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At least 19 recordsLinked to original sources
PubMed2026

Biological Parts in Yeast Synthetic Biology: From Regulatory Elements to Predictive Design Platforms.

Yeasts, particularly Saccharomyces cerevisiae, are important eukaryotic chassis for synthetic biology because of their tractable genetics, versatile toolkits, and broad utility in metabolic engineering and functional genomics. Progress in this field has been driven by biological parts that enable programmable control of gene expression and cellular behavior. Early efforts focused mainly on promoters, terminators, and other regulatory elements for tuning individual genes. However, as engineering expanded to multigene pathways, genetic circuits, and dynamic regulatory systems, the limits of part-centric design became clear. Part performance is often shaped by genomic context, chromatin state, host physiology, and interactions with other components, which restricts modularity and predictability. In response, yeast synthetic biology is shifting toward integrated design frameworks combining multilayer regulation, standardized assembly, automated experimentation, and computational modeling. This review provides an integrated perspective on the evolution of biological parts across DNA-, RNA-, and protein-level regulation, connecting these advances with assembly frameworks, biofoundries, and machine learning to trace the trajectory from part-centric engineering toward predictive, system-level design in yeast synthetic biology.

Biofoundry↗
PubMed2026

A Comprehensive Review on the Biosynthesis of Tropane Alkaloids.

Tropane alkaloids (TA) constitute a class of plant specialized metabolites with important pharmaceutical applications, including the anticholinergic agents hyoscyamine and scopolamine and the local anesthetic cocaine. Over the past decade, advances in genomics, structural biology, and synthetic biology have substantially revised our understanding of TA biosynthesis, leading to the identification of numerous key biosynthetic enzymes and evolutionary mechanisms. This review comprehensively summarizes current knowledge of TA biosynthesis from precursor formation to structurally diverse end products. We describe the pathway from putrescine to tropinone, the stereoselective metabolic branching mediated by Tropinone Reductases, and the downstream biosynthesis of medicinal tropane alkaloids, calystegines, and cocaine. Particular emphasis is placed on recent discoveries concerning catalytic mechanisms, structural determinants of substrate specificity, metabolic compartmentalization, and the convergent evolution of TA biosynthesis in Solanaceae and Erythroxylaceae. We further integrate advances in genomics, evolutionary biology, and metabolic engineering to highlight emerging strategies for microbial production and pathway redesign. By providing a comprehensive synthesis of recent progress and critical perspectives on unresolved questions, this review offers an updated framework for understanding TA biosynthesis and supports future research in plant specialized metabolism, synthetic biology, and natural product engineering.

Tropanes↗
PubMed2026

Application of emerging technologies in the antiviral field.

Viral diseases pose a serious threat to global public health, agriculture, and biosecurity. Conventional antiviral strategies are often limited by an incomplete understanding of disease mechanisms, poor targeting precision, and slow response times. Emerging technologies are now reshaping the landscape of antiviral research. This review examines the roles of four key frontiers, including organoid models, gene editing, AI-driven molecular design, and synthetic biology. Organoids provide physiologically relevant platforms that model virus-host interactions and disease progression. Viral infections remain a major challenge to human and animal health, agriculture, and biosecurity. Progress in antiviral research is constrained by the complexity of viral pathogenesis, the diversity and rapid evolution of viruses, and the limited translational relevance of some traditional model systems. Recent advances in organoid technology, gene editing, artificial intelligence, and synthetic biology are expanding the toolkit available for antiviral research and development. In this review, we discuss how these four technological frontiers contribute to disease modeling, target discovery, molecular design, and translational innovation. Organoids, in particular, provide physiologically relevant systems for investigating viral infection, tissue tropism, host responses, and pathogenesis. Gene editing tools, such as CRISPR, enable precise manipulation of host and viral genomes, facilitating the development of resistant organisms and next-generation vaccine platforms. AI technologies, including AlphaFold for structure prediction and platforms for de novo protein design, address long-standing bottlenecks in structural biology and offer powerful means to engineer antiviral proteins, antibodies, and vaccine antigens. Synthetic biology, guided by the Design-Build-Test-Learn cycle, integrates computational design, genetic assembly, and functional validation into a cohesive pipeline. Together, these technologies form a synergistic workflow that spans disease modeling, target discovery, molecular design, construction, testing, and iterative optimization. This integrated approach is shifting antiviral development from traditional empirical methods toward more precise, intelligent strategies. The review also highlights ongoing challenges in integration and scalability, stressing that high-quality biological datasets and stronger interdisciplinary collaboration are essential for realizing translational potential. By presenting a cohesive view of these converging methodologies, this review offers a framework to guide the intelligent evolution of antiviral strategies in both human and animal health.

Antiviral↗
PubMed2025

FluxRETAP: a REaction TArget Prioritization genome-scale modeling technique for selecting genetic targets.

MOTIVATION: Metabolic engineering is rapidly evolving as a result of new advances in synthetic biology tools and automation platforms that enable high throughput strain construction, as well as the development of machine learning tools (ML) for biology. However, selecting genetic engineering targets that effectively guide the metabolic engineering process is still challenging. ML can provide predictive power for synthetic biology, but current technical limitations prevent the independent use of ML approaches without previous biological knowledge. RESULTS: Here, we present FluxRETAP, a simple and computationally inexpensive method that leverages the prior mechanistic knowledge embedded in genome-scale models for suggesting targets for genetic overexpression, downregulation or deletion, with the final goal of increasing the production of a desired metabolite. This method can provide a list of desirable engineering targets that can be combined with current ML pipelines. FluxRETAP captured 100% of reaction targets experimentally verified to improve Escherichia coli isoprenol production, 50% of targets that experimentally improved taxadiene production in E. coli and ∼60% of genetic targets from a verified minimal constrained cut-set in Pseudomonas putida, while providing additional high priority targets that could be tested. Overall, FluxRETAP is an efficient algorithm for identifying a prioritized list of testable genetic and reaction targets. AVAILABILITY AND IMPLEMENTATION: FluxRETAP is implemented in python and released under the creative commons license. The implementation and code are freely available at: https://github.com/JBEI/FluxRETAP.

Escherichia coli↗
PubMed2026

CREAT: A CRISPR-Based Genome Trimming Strategy for Systematic Identification of Dispensable Regions and Rapid Genome Reduction.

The construction of minimal-genome microbes offers an ideal platform for understanding fundamental biological processes and synthetic biology, yet the research is hindered by incomplete lists of essential genes in microbes and by multiple rounds of genome trimming with a trial-and-error nature. To address this, we introduce CREAT (CRISPR-based genome trimming with a multi-homology-arm template)-a streamlined approach that integrates CRISPR-targeted genome cleavage and homology arm walking to classify essential from non-essential genomic subregions, thus providing the basis for predicting essential genes in a given organism. These essential genes were then assembled into synthetic gene cassettes for one-step replacement of the targeted non-deletable genomic regions for further genome trimming. Eight consecutive rounds of CREAT genome trimming achieved a 20.8% reduction in genome size in Saccharolobus islandicus. Furthermore, Cas9-based CREAT genome trimming was developed for Bacillus subtilis and Escherichia coli, with efficiency greatly enhanced by the λ-Red recombinase in the latter. Together, this iterative application of CREAT provides a scalable and generally applicable strategy for rapidly constructing minimal genomes across diverse microorganisms.

CRISPR-Cas Systems↗
PubMed2026

Modular synthetic cross-kingdom promoters enable coordinated expression in Escherichia coli and Saccharomyces cerevisiae.

Synthetic biology and metabolic engineering increasingly demand predictable and interoperable gene expression across phylogenetically distant organisms, as the need for portable genetic systems and transferable metabolic pathways continues to grow. However, fundamental differences in promoter architecture and transcriptional logic across kingdoms remain a key bottleneck in developing universal expression platforms. Here, we designed a set of modular hybrid promoters that enable tunable and quantitatively consistent gene expression in both Escherichia coli and Saccharomyces cerevisiae. These promoters integrate bacterial -10/-35 motifs and Shine-Dalgarno sequences with minimal yeast TATA boxes and Kozak sequences to ensure transcriptional and translational compatibility. The promoter set supported weak, moderate, and strong expression with high relative consistency across species. Applied to the biosynthetic pathway for the valuable pigment prodeoxyviolacein, the hybrid promoters enabled coordinated production in both hosts. This work establishes a broadly compatible promoter architecture and provides a foundational toolkit for cross-kingdom, multi-host synthetic biology.

Promoter Regions, Genetic↗
PubMed2026

Host-aware Identification of Intrinsic Gene Expression Biopart Parameters using Combinatorial Libraries.

Model-based design in synthetic biology is limited because bioparts are typically characterised by relative metrics that vary across genetic and physiological contexts. To address this, we introduce a host-aware framework for quantitatively characterising bioparts in combinatorial libraries of plasmid-based constitutive expression constructs. The approach integrates a digital twin of Escherichia coli, conditioned on measured growth rate, with model-in-the-loop parameter identification to separate biopart-associated properties from host-dependent effects. Using structured combinatorial libraries, we identify mechanistically interpretable, transferable parameters for plasmid origins, promoters and ribosome binding sites. In particular, we define an intrinsic translation initiation capacity that captures the dominant RBS-associated contribution to translation while context-dependent expression emerges from host physiology and local sequence context. The resulting parameterisation accurately predicts protein synthesis across physiological conditions, supports incremental library expansion, and reveals localised failures of modularity, providing a scalable foundation for predictive host-aware design in synthetic biology.

Escherichia coli↗
PubMed2025

Efficient site-specific integration of kilobase-length DNA fragments in plant cells via Kp03 recombinase.

Targeted insertion of large DNA sequences into plant genomes remains a major challenge in synthetic biology. Here, we evaluate the large serine recombinase Kp03 for site-specific integration of DNA fragments in rice and Arabidopsis. In transient protoplast assays, Kp03 mediates efficient insertion of donor DNA up to 27.3 kilobases (kb), with plasmid integration efficiencies reaching 99.1% for fragments up to 3.4 kb. Truncation experiments reveal that a minimal 15-bp attB sequence is necessary for integration. As a proof of concept, Kp03 successfully incorporates a 3.4-kb donor DNA into the rice genome at a locus containing this minimal attB sequence. Moreover, in rice callus, combining Kp03 with the NM-PE genome editing system to install a 26-bp attB site enables targeted integration of a 3.4-kb donor at the desired genomic locus. These findings establish Kp03 as a versatile tool for plant genome engineering, with broad applications for synthetic biology.

Oryza↗
PubMed2026

Synthetic transcriptional repression systems in plants.

Transcriptional repression is a fundamental regulatory mechanism that enables precise control of gene expression in response to developmental signals and environmental stimuli. Synthetic biology can leverage this process within plants to engineer programmable transgene repression systems. This review examines strategies for harnessing prokaryotic repressors in eukaryotic systems to develop synthetic repression systems in plants. These systems utilize modular promoter and repressor architectures that can be tuned through operator placement and repression-domain fusion, respectively, to adjust transcriptional regulation. Chemically dependent inducibility can also be introduced either through use of native derepression mechanisms of the prokaryotic repressors or the incorporation of ligand-binding domains. Finally, this review explores key challenges in designing synthetic repression systems, including kinetics constraints, balancing ON and OFF states, and differences between transient and transgenic expression contexts. Overall, this review highlights modular design frameworks for tunable transgene expression in plants.

Gene Expression Regulation, Plant↗
PubMed2026

Bacterial R-bodies with common morphologies and unrolling dynamics are phylogenetically scattered, indicating extensive lateral gene transfer and wide application potential.

Refractile bodies (R-bodies) of gram-negative bacteria are large proteinaceous assemblies, rolled up in the form of an Archimedean spiral. They exhibit rapid rod-like reversible extension in the micrometer range when cued by chemical environmental triggers and have potential for synthetic biology and biochip applications. Initially described for the Paramecium endosymbionts Caedibacter taeniospiralis and Caedimonas varicaedens, R-bodies have since been discovered in many classes of Pseudomonadota, both in endosymbionts and in non-endosymbionts. However, despite the fact that the genetics and morphologies, as well as the unrolling kinetics of R-bodies from different species, show considerable diversity, no recent study has integrated these aspects into a single framework. The latter would be advantageous for the creation of an R-body biotechnology toolbox, where different properties determine the application area. Here, we have examined the R-bodies from six different Pseudomonadota, comprising both phylogenetically diverse endosymbionts and non-endosymbionts. Comparison of the morphologies of the rolled-up and unrolled forms, obtained using electron microscopy and high-quality images, to their corresponding genetic data indicates that extensive lateral gene transfer has occurred, which confounds a common framework based on these data. However, we have also studied the R-body extension and retraction kinetics using high frame-rate light microscopic video recordings, where we show for the first time that R-bodies can be classified into two classes, showing "fast burst" or "slow" acid-induced extension kinetics, respectively. We propose that this criterion may, in fact, be the most useful for the choice of an R-body tool for biotechnological purposes.IMPORTANCER-bodies are unique proteinaceous macromolecular structures capable of massive reversible extension in response to external environmental triggers without the input of chemical energy. They comprise only a few small polypeptides, which makes them potentially highly amenable to tuning via genetic engineering, as well as being exceptionally stable. These properties would be highly desirable in biotechnology and synthetic biology, as well as in biochip applications, where a controlled mechanical extensor might play an integral part in a nanoscale molecular machine. So far, only R-bodies from a single species, Caedibacter taeniospiralis, have been characterized extensively. However, in recent years, genomic information has revealed that a panoply of R-bodies are widely distributed among gram-negative phyla, although studies have generally not included morphological data. This study brings these two areas together to provide a holistic overview of the field and also reveals new insights into key dynamic aspects of R-body extension.

R-bodies↗
PubMed2025

Analog epigenetic memory revealed by targeted chromatin editing.

Cells store information by means of chromatin modifications that persist through cell divisions and can hold gene expression silenced over generations. However, how these modifications may maintain other gene expression states has remained unclear. This study shows that chromatin modifications can maintain a wide range of gene expression levels over time, thus uncovering analog epigenetic memory. By engineering a genomic reporter and epigenetic effectors, we tracked the gene expression dynamics following targeted perturbations to the chromatin state. We found that distinct grades of DNA methylation led to corresponding, persistent gene expression levels. Altering the DNA methylation grade, in turn, resulted in permanent loss of gene expression memory. Consistent with experiments, our chromatin modification model indicates that analog memory arises when the positive feedback between DNA methylation and repressive histone modifications is lacking. This discovery will lead to a deeper understanding of epigenetic memory and to new tools for synthetic biology.

Epigenesis, Genetic↗
PubMed2024

Strategies in engineering sustainable biochemical synthesis through microbial systems.

Growing environmental concerns and the urgency to address climate change have increased demand for the development of sustainable alternatives to fossil-derived fuels and chemicals. Microbial systems, possessing inherent biosynthetic capabilities, present a promising approach for achieving this goal. This review discusses the coupling of systems and synthetic biology to enable the elucidation and manipulation of microbial phenotypes for the production of chemicals that can substitute for petroleum-derived counterparts and contribute to advancing green biotechnology. The integration of artificial intelligence with metabolic engineering to facilitate precise and data-driven design of biosynthetic pathways is also discussed, along with the identification of current limitations and proposition of strategies for optimizing biosystems, thereby propelling the field of chemical biology towards sustainable chemical production.

Metabolic Engineering↗
PubMed2025

A comprehensive review of genomic-scale genetic engineering as a strategy to improve bacterial productivity.

Bacterial genome engineering has evolved to provide increasingly precise, robust and rapid tools, driving the development and optimization of bacterial production of numerous compounds. The field has progressed from early random mutagenesis methods, labour-intensive and inefficient, to rational and multiplexed strategies enabled by advances in genomics and synthetic biology. Among these tools, CRISPR/Cas has stood out for its versatility and its ability to achieve precision levels ranging from 50% to 90%, compared to the 10-40% obtained with earlier techniques, thereby enabling remarkable improvements in bacterial productivity. Nevertheless, like its predecessors, it still demands continuous refinement to reach full maturity. In this context, the present review addresses the lack of a unified overview by summarizing historical milestones and practical applications of genomic engineering tools in bacteria. It integrates diverse approaches to provide a comprehensive perspective on the evolution and prospects of these fundamental biotechnological tools.

Bacteria↗
PubMed2026

Multichannel genomic recording of biological information with ENGRAM.

Molecular recording is an emerging paradigm for measuring biology over time. Enhancer-mediated genomic recording of activity in multiplex (ENGRAM) is a recently described synthetic biology circuit architecture that converts the transient activity of cis-regulatory elements (CREs) into stable genomic records that can be retrospectively recovered via DNA sequencing. Here we provide a step-by-step protocol for conducting ENGRAM experiments and analyzing the resulting data. We also describe key design considerations for ENGRAM recorders, summarize the strengths and limitations of ENGRAM, and highlight applications, including multiplex signal recording and high-throughput CRE screening. In contrast to other systems for DNA-based recording in mammalian systems, ENGRAM relies on prime editing-mediated insertions to record the activity of a given CRE, such that it is inherently multiplexable-for example, four-base-pair insertions can represent the activities of up to 256 distinct CREs. A further contrast lies with ENGRAM's compatibility with DNA Typewriter, which facilitates the capture of signal order. For users with basic skills in molecular biology, mammalian cell culture and DNA sequencing analysis, ENGRAM experiments can typically be completed within 5-6 weeks.

Genomics↗
PubMed2026

Integrative quantum and systems biology of cancer: From molecular fluctuations to ecological outcomes.

This review treats cancer as a multiscale adaptive system, asks what the framework must predict to be worth adopting, and separates at each scale what the evidence establishes from what is proposed. It is an expert narrative synthesis, not a systematic review, and states the limits of that design. Proton transfer and tautomeric shifts contribute to spontaneous mispairing but do not license claims of directed or non-random mutation: replication timing, three-dimensional chromatin organization, sequence context and known mutagenic processes explain most mutational heterogeneity, leaving any quantum contribution as a residual against that baseline. The Waddington quasi-potential is bounded: outside detailed balance the dynamics are not gradient-derivable and require a probability-flux term. Hysteresis, rate-limited bimodality and return to state after perturbation distinguish an attractor from a transcriptomic cluster. Single-cell karyotype and live-imaging evidence supports whole-genome doubling as an unstable intermediate of heterogeneous origin and context-dependent consequence, not a uniform adaptive strategy. Systems and synthetic biology, virtual cells and digital twins are assessed against benchmarks, not promise. Tissue-scale ecology is reported with the spatial measurements now quantifying it, including evidence that stromal niche construction is not uniformly tumor-supporting. RNA modification is a layer in its own right, showing that the interpretation of a regulatory signal, not its magnitude, is biologically decisive. A dedicated section states the framework's commitments, the observable and evidence at each scale, and what would falsify them, asking what this adds to somatic mutation theory with clonal evolution and plasticity.

Neoplasms↗
PubMed2026

Engineering Bacillus Subtilis for Efficient Biosynthesis of Riboflavin: Current Knowledge and Future Perspectives.

Riboflavin is an essential water-soluble vitamin that serves as a precursor for the biosynthesis of the flavin cofactors FMN and FAD, which play pivotal roles in numerous redox and energy metabolism reactions. With the growing global demand for sustainable vitamin production, microbial fermentation has become an attractive alternative to chemical synthesis due to its environmental and economic advantages. Among microbial hosts, Bacillus subtilis has emerged as a leading cell factory for riboflavin production owing to its GRAS status, well-characterized genetics, and efficient protein secretion system. This review provides a comprehensive overview of recent advances in metabolic engineering strategies to enhance riboflavin biosynthesis in B. subtilis. Key topics include strengthening biosynthetic and precursor pathways, relieving feedback inhibition, balancing metabolic flux and cell growth, employing adaptive laboratory evolution, and utilizing omics-guided optimization and 13C metabolic flux analysis. Moreover, the integration of synthetic biology tools such as riboswitch engineering, regulatory element design, and high-throughput screening has significantly accelerated strain improvement. Despite remarkable progress, challenges remain in achieving precise regulatory control, optimizing multi-gene expression, and enhancing genome integration efficiency. Future research combining multi-omics data, synthetic regulatory design, and machine learning-driven predictive modeling is expected to further advance the development of intelligent B. subtilis cell factories. However, the practical implementation of these systems remains constrained by the metabolic burden of overproduction and the lack of universal regulatory models that can predict strain performance across varying industrial scales.

Bacillus subtilis↗
PubMed2026

A dual-dimensional CRISPR toolkit enables one-step high-efficiency multiplex genome editing in Komagataella phaffii.

Against the backdrop of green biomanufacturing, engineering methanol-utilizing Komagataella phaffii (K. phaffii) represents an effective strategy to expand the one carbon (C1) product profile and speed up the industrialization of C1-based bioeconomy. To address the technical challenges of low efficiency and cumbersome experimental procedures for multiplex gene editing and precise large-fragment integration during the reconstruction of complex metabolic pathways in K. phaffii, this study established a CRISPR toolkit - Efficient Multi-Gene Editing System 3.0 (EMGES 3.0) - which enabled one-step large-fragment integration coupled with multiplex gene knockout. EMGES 3.0 was constructed through the synergistic optimization of a repair-engineered chassis and an episomal CRISPR vector. For chassis engineering, five DNA repair modules: Δlig4 (DNA Ligase IV, non-homologous end joining end ligation), ppMRE11(The endogenous MRE11 gene from Pichia pastoris) overexpression (The Meiotic Recombination 11, DNA double-strand break end resection), Δrad9 (Radiation-Sensitive 9, DNA damage checkpoint regulation), Δmph1 (Mutator Phenotype Helicase 1, improvement of homologous recombinant strand extension), and PapRecT-PaSSB co-expression (stabilization of recombination intermediates) were integrated to generate the highly recombinogenic strain Y09. For vector engineering, cenARS was replaced by panARS and the endogenous promoter PGAP was employed to drive the double hammerhead ribozyme-single guide RNA-hepatitis delta virus ribozyme (double HH-sgRNA-HDV: dHgH)-mediated sgRNA expression, yielding the optimized vector Nov_pGAP_panARS_pLAT1_Cas9. These two features on K. phaffii together enhanced the EMGES 3.0 to a higher standard of transformation rate and editing efficiency. According to our results, EMGES 3.0 achieved dual-functional gene knockout efficiencies between 76.6% and 100%. For insertion of medium-long fragments (>4.5 kb), the efficiency achieved 93.3%. In addition, the one-step integration of ultra-long fragments (>16 kb) achieved 14.8%, which was reported for the first time. Furthermore, the efficiency of simultaneous long-fragment integration at three neutral loci reached 38.4% (>15 kb). We applied the system for one-step production of free fatty acids (FFAs, yield: 5.82 ∼ 7.30 mg/L/OD600) and resveratrol (yield: 1.14 ∼ 1.28 mg/L) using methanol as the sole carbon source. EMGES 3.0 provides a robust technical foundation for complex compounds biosynthesis and high-yield industrial strains, while also advancing K. phaffii as an industrial synthetic biology chassis for efficient C1 utilization.

CRISPR-Cas Systems↗
PubMed2026

Identification and Catalytic Optimization of Pinene Oxidases in Paeoniflorin Biosynthetic Pathway.

Paeoniflorin is a pharmacologically important cage-like monoterpene glycoside characteristic of Paeonia plants, yet its biosynthetic pathway has remained largely unresolved, hindering sustainable production. Here, we confirmed that paeoniflorin biosynthesis originates from α-pinene and identified three novel cytochrome P450 enzymes that catalyze pinene oxidation. CYP71AN126 catalyzes the hydroxylation of α-pinene at positions C4 and C10, followed by further oxidation of the alcohol to a ketone at C4, whereas CYP76A225/226 exclusively catalyze C10 hydroxylation. Virus-induced gene silencing (VIGS) assays demonstrated that silencing CYP71AN126, but not CYP76A225 and CYP76A226, significantly reduced the paeoniflorin content, indicating that C4 hydroxylation plays an important role in paeoniflorin biosynthesis, whereas C10 hydroxylation is not. Through the analysis of natural sequence and activity divergence among CYP71AN126 and CYP76A225/226, combined with protein structure prediction and site-directed mutagenesis, we identified L493 as a critical residue involved in regulating catalytic site specificity and substrate specificity of CYP71AN126. Mutation of L493 reduced or eliminated the formation of undesired C10 hydroxylation side-product and enhanced substrate specificity. These findings establish C4 oxidation of α-pinene as the critical committed step in paeoniflorin biosynthesis. Our study lays a foundation for elucidating the complete biosynthetic pathway of paeoniflorin in Paeonia and provides a target for enzyme engineering of CYP71AN126 aimed at the efficient production of paeoniflorin via synthetic biology approaches.

Paeonia genus↗