ACS Synthetic Biology最新文献

Genetic manipulation of core gut probiotics remains challenging due to endogenous cellular barriers and a scarcity of efficient molecular tools, limiting progress in live biotherapeutic development. Here, we characterized the native type II-C CRISPR-Cas system in Bifidobacterium longum subsp. longum GNB (B. longum GNB). Through integrated bioinformatic analysis and high-throughput protospacer adjacent motif (PAM) screening, we identified a novel 5'-NNRMAT-3' (where R = A/G, M = A/C) motif recognized by its compact Cas9 nuclease (BLCas9). The stringent PAM dependency of BLCas9 was unequivocally confirmed by in vitro cleavage assays. Leveraging this endogenous mechanism, we developed a dual-plasmid editing platform for robust and multiplex genome engineering in the probiotic strain Escherichia coli Nissle 1917 (EcN). Application of this system notably enhanced extracellular γ-aminobutyric acid (GABA) production in EcN through targeted metabolic engineering. Our work provides the first molecular dissection of a type II-C system in Bifidobacterium longum and establishes a generalizable framework for the discovery and application of compact programmable nucleases, suggesting a viable strategy for modulating host physiology via the gut-brain axis.

Inducible translocation to subcellular compartments is a common strategy for protein switches that control a variety of cell behaviors. However, existing switches achieve translocation through induced dimerization, requiring constitutive anchoring of one component into the target compartment and optimization of relative expression levels between the two components. We present a simpler, single-component strategy called Avidity-assisted targeting (Aviatar). Aviatar achieves translocation with only a single protein by converting low-affinity monomers into high-avidity assemblies through inducible clustering. We demonstrated the Aviatar concept and its generality using optogenetic clustering to drive translocation to the plasma membrane, endosomes, golgi, endoplasmic reticulum, and microtubules using binding domains for lipids or endogenous proteins that were specific to those compartments. Aviatar recruitment regulated actin polymerization at the cell periphery and revealed compartment-specific signaling of receptor tyrosine kinase fusions associated with cancer. Finally, GFP-targeting Aviatar probes allowed inducible localization to any GFP-tagged target, including endogenously tagged stress granule proteins. Aviatar is a straightforward platform that can be rapidly adapted to a broad array of targets without the need for their prior modification or disruption.

Saccharomyces cerevisiae is widely adopted as a chassis in synthetic biology. However, heterologous constructs often disrupt proteostasis, metabolism, redox balance, and secretory processes. These disruptions activate a complex network of stress pathways. These include the heat shock response, unfolded protein response, oxidative stress defenses, cell wall integrity signaling, the high-osmolarity glycerol pathway, and Snf1/AMPK-mediated energy regulation. Collectively, these pathways form a stressome that maintains cellular homeostasis but constrains productive capacity. A comprehensive understanding of how synthetic designs interact with these pathways is essential for developing robust yeast systems. Strategies such as promoter tuning, chaperone augmentation, redox and cofactor balancing, lipid and membrane optimization, dynamic regulation, and pathway compartmentalization can reduce cellular burden. Emerging methods also improve stress mitigation. These include CRISPR-based circuit rewiring, adaptive laboratory evolution, synthetic organelle construction, and data-driven strain engineering. This review summarizes construct-induced stress in engineered yeast and presents stress-aware design principles to advance more resilient, higher-yielding S. cerevisiae strains for biotechnology.

Cupriavidus necator is a well-established microbial platform for polyhydroxyalkanoate (PHA) production and numerous metabolic engineering strategies have been applied to improve its performance. However, little is known of the relationship between cell size and the production of the poly-3-hydroxybutyrate (PHB), with studies mainly focusing on cell division markers such as ftsZ, mreB, and minCD. These genes are influenced by multiple regulatory factors, though the specific regulators have not been characterized. This study investigated the role of a novel regulatory gene mraZ, which is located upstream of ftsZ and acts as a transcriptional repressor of cell growth and division, in PHA production by C. necator. We constructed an mraZ-deleted strain to assess the physiological and metabolic changes. qRT-PCR revealed the upregulated expression of ftsZ and PHA biosynthesis genes, along with ∼20% higher cell growth and PHA accumulation in the mutant across various culture conditions. Simultaneously, the cell length was reduced by >5 times that of the wild type, as confirmed by scanning and transmission electron microscopy. Although the smaller cell size increased the surface-to-volume ratio and enhanced the sugar utilization efficiency, it did not alter the molecular weight or mechanical properties of PHB. These findings highlight the value of targeting upstream regulators such as mraZ and provide new insights into the significance of reduced cell size in relation to PHB production. Considering that regulators constitute ∼12% of all genes in C. necator, our results highlight the untapped potential of regulator-based molecular engineering for optimizing PHA production.

The nonconventional yeast, Yarrowia lipolytica, is a promising protein expression host, having achieved recombinant protein expression yield on par with the commonly used host, Komagatella phaffii (Pichia pastoris). However, strong, fully constitutive genetic elements and expression cassettes for protein expression in Y. lipolytica remain limited. In this study, we leveraged genome-wide transcriptomics to uncover five strong promoters and four terminators. Among these, the promoter of ribosomal protein L41 demonstrated superior activity to the strongest previously reported promoters. We further demonstrated the functionality of pL41 across different media conditions and by using it to express diverse heterologous proteins. Similarly, we showed that the terminator of glutathione-S-transferase (tGST) supported higher protein expression and low transcriptional readthrough compared to commonly used terminators. To support protein secretion efforts, we utilized a secretomics-guided signal peptide screen to unveil three signal peptides, demonstrating broad applicability to different proteins. Integrating these genetic elements into a new expression cassette (YALI-pSTOmics1) resulted in a 3-fold increase in secretory expression of bovine fibroblast growth factor 2 compared to a combination of the best available state-of-the-art genetic tools for gene expression in Y. lipolytica. This expression cassette represents an open-source alternative to expensive commercial ones. Furthermore, the novel promoters and terminators provide options for metabolic engineering, where reuse of existing genetic parts is often a limitation.

Hemicelluloses are a group of plant cell wall polysaccharides characterized by their high structural complexity. These glycans are part of an intricate composite polymer network that contribute to the mechanical strength and flexibility of plant cell walls. Hemicellulose structural and functional diversity is further enhanced by the presence of chemical modifications, such as O-acetylation, altering the polysaccharide's physicochemical properties and the overall functionality. Plant-derived hemicellulose glycans hold great promise for a range of biotechnological applications in a bioeconomy including biomaterials and pharmaceuticals. Synthetic biology approaches have the potential to produce hemicellulose polymers in microbial factories replicating the biosynthetic pathways observed in plants. In this study, we successfully reconstructed in the yeast Yarrowia lipolytica the biosynthesis of two hemicellulose backbone structures i.e., β-glucomannan (GM) and β-glucan, by the expression of glycosyltransferases of diverse plant origins. Oligosaccharide mass profiling combined with compositional and glycosidic linkage analysis confirmed the production of hemicellulose structures analogous to those found in the original plant systems. Furthermore, the additional expression of plant hemicellulose-specific O-acetyltransferases resulted in the biosynthesis of O-acetylated GM and O-acetylated glucan polymers, expanding the repertoire of hemicellulose structures produced in this yeast. These findings demonstrate the feasibility of generating not only compositionally diverse plant-like hemicellulose backbone polymers in microbial systems, but also more structurally complex O-acetylated variants beyond what is found in nature. The use of Y. lipolytica as a biofactory for designer glycans expands the potential of microbial glycoengineering and provides a platform for sustainable production of functionalized polysaccharides with tailored physicochemical properties optimized for specific biotechnological applications.

Promoters are essential in transcriptional regulation, with the -10 and -35 boxes playing a critical role in determining their strength. Modulating these regions can effectively fine-tune promoter strength. However, the lack of a clear quantitative relationship between sequence composition and transcriptional output impedes the rational design of promoters. To address this, we developed a synthetic promoter library by varying RNA polymerase binding energies at the -10 and -35 boxes. The library was partitioned into four sublibraries with expression strengths spanning an 80-fold range. Using fluorescence-activated cell sorting followed by sequencing, we identified 20,799 distinct promoters. Analysis of this library uncovered distinct sequence-activity patterns, including a small subset of -35 box sequences that consistently conferred high transcriptional output across diverse -10 partners. Based on this, we developed an artificial intelligence platform that integrates a convolutional neural network for strength prediction (Pearson's r = 0.84) with a balanced generative adversarial network incorporating a gradient penalty for de novo promoter design. By coupling these models, we achieved a precise design of promoters with user-defined strengths (r = 0.85), establishing a bidirectional framework that links -10/-35 boxes to transcriptional activity through deep learning. This study expands the sequence diversity of functional -10 and -35 boxes in E. coli, provides a predictive platform for rational promoter engineering, and deciphers combinatorial motif interactions governing transcriptional regulation.

Genetic circuits are a cornerstone of synthetic biology, enabling programmable control of cellular behavior for applications in health, sustainability, and biotechnology. While Genetic Design Automation (GDA) tools have optimized and streamlined the design of such circuits, rapid and efficient assembly of DNA remains a bottleneck in the Design-Build-Test-Learn (DBTL) cycle. Here, we present the Coli Toolkit (CTK), a modular Golden Gate-based cloning system, adapted from the Yeast Toolkit (YTK) for use in Escherichia coli. The CTK expands on the original YTK architecture by introducing a more flexible control of transcription and translation through subdividing the former promoter part into subparts: promoter, insulating ribozyme, and ribosome binding site (RBS). We provide a range of basic parts that enable the assembly of a wide range of constructs as well as characterization data for all constitutive and inducible promoters provided. Additionally, we provide characterization data, as well as calibrated models, for all 20 NOT gates from the Cello library, and we provide the NOT gates as preassembled basic parts, which enables rapid cloning of larger genetic circuits. With this toolkit, we leverage the strengths of the YTK architecture to enable rapid and high-efficiency assembly of genetic circuits in E. coli, filling a key gap in the infrastructure of bacterial synthetic biology.

Indigoidine, a microbial-derived pyridone pigment, has emerged as a promising sustainable alternative to synthetic blue dyes in textile industry. Its biosynthesis is mediated by nonribosomal peptide synthetases (NRPSs), offering a favorable alternative to conventional chemical synthesis for pigment production. Recent advances in synthetic biology have facilitated the scalable engineering of microbial chassis strains, enabling industrial level pigment production. However, challenges persist in pathway optimization, titer improvement, and application diversity. In this review, we systematically examine microbial host systems, metabolic and enzyme engineering approaches, and synthetic biology techniques to enhance indigoidine production. This review offers a roadmap for advancing next-generation microbial cell factories for pigment manufacturing.

Nitrogenase catalyzes the reduction of atmospheric nitrogen gas to ammonia, forming the foundation of biological nitrogen fixation in diazotrophic microbes. While functional nitrogenase can be assembled in non-native hosts, its activity is severely limited. This is partially due to the O₂ sensitivity, which irreversibly inactivates the enzyme. Here, we aimed to address this challenge by compartmentalizing nitrogenase into carboxysomes-bacterial microcompartments that restrict O₂ diffusion. We demonstrate that nitrogenase subunit NifH can be selectively localized to the carboxysomes of Nostoc punctiforme. Electron microscopy indicated normal assembly of these NifH-loaded carboxysomes, while growth experiments suggested minimal impact to the carboxysome function. Mass spectrometry confirmed accumulation of the fusion proteins in purified carboxysomes. These data set the stage for further development of nitroxysomes, exploring integration of fully active nitrogenase complexes into these carboxysomes. If successful, this approach will pave the way to engineer nitrogen fixation directly into crops, promoting sustainable agriculture to enhance global food security.