Search Results (54)

Cell-free biosensors harness the selectivity of cellular machinery without living cells’ constraints, offering advantages in environmental monitoring, medical diagnostics, and biotechnological applications. This review examines recent advances in cell-free biosensor development, highlighting their ability to detect diverse analytes including heavy metals, organic pollutants, pathogens, and clinical biomarkers with high sensitivity and specificity. We analyze technological innovations in cell-free protein synthesis optimization, preservation strategies, and field deployment methods that have enhanced sensitivity, and practical applicability. The integration of synthetic biology approaches has enabled complex signal processing, multiplexed detection, and novel sensor designs including riboswitches, split reporter systems, and metabolic sensing modules. Emerging materials such as supported lipid bilayers, hydrogels, and artificial cells are expanding biosensor capabilities through microcompartmentalization and electronic integration. Despite significant progress, challenges remain in standardization, sample interference mitigation, and cost reduction. Future opportunities include smartphone integration, enhanced preservation methods, and hybrid sensing platforms. Cell-free biosensors hold particular promise for point-of-care diagnostics in resource-limited settings, environmental monitoring applications, and food safety testing, representing essential tools for addressing global challenges in healthcare, environmental protection, and biosecurity. Full article

Microalgae, with their unparalleled capabilities for sunlight-driven growth, CO₂ fixation, and synthesis of diverse high-value compounds, represent sustainable cell factories for a circular bioeconomy. However, industrial deployment has been hindered by biological constraints and the inadequacy of conventional genetic tools. The advent of CRISPR-Cas systems initially provided precise gene editing via targeted DNA cleavage. This review argues that the true transformative potential lies in moving decisively beyond cutting to harness CRISPR as a versatile synthetic biology “Swiss Army Knife”. We synthesize the rapid evolution of CRISPR-derived tools—including transcriptional modulators (CRISPRa/i), epigenome editors, base/prime editors, multiplexed systems, and biosensor-integrated logic gates—and their revolutionary applications in microalgal engineering. These tools enable tunable gene expression, stable epigenetic reprogramming, DSB-free nucleotide-level precision editing, coordinated rewiring of complex metabolic networks, and dynamic, autonomous control in response to environmental cues. We critically evaluate their deployment to enhance photosynthesis, boost lipid/biofuel production, engineer high-value compound pathways (carotenoids, PUFAs, proteins), improve stress resilience, and optimize carbon utilization. Persistent challenges—species-specific tool optimization, delivery efficiency, genetic stability, scalability, and biosafety—are analyzed, alongside emerging solutions and future directions integrating AI, automation, and multi-omics. The strategic integration of this CRISPR toolkit unlocks the potential to engineer robust, high-productivity microalgal cell factories, finally realizing their promise as sustainable platforms for next-generation biomanufacturing. Full article

Bacteriophages (phages), the most abundant biological entities on Earth, have long served as both model systems and therapeutic tools. Recent advances in synthetic biology and genetic engineering have revolutionized the capacity to tailor phages with enhanced functionality beyond their natural capabilities. This review outlines the current landscape of synthetic and functional engineering of phages, encompassing both in-vivo and in-vitro strategies. We describe in-vivo approaches such as phage recombineering systems, CRISPR-Cas-assisted editing, and bacterial retron-based methods, as well as synthetic assembly platforms including yeast-based artificial chromosomes, Gibson, Golden Gate, and iPac assemblies. In addition, we explore in-vitro rebooting using TXTL (transcription–translation) systems, which offer a flexible alternative to cell-based rebooting but are less effective for large genomes or structurally complex phages. Special focus is given to the design of customized phages for targeted applications, including host range expansion via receptor-binding protein modifications, delivery of antimicrobial proteins or CRISPR payloads, and the construction of biocontained, non-replicative capsid systems for safe clinical use. Through illustrative examples, we highlight how these technologies enable the transformation of phages into programmable bactericidal agents, precision diagnostic tools, and drug delivery vehicles. Together, these advances establish a powerful foundation for next-generation antimicrobial platforms and synthetic microbiology. Full article

Accurate and sensitive detection of protein biomarkers is critical for advancing in vitro diagnostics (IVD), yet conventional enzyme-linked immunosorbent assays (ELISA) often fall short in terms of sensitivity compared to nucleic acid-based tests. Bridging this sensitivity gap is essential for improving diagnostic accuracy, particularly in diseases where protein levels better reflect disease progression than nucleic acid biomarkers. In this review, we present strategies developed to enhance the sensitivity of ELISA, structured according to the sequential steps of the assay workflow. Beginning with surface modifications, we then discuss the methodologies to improve mixing and washing efficiency, followed by a summary of recent advances in signal generation and amplification techniques. In particular, we highlight the emerging role of cell-free synthetic biology in augmenting ELISA sensitivity. Recent developments such as expression immunoassays, CRISPR-linked immunoassays (CLISA), and T7 RNA polymerase–linked immunosensing assays (TLISA) demonstrate how programmable nucleic acid and protein synthesis systems can be integrated into ELISA workflows to surpass the present sensitivity, affordability, and accessibility. By combining synthetic biology-driven amplification and signal generation mechanisms with traditional immunoassay formats, ELISA is poised to evolve into a highly modular and adaptable diagnostic platform, representing a significant step toward the next generation of highly sensitive and programmable immunoassays. Full article

The ester of caffeic acid with α-hydroxydihydrocaffeic acid, named rosmarinic acid (α-o-caffeoyl-3,4-dihydroxyphenyllactic acid; RA) can occur as oligomeric molecules, or in free, esterified, and glycosidic forms. Although it is commonly found among the members of the plants from the Lamiaceae (mints) and Boraginaceae (borages) families, only certain plant species produce a comparatively high concentration of RA. This valuable bioactive compound exhibits anti-cancer, anti-angiogenic, antioxidant, anti-inflammatory, antiviral, and antimicrobial properties, among others. As it is difficult to obtain high quantities of RA from natural sources, and since chemical manufacturing is costly and challenging, various biotechnological methods have recently been investigated to boost RA production. Plant cell tissue culture has been used to promote RA production in various plant species, particularly medicinal ones, with elicitation being the most commonly used technique. This review explores the main steps involved in RA biosynthesis in plants, including the molecular mechanisms and physiological alterations underlying its function, along with the primary mechanisms of RA accumulation in response to elicitation. Recent progress in synthetic biology-based RA synthesis, as well as metabolic engineering techniques to enhance the industrial production of this valuable bioactive constituent, are also discussed. Full article

Biosimilars represent medicinal products that exhibit a high degree of similarity to an already sanctioned reference biologic agent, with negligible clinically significant disparities concerning safety, purity, or potency. These therapeutic modalities are formulated as economically viable substitutes for established biologics, thereby facilitating increased accessibility to sophisticated treatments for a range of medical conditions, including infectious diseases caused by bacterial, fungal, and viral pathogens. The current landscape of biosimilars includes therapeutic proteins, such as monoclonal antibodies, antimicrobial peptides, antiviral peptides, and antifungal peptides. Here, we discuss the obstacles inherent in the development of biosimilars, including the rapid mutation rates of pathogens. Furthermore, we discuss innovative technologies within the domain, including antibody engineering, synthetic biology, and cell-free protein synthesis, which exhibit potential for improving the potency and production efficiency of biosimilars. We end with a prospective outlook to highlight the importance and capacity of biosimilars to tackle emerging infectious diseases, highlighting the imperative need for ongoing research and financial commitment. Full article

HUG is the HELP-UnaG recombinant fusion protein featuring the typical functions of both HELP and UnaG. In HUG, the HELP domain is a thermoresponsive human elastin-like polypeptide. It forms a shield enwrapping the UnaG domain that emits bilirubin-dependent fluorescence. Here, we recapitulate the technological development of this bifunctional synthetic protein from the theoretical background of its distinct protein moieties to the detailed characterization of its macromolecular and functional properties. These pieces of knowledge are the foundations for HUG production and application in the fluorometric analysis of bilirubin and its congeners, biliverdin and bilirubin glucuronide. These bile pigments are metabolites that arise from the catabolism of heme, the prosthetic group of cytochromes, hemoglobin and several other intracellular enzymes engaged in electron transfer, oxygen transport and protection against oxygen free radicals. The HUG assay is a powerful, user-friendly and affordable analytical tool that alone supports research at each level of complexity or taxonomy of living entities, from enzymology, cell biology and pathophysiology to veterinary and clinical sciences. Full article

Nicotinamide mononucleotide (NMN) has emerged as a promising non-natural cofactor with significant potential to transform biocatalysis, synthetic biology, and therapeutic applications. By modulating NAD⁺ metabolism, NMN offers unique advantages in enzymatic reactions, metabolic engineering, and regenerative medicine. This review provides a comprehensive analysis of NMN’s biochemical properties, mechanisms of action, and diverse applications. Emphasis is placed on its role in addressing challenges in multi-enzyme cascades, biofuel production, and the synthesis of high-value chemicals. The paper also highlights critical research gaps, including the need for scalable NMN synthesis methods, improved integration into enzymatic systems, and comprehensive toxicity studies for therapeutic use. Emerging technologies such as AI-driven enzyme design and CRISPR-based genome engineering are discussed as transformative tools for optimizing NMN-dependent pathways. Furthermore, the synergistic potential of NMN with synthetic biology innovations, such as cell-free systems and dynamic regulatory networks, is explored, paving the way for precise and modular biotechnological solutions. Looking forward, NMN’s versatility as a cofactor positions it as a pivotal tool in advancing sustainable bioprocessing and precision medicine. Addressing current limitations through interdisciplinary approaches will enable NMN to redefine the boundaries of metabolic engineering and therapeutic innovation. This review serves as a roadmap for leveraging NMN’s potential across diverse scientific and industrial domains. Full article

Monolignols represent pivotal alcohol-based constituents in lignin synthesis, playing indispensable roles in plant growth and development with profound implications for industries reliant on wood and paper. Monolignols and their derivates have multiple applications in several industries. Monolignols exhibit antioxidant activity due to their ability to donate hydrogen atoms or electrons to neutralize free radicals, thus preventing oxidative stress and damage to cells. Characterized by their alcohol functionalities, monolignols present three main forms: p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. In nature, particularly in plants, monolignols with geometry (E) predominate over their Z counterparts. The methods for obtaining the three canonical monolignols, two less-common monolignols, and a monolignol analogue are addressed to present an overview of these phenol-based compounds, particularly from a synthetic standpoint. A SWOT (Strengths, Weaknesses, Opportunities, and Threats) analysis is used to explain the advantages and disadvantages of synthesizing monolignols, key alcohol-containing raw materials with enormous significance in both plant biology and industrial applications, using bench chemical methods. The uniqueness of this work is that it provides an overview of the synthetic pathways of monolignols to assist researchers in pharmaceutical and biological fields in selecting an appropriate procedure for the preparation of their lignin models. Moreover, we aim to inspire scientists, particularly chemists, to develop more sustainable synthetic protocols for monolignols. Full article

Cell-free protein synthesis (CFPS) has emerged as a powerful tool for protein production, with applications ranging from basic research to biotechnology and pharmaceutical development. However, enhancing the efficiency of CFPS systems remains a crucial challenge for realizing their full potential. Computational strategies offer promising avenues for optimizing CFPS efficiency by providing insights into complex biological processes and enabling rational design approaches. This review provides a comprehensive overview of the computational approaches aimed at enhancing CFPS efficiency. The introduction outlines the significance of CFPS and the role of computational methods in addressing efficiency limitations. It discusses mathematical modeling and simulation-based approaches for predicting protein synthesis kinetics and optimizing CFPS reactions. The review also delves into the design of DNA templates, including codon optimization strategies and mRNA secondary structure prediction tools, to improve protein synthesis efficiency. Furthermore, it explores computational techniques for engineering cell-free transcription and translation machinery, such as the rational design of expression systems and the predictive modeling of ribosome dynamics. The predictive modeling of metabolic pathways and the energy utilization in CFPS systems is also discussed, highlighting metabolic flux analysis and resource allocation strategies. Machine learning and artificial intelligence approaches are being increasingly employed for CFPS optimization, including neural network models, deep learning algorithms, and reinforcement learning for adaptive control. This review presents case studies showcasing successful CFPS optimization using computational methods and discusses applications in synthetic biology, biotechnology, and pharmaceuticals. The challenges and limitations of current computational approaches are addressed, along with future perspectives and emerging trends, such as the integration of multi-omics data and advances in high-throughput screening. The conclusion summarizes key findings, discusses implications for future research directions and applications, and emphasizes opportunities for interdisciplinary collaboration. This review offers valuable insights and prospects regarding computational strategies to enhance CFPS efficiency. It serves as a comprehensive resource, consolidating current knowledge in the field and guiding further advancements. Full article

Since the development of recombinant DNA technologies, the need to establish biosafety and biosecurity measures to control genetically modified organisms has been clear. Auxotrophies, or conditional suicide switches, have been used as firewalls to avoid horizontal or vertical gene transfer, but their efficacy has important limitations. The use of xenobiological systems has been proposed as the ultimate biosafety tool to circumvent biosafety problems in genetically modified organisms. Xenobiology is a subfield of Synthetic Biology that aims to construct orthogonal biological systems based on alternative biochemistries. Establishing true orthogonality in cell-based or cell-free systems promises to improve and assure that we can progress in synthetic biology safely. Although a wide array of strategies for orthogonal genetic systems have been tested, the construction of a host harboring fully orthogonal genetic system, with all parts operating in an orchestrated, integrated, and controlled manner, still poses an extraordinary challenge for researchers. In this study, we have performed a thorough review of the current literature to present the main advances in the use of xenobiology as a strategy for biocontainment, expanding on the opportunities and challenges of this field of research. Full article

Cooperation between catabolism and anabolism is crucial for maintaining homeostasis in living cells. The most fundamental systems for catabolism and anabolism are the glycolysis of sugars and the transcription–translation (TX-TL) of DNA, respectively. Despite their importance in living cells, the in vitro reconstitution of their cooperation through purified factors has not been achieved, which hinders the elucidation of the design principle in living cells. Here, we reconstituted glycolysis using sugars and integrated it with the PURE system, a commercial in vitro TX-TL kit composed of purified factors. By optimizing key parameters, such as glucokinase and initial phosphate concentrations, we determined suitable conditions for their cooperation. The optimized system showed protein synthesis at up to 33% of that of the original PURE system. We observed that ATP consumption in upstream glycolysis inhibits TX-TL and that this inhibition can be alleviated by the co-addition of glycolytic intermediates, such as glyceraldehyde 3-phosphate, with glucose. Moreover, the system developed here simultaneously synthesizes a subset of its own enzymes, that is, glycolytic enzymes, in a single test tube, which is a necessary step toward self-replication. As glycolysis and TX-TL provide building blocks for constructing cells, the integrated system can be a fundamental material for reconstituting living cells from purified factors. Full article

The Escherichia coli (E. coli)-based protein synthesis using recombinant elements (PURE) system is a cell-free protein synthesis system reconstituted from purified factors essential for E. coli translation. The PURE system is widely used for basic and synthetic biology applications. One of the major challenges associated with the PURE system is that the protein yield of the system varies depending on the protein. Studies have reported that the efficiency of translation is significantly affected by nucleotide and amino acid sequences, especially in the N-terminal region. Here, we investigated the inherent effect of various N-terminal sequences on protein synthesis using the PURE system. We found that a single amino acid substitution in the N-terminal region significantly altered translation efficiency in the PURE system, especially at low temperatures. This result gives us useful suggestions for the expression of the protein of interest in vitro and in vivo. Full article

The increasing demand for sustainable alternatives underscores the critical need for a shift away from traditional hydrocarbon-dependent processes. In this landscape, biomanufacturing emerges as a compelling solution, offering a pathway to produce essential chemical materials with significantly reduced environmental impacts. By utilizing engineered microorganisms and biomass as raw materials, biomanufacturing seeks to achieve a carbon-neutral footprint, effectively counteracting the carbon dioxide emissions associated with fossil fuel use. The efficiency and specificity of biocatalysts further contribute to lowering energy consumption and enhancing the sustainability of the production process. Within this context, cell-free synthesis emerges as a promising approach to accelerate the shift towards biomanufacturing. Operating with cellular machinery in a controlled environment, cell-free synthesis offers multiple advantages: it enables the rapid evaluation of biosynthetic pathways and optimization of the conditions for the synthesis of specific chemicals. It also holds potential as an on-demand platform for the production of personalized and specialized products. This review explores recent progress in cell-free synthesis, highlighting its potential to expedite the transformation of chemical processes into more sustainable biomanufacturing practices. We discuss how cell-free techniques not only accelerate the development of new bioproducts but also broaden the horizons for sustainable chemical production. Additionally, we address the challenges of scaling these technologies for commercial use and ensuring their affordability, which are critical for cell-free systems to meet the future demands of industries and fully realize their potential. Full article

With the increasing demand for therapeutic antibodies, CHO cells have become the de facto standard as producer host cells for biopharmaceutical production. High production yields are required for antibody production, and developing a high-titer production system is increasingly crucial. This study was established to develop a high-production system using a synthetic biology approach by designing a gene expression system based on an artificial transcription factor that can strongly induce the high expression of target genes in CHO cells. To demonstrate the functionality of this artificial gene expression system and its ability to induce the high expression of target genes in CHO cells, a model antibody (scFv-Fc) was produced using this system. Excellent results were obtained with the plate scale, and when attempting continuous production in semi-continuous cultures using bioreactor tubes with high-cell-density suspension culture using a serum-free medium, high-titer antibody production at the gram-per-liter level was achieved. Shifting the culture temperature to a low temperature of 33 °C achieved scFv-Fc concentrations of up to 5.5 g/L with a specific production rate of 262 pg/(cell∙day). This artificial gene expression system should be a powerful tool for CHO cell engineering aimed at constructing high-yield production systems. Full article