Synthetic Biology最新文献

Biological research often involves complex, repetitive, and high-throughput manipulations that are well-suited to automation. However, current robotic systems generally excel only at narrowly defined tasks or standardized workflows and remain expensive, inflexible, and dependent on proprietary modules or reagents. To address these limitations, we developed the Open-Source Collaborative Automation & Robotics (OSCAR) platform, a flexible and low-cost system designed to perform common laboratory manipulations using standard, human-operated equipment. OSCAR incorporates open-source software and modular hardware to maximize accessibility and affordability. The platform features a robotic arm equipped with a dual-function end-effector: a pipetting module for precise liquid handling and a vision-enabled gripper for manipulating laboratory tools. To demonstrate the platform's versatility, we implemented a representative plasmid assembly workflow, from PCR amplification and enzymatic assembly to transformation, plating, colony picking, PCR screening, and validation by agarose gel electrophoresis. By making this system open-source and compatible with widely used consumables and equipment, we aim to democratize access to automation and broaden its adoption across diverse research environments.

Corynebacterium glutamicum is a key industrial chassis for producing high-value chemicals, particularly amino acids. However, integration of large DNA fragments remains either inefficient or labor-intensive. Here, we optimized a RecET variant-assisted homologous recombination system to achieve single-step integration of DNA fragments larger than 11.3 kb by transforming donor DNA derived from either chromosome or linear DNA fragments. To further expand the size limit, we developed the One-Step Multi-Fragment Assembly Integration (OMAI) strategy, in which multiple overlapping PCR fragments are cotransformed and assembled in vivo, permitting integration of heterologous sequences with a length approximately 50% longer than the conventional single-fragment limit. Each editing cycle of OMAI is completed within 3 days, which is expected to be the most rapid method for large-fragment insertion in C. glutamicum.

Natural products (NPs) produced by actinobacteria, particularly Streptomyces species, represent a rich source of bioactive compounds and have yielded many clinically important compounds. Actinobacterial genomes are characterized by high GC content and typically harbor 20-40 biosynthetic gene clusters (BGCs) per genome, which encode diverse NPs such as polyketides, peptides, and glycosides. CRISPR/Cas-based genome editing has emerged as a promising tool to activate silent BGCs and engineer NP biosynthesis. However, the efficiency of multiplex editing drastically decreases as the number of targeted sites increases. Here, we report a novel one-pot DNA assembly method, termed direct pathway synthesis and editing (DiPaSE), for the efficient synthesis and multiplex editing of long, high-GC BGCs. DiPaSE accurately assembles multiple high-GC DNA fragments up to 60 kb and enables simultaneous deletions and insertions within a target BGC without compromising the assembly efficiency. Using this approach, we identified functions of previously uncharacterized genes in the aureothin BGC and significantly enhanced the titer of the corresponding NP. The workflow employs conventional polymerase chain reaction, type IIP restriction enzymes, commercially available DNA assembly reagents, and Escherichia coli, providing a simple, cost-effective, and broadly applicable platform for genome mining, BGC refactoring, and rational design of artificial biosynthetic pathways.

Chondroitin sulfate C (CS-C) is a biologically significant glycosaminoglycan in which a precise 6-O-sulfation pattern confers critical structural and signaling functions in connective and neural tissues. Here, we report the first functional expression of human chondroitin 6-O-sulfotransferase-1 (C6ST-1) in Escherichia coli, enabling cell-free biosynthesis of CS-C. By applying structure-guided protein engineering combining transmembrane truncation, His₆ -MBP fusion, and PROSS-directed stabilizing mutations, we generated a soluble and catalytically active variant in E. coli Origami B (DE3) and modified Shuffle T7 Express. Under optimized reaction conditions (MES buffer pH 5.5, 30 °C, Mg²⁺/Ca²⁺/Mn²⁺ with protamine sulfate), the engineered enzyme catalyzed up to 67% sulfation of chondroitin (CS-O) to CS-C, verified via SAX-HPLC. Kinetic and molecular dynamics analyses revealed enhanced substrate affinity and catalytic efficiency for the M9 Δ131 mutant. This study establishes a sustainable, animal-free platform for high-purity CS-C biomanufacturing and provides a generalizable strategy for engineering eukaryotic sulfotransferases for functional expression in bacterial hosts.

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is a key enzyme in the mevalonate (MVA) pathway that catalyzes the formation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) from acetoacetyl-CoA and acetyl-CoA. Recently, a novel class of HMGS-sesquiterpene synthase (STS) fusion enzymes has been identified. In this study, we discovered a natural fusion enzyme, GihirA, in Gloeostereum incarnatum, which contains both STS and HMGS domains and synthesizes the sesquiterpenoid hirsutene. Our investigation revealed that the HMGS domain significantly enhances the cyclization activity of the STS domain, resulting in an 8.87-fold increase in sesquiterpene production with a final yield of 121.3 mg/kg, highlighting HMGS's critical role in catalytic efficiency. Additionally, domain-swapping experiments were performed by replacing the HMGS domain of G. incarnatum with the native HMGS domain from Flammulina velutipes sesquiterpene synthase Fla2. The results demonstrated that Fla2 fused with its cognate HMGS domain exhibited a significant yield enhancement from 11.5 to 54.5 mg/kg, underscoring the importance of metabolic compatibility in enzyme performance. This study not only reveals the unique advantages of natural fusion enzymes in sesquiterpene biosynthesis but also provides an important theoretical foundation for enhancing sesquiterpene production through the optimization of enzyme fusion strategies and metabolic pathway design. These findings offer a rational strategy for engineering high-efficiency terpenoid biosynthesis.

Considerable and increasing attention is being directed toward the application of halogenases for the biocatalytic functionalization of C-H bonds. Biocatalytic approaches offer distinct advantages over conventional chemical methods, including excellent catalyst-controlled selectivity and the avoidance of protecting groups and hazardous reagents. Ongoing discovery of natural halogenation pathways has led to the identification and characterization of a growing number of halogenases. Flavin-dependent halogenases (FDHs) have emerged as a major research focus, driven by their utility in the biohalogenation of aromatic scaffolds. These enzymes exhibit high regioselectivity and operate under environmentally benign conditions, making them attractive catalysts for the sustainable synthesis of halogenated compounds. Nevertheless, the activity of FDHs depends on reduced flavin adenine dinucleotide (FADH₂), and the efficient regeneration of this cofactor remains a critical bottleneck hindering industrial implementation. This review comprehensively summarizes recent advances in cofactor regeneration strategies for FDH. Herein, we discuss the mechanisms, advantages, and applicability of various regeneration systems, including traditional multienzyme cascades and emerging single-component self-sufficient architectures, finally concluding with an outlook on future research directions.

Precise packaging of diverse cargo within self-assembling protein cages of defined size and shape is essential for many biotechnological applications, yet cellular expression offers limited control over loading. Here, we developed an in vitro cargo-directed reconstitution system of a split, artificial nucleocapsid (spNC-4). Two spNC-4 capsid protein subunits were prepared independently and assembled with cargos cooperatively. As an authentic cargo, a nucleocapsid mRNA is packaged into a 30 nm-spheric nucleocapsid in vitro, closely matching to spNC-4 expressed in cells. In this system, a diverse range of cargos are encapsulated, including noncognate RNA, RNA-positively supercharged fluorescent protein complex, and linear double-stranded DNA. Moreover, by packaging 30 nm-spherical or rod-shaped DNA origamis as templates, the nucleocapsid morphology was altered to an enlarged 60 nm-spherical structure or rod-shaped structure. The developed system accepts versatile composition and programmable control over the artificial nucleocapsid architecture, creating a general platform for enzyme nanoreactors, targeted delivery, and vaccine development.

Dynamic metabolic regulation facilitates real-time sensing of pathway states and precise redirection of metabolic flux, thereby enhancing the biosynthesis of diverse value-added compounds. Here, we designed an autoinducible molecular switch in Bacillus amyloliquefaciens by integrating a Pantoea alhagi-derived PaSspB-ssrA* proteolytic system with the DegU-RapG-PhrG quorum-sensing (QS) module. The OFF module employs the novel PaSspB-ssrA* system for targeted degradation of ssrA*-tagged proteins with 86% efficiency, and the ON module utilizes a DegU-activated P_ispA promoter for cell density-dependent gene expression. To improve hyaluronic acid (HA) production and reduce byproduct accumulation, this system was applied by fusing ssrA* tags into the glycolysis-critical genes pfkA and fruA to attenuate glycolytic flux, together with QS-regulated coexpression of PaSspB and SthHL in engineered Δldh/ΔwbpA/ΔnagB strains, achieving 13.5 ± 0.19 g/L of low-molecular-weight HA (2.89 × 10⁴ Da). The autonomous, inducer-free platform enables cost-effective HA biosynthesis in B. amyloliquefaciens, with broad applicability for synthesizing high-value bioproducts.

The gut microbiome and its effects on human health have generated considerable scientific, veterinary, and medical interest in recent years. Several gut bacterial species have emerged as potential chassis organisms for the delivery of therapeutics in this milieu. Among these, E. coli Nissle 1917 (EcN), a nonpathogenic gut isolate bacterium, is quickly gaining popularity. However, a bottleneck in harnessing EcN's potential has been its poor transformation efficiency relative to other bacterial strains. In this study, we present the use of adaptive laboratory evolution to increase EcN's transformation efficiency by subjecting the strain to repeated cycles of electroporation and recovery. This new strain has been comprehensively characterized in comparison to the wild-type EcN, including assessments of growth under gut-mimicking duress conditions, permeability, motility, hydrophobicity, and plasmid replication. Since EcN is known to compete with pathogenic strains in the gut for iron, the competition dynamics and iron consumption of the strain were also significant factors to consider. Furthermore, we conducted genome sequencing and gene ontology enrichment analysis to identify affected genes and pathways to probe the potential mechanisms of the improved phenotype. Overall, the strain shows improved transformation efficiency and robustness while preserving its key biological functionality.

Open-chain flavonoids constitute a structurally distinct subgroup of the flavonoid family, characterized by two aromatic rings (A and B) connected through an uncyclized three-carbon bridge lacking the canonical heterocyclic C-ring. As key intermediates within the broader flavonoid biosynthetic network, they play essential roles in plant physiology and also exhibit diverse pharmacological properties. Recent advances in metabolomics, structural enzymology, and synthetic biology have substantially deepened our understanding of their biosynthetic logic, catalytic mechanisms, and regulatory features. In this review, we summarize the structural characteristics and classification of open-chain flavonoids, highlight recent progress in elucidating their biosynthetic pathways and tailoring enzymes, discuss emerging metabolic engineering and microbial production strategies, and provide perspectives on future research and biotechnological applications.