Cloning methods are fundamental to synthetic biology research. The capability to generate custom DNA constructs exhibiting predictable protein expression levels is crucial to the engineering of biology. Golden Gate cloning, a modular cloning (MoClo) technique, enables rapid and reliable one-pot assembly of genetic parts. In this study, we expand on the existing MoClo toolkits by constructing and characterizing compatible low- (p15A) and medium-copy (pBR322) destination vectors. Together with existing high-copy vectors, these backbones enable a protein expression range covering a 500-fold difference in normalized fluorescence output. We further characterize the expression- and burden profiles of each vector and demonstrate their use for the optimization of growth-coupled enzyme expression. The optimal expression of (encoding alcohol dehydrogenase) for ethanol-dependent growth of is determined using randomized Golden Gate Assembly, creating a diverse library of constructs with varying expression strengths and plasmid copy numbers. Through selective growth experiments, we show that relatively low expression levels of facilitated optimal growth using ethanol as the sole carbon source, demonstrating the importance of adding low-copy vectors to the MoClo vector repertoire. This study emphasizes the importance of varying vector copy numbers in selection experiments to balance expression levels and burden, ensuring accurate identification of optimal conditions for growth. The vectors developed in this work are publicly available via Addgene (catalog #217582-217609).

is a common microorganism in the human gut that has been linked to health benefits. Furthermore, it is an emerging synthetic biology chassis with the potential to be modified into diagnostic or therapeutic engineered probiotics. However, the absence of biological components limits its further applications. In this study, we developed an antiterminator microbial whole-cell biosensor (MWCB) based on . The antiterminator-based element allows the chassis to detect colitis in mice by responding to nitrate and nitrite in an inflammatory environment. In particular, the nitrate/nitrite-inducible promoter was obtained by combining the constitutive promoter with the inducible terminator. Subsequently, the promoter and RBS were replaced to optimize a sensitive and specific response to nitrate/nitrite. A preliminary assessment was conducted to ascertain the functionality of the biosensor. Its sensing ability was evaluated in a chemically induced mouse model of ulcerative colitis (UC). The results demonstrated that the MWCB exhibited a robust response to colitis, with a notable positive correlation between the intensity of the response and the level of inflammation. This novel sensing element may provide a new avenue for the development of components for unconventional chassis, like . It will also facilitate the development of engineered probiotics based on , thereby providing patients with a wider range of medical treatment options.

Naturally evolved and synthetically designed forms of compartmentalization benefit encapsulated function by increasing local concentrations of substrates and protecting cargo from destabilizing environments and inhibitors. Crucial to understanding the fundamental principles of compartmentalization are experimental systems enabling the measurement of the permeability rates of small molecules. Here, we report the experimental measurement of the small-molecule permeability of a 40 nm icosahedral bacterial microcompartment shell. This was accomplished by heterologous loading of light-producing luciferase enzymes and kinetic measurement of luminescence using stopped-flow spectrophotometry. Compared to free enzyme, the luminescence signal kinetics was slower when the luciferase was encapsulated in bacterial microcompartment shells. The results indicate that substrates and products can still exchange across the shell, and modeling of the experimental data suggest that a 50× permeability rate increase occurs when shell vertices were vacant. Overall, our results suggest design considerations for the construction of heterologous bacterial microcompartment shell systems and compartmentalized function at the nanoscale.

Cannabichromene (CBC), a valuable but extremely low-abundance component of cannabinoids in L., is known for its ability to promote neurogenesis. The scarcity of CBC in natural is primarily attributed to the inefficiency of the 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4 phosphate (DOXP/MEP) and fatty acid metabolism pathways, along with the limited competitive advantage of cannabichromenic acid synthetase (CBCAS) compared to other cannabinoid synthases. In this study, we constructed capable of biosynthesizing cannabichromenic acid (CBCA) from glucose and olivetolic acid. First, we enhanced the supply of the precursor isopentenyl diphosphate/dimethylallyl diphosphate by introducing a two-step isopentenol utilization pathway (IUP). Additionally, we increased the CBCA titer by co-overexpressing endoplasmic reticulum auxiliary protein genes. Moreover, we improved the selectivity and catalytic activity of CBCAS through rational design. By localizing the IUP to peroxisomes, geranylgeranyl pyrophosphate and CBCA titers were further increased by 1.6-fold and 28%, respectively. Notably, the yeast strain synthesized CBCA at a rate 25.8% higher than that of . Our findings suggest that microbial synthesis offers a promising alternative to traditional for sustainable CBCA production.

Bacterial outer membrane vesicles (OMVs) have emerged as promising vehicles for anticancer drug delivery due to their inherent tumor tropism, immune-stimulatory properties, and potential for functionalization with therapeutic proteins. Despite their advantages, the high lipopolysaccharide (LPS) endotoxin content in the OMVs raises significant safety and regulatory challenges. In this work, we produce LPS-attenuated and LPS-free OMVs and systematically assess the effects of LPS modification on OMVs' physicochemical characteristics, membrane protein content, immune-stimulatory capacity, tolerability, and anticancer efficacy. Our findings reveal that LPS removal increased the maximal tolerated dose of the OMVs by over 25-fold. When adjusted for comparable safety profiles, LPS-free OMVs exhibit superior anticancer effects compared with wild-type OMVs. Mechanistic investigations indicate that the LPS removal obviates immune cell death caused by LPS and reduces the negatory effects of wild type of OMVs on tumor immune cell infiltrates. We further show the functionality of the LPS-free OMV through the incorporation of an IL-2 variant protein (Neo-2/15). This functionalization augments OMV's ability of the OMV to inhibit tumor growth and promote lymphocyte infiltration into the tumor microenvironment. This study presents a safe and functionalizable OMV with improved translational prospect.

Fungi, especially filamentous fungi, are a relatively understudied, biotechnologically useful resource with incredible potential for commercial applications. These multicellular eukaryotic organisms have long been exploited for their natural production of useful commodity chemicals and proteins such as enzymes used in starch processing, detergents, food and feed production, pulping and paper making and biofuels production. The ability of filamentous fungi to use a wide range of feedstocks is another key advantage. As chassis organisms, filamentous fungi can express cellular machinery, and metabolic and signal transduction pathways from both prokaryotic and eukaryotic origins. Their genomes abound with novel genetic elements and metabolic processes that can be harnessed for biotechnology applications. Synthetic biology tools are becoming inexpensive, modular, and expansive while systems biology is beginning to provide the level of understanding required to design increasingly complex synthetic systems. This review covers the challenges of working in filamentous fungi and offers a perspective on the approaches needed to exploit fungi as microbial cell factories.

Ectoine is an important natural secondary metabolite widely used in biomedical fields, novel cosmetics development, and the food industry. Due to the increasing market demand for ectoine, more cost-effective production methods are being explored. With the rapid development of synthetic biology and metabolic engineering technologies, the production of ectoine using traditional halophilic bacteria is gradually being replaced by higher-yielding and environmentally friendly nonhalophilic engineered strains. By introducing the ectoine synthesis pathway into model strains and optimizing the fermentation process through various metabolic regulations, high-level production of ectoine can be achieved. This review focuses on strategies for the microbial production of ectoine, including screening of wild strains, mutation breeding, and metabolic engineering of model strains, to elucidate the current research status and provide insights for the industrial production of ectoine.

The COVID-19 pandemic has highlighted the critical need for pathogen detection methods that offer both low detection limits and rapid results. Despite advancements in simplifying and enhancing nucleic acid amplification techniques, immunochemical methods remain the preferred methods for mass testing. These methods eliminate the need for specialized laboratories and highly skilled personnel, making home testing feasible. Here, we developed nanobody-based lateral flow assays (LFAs) for the rapid detection of SARS-CoV-2 and MERS-CoV in single and dual formats as point-of-care diagnostic tools. The developed LFAs are highly sensitive and successfully detected analytes at clinically relevant diagnostic cutoff values. Additionally, our results confirmed that the LFAs have a long shelf life and can be produced cost-effectively and with ease.

Filamentous fungi are important cell factories for producing chemicals, organic acids, and enzymes. Although several genome editing tools are available for filamentous fungi, few effectively enable continuous evolution for rational engineering of complex phenotype. Here, we present CRISPR-Cas9 cytidine-base-editor (CBE) assisted evolution by continuously delivering a combinatorial sgRNA library to filamentous fungi. The method was validated by targeting core genes of 46 natural product biosynthetic gene clusters in NRRL 8112 to eliminate fungal toxins via six rounds of evolution. NGS analysis revealed the average C-to-T conversion rates in the first, third, and sixth rounds were 2.02%, 5.25%, and 9.34%, respectively. Metabolic profiles of the evolved mutants exhibited significant changes, allowing for the isolation of clean-background strains with enhanced production of an antifungal compound Echinocandin B. This study demonstrates that CBE-mediated evolution greatly facilitates the iterative refinement of complex morphogenetic traits in filamentous fungi.

We herein developed an ultrasensitive and rapid strategy to identify genomic nucleic acids by integrating a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 13a (Cas13a) into our recently developed isothermal technique, nicking and extension chain reaction system-based amplification (NESBA) reaction. In this technique, named CESBA, the NESBA reaction isothermally produces a large amount of RNA amplicons from the initial target genomic RNA (gRNA). The RNA amplicons bind to the crispr RNA (crRNA) and activate the collateral cleavage activity of Cas13a, which would then cleave the reporter probe nearby, consequently producing the final signals. Based on this design principle, we successfully detected SARS-CoV-2 gRNA as a model target very sensitively down to even a single copy (0.05 copies/μL) in both fluorescence- and lateral flow assay (LFA)-based modes with excellent specificity against other human coronaviruses (H-CoVs). We further validated the clinical applicability of CESBA by testing the 20 clinical samples with 100% clinical sensitivity and specificity. This work represents a potent and innovative strategy for the identification of genomic nucleic acids in molecular diagnostics, delivering exceptional levels of sensitivity.

Naturally occurring DNA inversion systems play an important role in the generation of genetic variation and adaptation in prokaryotes. Shufflon invertase (SI) from plasmid R64, recognizing asymmetric sites, has been adopted as a tool for synthetic biology. However, the availability of a single enzyme with moderate rates of recombination has hampered the more widespread use of SIs. We identified 14 previously untested SI genes and their sites in public databases. We established an assay based on single-molecule sequencing that allows the quantification of the inversion rates of these enzymes and determined cross-recognition to identify orthogonal SI/ pairs. We describe SI enzymes with substantially improved shuffling rates when expressed in an inducible manner in . Our findings will facilitate the use of SIs in engineering biology where synthetic shufflons enable the generation of millions of sequence variants for applications such as barcoding or experimental selection.

Cell-free synthetic biology incorporates purified components and/or crude cell extracts to carry out metabolic and genetic programs. While protein synthesis has historically been the primary focus, more metabolism researchers are now turning toward cell-free systems either to prototype pathways for cellular implementation or to design new-to-nature reaction networks that incorporate environmentally relevant substrates or new energy sources. The ability to design, build, and test enzyme combinations has accelerated efforts to understand metabolic bottlenecks and engineer high-yielding pathways. However, only a small fraction of metabolic possibilities has been explored in cell-free systems, and extracts from model organisms remain the most common starting points. Expanding the scope of cell-free metabolism to include extracts from new organisms, alternative metabolic pathways, and non-natural chemistries will enhance our ability to understand and engineer bio-based chemical conversions.

Advancements in molecular diagnostics, such as polymerase chain reaction and next-generation sequencing, have revolutionized disease management and prognosis. Despite these advancements in molecular diagnostics, the field faces challenges due to high operational costs and the need for sophisticated equipment and highly trained personnel besides having several technical limitations. The emergent field of CRISPR/Cas sensing technology is showing promise as a new paradigm in clinical diagnostics, although widespread clinical adoption remains limited. This perspective paper discusses specific cases where CRISPR/Cas technology can surmount the challenges of existing diagnostic methods by stressing the significant role that CRISPR/Cas technology can play in revolutionizing clinical diagnostics. It underscores the urgency and importance of addressing the technological and regulatory hurdles that must be overcome to harness this technology effectively in clinical laboratories.

In mammals, Trimethylamine N-oxide (TMAO) is involved in various physiological processes, and is considered a biomarker for multiple diseases. As a natural molecule found in marine organisms, TMAO is also an important indicator of seafood freshness. In this study, a TMAO biosensor was developed in harnessing TorRST two-component system. By using a cascade amplification circuit based on HrpRS-P, the biosensor's dynamic range was increased from 4.1- to 10.3-fold. By optimizing the affinity between the regulatory protein TorR and DNA binding sites in promoters, the concentration for 50% of maximal effect (EC) value was reduced from 1008 to 141 μM. The biosensor was successfully used for aquatic sample detection. By introducing an exogenous TMAO degradation pathway into Nissle 1917, a probiotic chassis capable of TMAO detection, transportation, and degradation was constructed, providing an effective tool for rapid detection of TMAO and prevention of multiple diseases.

The development of an engineered strain for efficient cytidine production holds significant value for both research and industrial applications. In this study, the and genes were knocked out to reveal their roles involved in the regulation of efficient cytidine synthesis in . The results showed that after 36 h of shaking flask fermentation, the knockout strain NXBG-14 produced a cytidine concentration of 2.57 ± 0.04 g/L, and the and double knockout strain NXBG-15 produced a cytidine titer of 2.68 ± 0.03 g/L, which represented enhancements of 1.68 and 1.75 times over the start strain, respectively. Transcriptome analysis revealed that the differentially expressed genes (DEGs) in the NXBG-14 strain were mainly enriched in the glycolytic pathway and the tricarboxylic acid (TCA) cycle. Additionally, C metabolic flow distribution indicated a significant increase in 6-phosphogluconate in the pentose phosphate pathway (PPP) for NXBG-15. These findings suggest that modifications of the and genes redirect central carbon metabolism and promote cytidine accumulation.

The fusion expression of deoxyribonucleic acid (DNA) replication-related proteins with nucleotide deaminase enzymes promotes random mutations in bacterial genomes, thereby increasing genetic diversity among the population. Most previous studies have focused on cytosine deaminase, which produces only C → T mutations, significantly limiting the variety of mutation types. In this study, we developed a fusion expression system by combining DnaG (RNA primase) with adenine deaminase TadA-8e (DnaG-TadA) in , which is capable of rapidly introducing A → G mutations into the genome, resulting in a 664-fold increase in terms of mutation rate. Additionally, we tested a dual-functional TadA variant, TadAD, and then fused it with DnaG. This construct introduced both C → T and A → G mutations into the genome, with the mutation rate increased by 370-fold upon coexpression with a uracil glycosylase inhibitor (DnaG-TadAD-UGI). We applied DnaG-TadA and DnaG-TadAD-UGI systems to the adaptive laboratory evolution for Cd and kanamycin resistance, achieving an 8.0 mM Cd and 200 μg/mL kanamycin tolerance within just 17 days and 132 h, respectively. Compared to conventional evolution methods, the final tolerance levels were increased by 320 and 266%, respectively. Our work offers a novel strategy for random mutagenesis in and potentially other prokaryotic species.

Smart textiles that integrate multiple environmental sensing capabilities are an emerging frontier in wearable technology. In this study, we developed dual pH- and temperature-responsive textiles by combining engineered bacterial systems with bacterially derived proteins. For temperature sensing, we characterized the properties of a heat sensitive promoter, P, in () using enhanced green fluorescent protein as a reporter. Our findings demonstrate that the P promoter drives elevated gene expression at temperatures between 37 and 43 °C, maintaining sustained activity for several hours. Moreover, we found that short heat shocks can significantly boost expression levels of the P promoter. We successfully integrated expressing P-EGFP cells onto textiles and confirmed their ability to retain heat-responsive behavior after integration. To achieve pH responsiveness, we utilized curli fibers, genetically engineered to incorporate a pH-sensitive fluorescent protein, pHuji. pH-sensing curli fibers are bacterial proteins that have a proven track record of creating stable bioresponsive textile coatings. By embedding P-EGFP-expressing bacteria within curli fiber coatings, we created a dual-responsive textile capable of differentiating between acidic and alkaline environments while simultaneously responding to thermal stimuli. These multifunctional textiles exhibited dual environmental response and sensing capabilities. This work establishes a proof-of-concept for creating smart living textiles with modular functionalities, paving the way toward advanced bioresponsive materials.

The probiotic Nissle (EcN) is an exceptional strain that has attracted significant attention not only for its clinical efficacy in the treatment and prevention of gastrointestinal disorders but also as a burgeoning microbial chassis for living therapeutic applications. However, there is an immediate necessity to develop conditional expression systems that confine the activity of EcN specifically in the gastrointestinal tract, to avoid influencing the environment. Here, we constructed two genetically encoded interchangeable sensors responsive to body temperature at 37 °C, and small molecules such as protocatechuic acid (PCA), a metabolite found in green tea. By employing dCpf1 targeted deactivation of the gene, we thereby coupled the above sensing modules with the P-lacO system and achieved improved signal outputs and relatively high ON/OFF ratios. Subsequently, we validated the biological function of engineering EcN using the enhanced green fluorescent protein (eGFP) in an animal model of mice. Taken together, the construction of genetically encoded sensors to restrict the biological functions of EcN would be applicable for the real-world implementation of living therapeutics or drug delivery.

2'-Fucosyllactose (2'-FL) is the most abundant human milk oligosaccharides (HMOs). 2'-FL exhibits great benefits for infant health, such as preventing infantile diarrhea and promoting the growth of intestinal probiotics. The microbial cell factory technique has shown promise for the massive production of 2'-FL. Here, we aimed to construct a recombinant BL21(DE3) strain for the hyperproduction of 2'-FL. Initially, multicopy genomic integration and expression of the lactose permease gene reduced the formation of byproducts. Furthermore, a more efficient Shine-Dalgarno sequence was used to replace the wild-type sequence in the - and - gene clusters, which significantly increased the 2'-FL titer. Based on these results, we overexpressed the sugar efflux transporter SetA and knocked out the gene. This further improved 2'-FL synthesis when glycerol was used as the sole carbon source. Finally, a new α-1,2-fucosyltransferase was identified in sp., which exhibited a higher capacity for 2'-FL production. Fed-batch fermentation produced 141.27 g/L 2'-FL in 45 h with a productivity of 3.14 g/L × h. This productivity rate achieved the highest recorded 2'-FL levels, indicating the potential of engineered BL21 (DE3) strains for use in the industrial production of 2'-FL.

The field of healthcare diagnostics is navigating complex challenges driven by evolving patient demographics and the rapid advancement of new technologies worldwide. In response to these challenges, these biosensors offer distinctive advantages over traditional diagnostic methods, such as cost-effectiveness, enhanced specificity, and adaptability, making their integration with point-of-care (POC) platforms more feasible. In recent years, aptasensors have significantly evolved in diagnostic capabilities through the integration of emerging technologies such as microfluidics, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems, wearable devices, and machine learning (ML), driving progress in precision medicine and global healthcare solutions. Moreover, these advancements not only improve diagnostic accuracy but also hold the potential to revolutionize early detection, reduce healthcare costs, and improve patient outcomes, especially in resource-limited settings. This Account examines key advancements, focusing on how scientific breakthroughs, including artificial intelligence (AI), have improved sensitivity and precision. Additionally, the integration of aptasensors with these technologies has enabled real-time monitoring and data analysis, fostering advances in personalized healthcare. Furthermore, the potential commercialization of aptasensor technologies could increase their availability in clinical settings and support their use as widespread solutions for global health challenges. Hence, this review discusses technological improvements, practical uses, and prospects while also focusing on the challenges surrounding standardization, clinical validation, and interdisciplinary collaboration for widespread application. Finally, ongoing efforts to address these challenges are key to ensure that aptasensors can be effectively implemented in diverse healthcare systems.

The continuous advancement of single-cell multimodal omics (scMulti-omics) technologies offers unprecedented opportunities to measure various modalities, including RNA expression, protein abundance, gene perturbation, DNA methylation, and chromatin accessibility at single-cell resolution. These advances hold significant potential for breakthroughs by integrating diverse omics modalities. However, the data generated from different omics layers often face challenges due to high dimensionality, heterogeneity, and sparsity, which can adversely impact the accuracy and efficiency of data integration analyses. To address these challenges, we propose a high-precision analysis method called scMGAT (single-cell multiomics data analysis based on multihead graph attention networks). This method effectively coordinates reliable information across multiomics data sets using a multihead attention mechanism, allowing for better management of the heterogeneous characteristics inherent in scMulti-omics data. We evaluated scMGAT's performance on eight sets of real scMulti-omics data, including samples from both human and mouse. The experimental results demonstrate that scMGAT significantly enhances the quality of multiomics data and improves the accuracy of cell-type annotation compared to state-of-the-art methods. scMGAT is now freely accessible at https://github.com/Xingyu-Liao/scMGAT.